Anti-influenza virus pharmaceutical composition, preparation and application thereof

Abstract

The invention provides an anti-influenza virus pharmaceutical composition, a preparation and application thereof, and belongs to the technical field of biological medicine. The pharmaceutical composition comprises andrographolide and tetrandrine, has double effects, not only can inhibit in-vivo and in-vitro replication of influenza viruses, but also can relieve inflammation caused by the influenza viruses, and has a synergistic interaction effect. The andrographolide and the tetrandrine in the pharmaceutical composition are high in safety, the raw materials are rich and easy to obtain and prepare, and the pharmaceutical composition is suitable for industrial production. The invention provides a choice for clinical anti-influenza virus medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a drug combination, a preparation and uses thereof for anti-influenza virus. Background Art

[0002] Tetrandrine is a bisbenzylisoquinoline alkaloid extracted from the root tuber of Stephania tetrandra S. Moore, which is one of the main active ingredients of Stephania tetrandra S. Moore and has the effects of immunosuppression, antipyretic analgesia, diuresis and detumescence, anti-inflammation, etc.

[0003] Andrographolide is extracted from Andrographis paniculata, which belongs to an annual herbaceous plant. Its secondary metabolite andrographolide belongs to diterpenoid lactone compounds. Andrographolide exhibits antibacterial, antiviral, anti-inflammatory, immunomodulatory and anticancer and other biological activities in in vivo and in vitro studies.

[0004] Influenza A virus (IAV) is a virus that can cause global pandemics and has been plaguing human life and health. The outbreak of influenza virus is closely related to seasons and geographical environments. Most influenza viruses will continuously evolve through antigenic drift and antigenic shift, and can infect multiple age groups. The clinical manifestations are high fever, headache, cough, limb soreness and other systemic and respiratory symptoms of varying degrees. In severe cases, it can cause complications and even death. The genetic material instability of influenza virus and its adaptability to the environment enable it to evolve rapidly. Although there are vaccines against the influenza virus prevalent in the current season, the rapid evolution of influenza virus makes it resistant to antiviral drugs, thus increasing the difficulty of preventing influenza virus. With the evolution of influenza virus, it is difficult to guarantee that there will be no more drug-resistant influenza virus strains in the future. Moreover, the complications caused by cytokine storms induced by influenza virus also need to be alleviated. Therefore, an effective anti-influenza virus drug is needed. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a drug combination, a preparation and uses thereof for anti-influenza virus, and this drug combination can synergistically inhibit influenza virus and the inflammation caused by it.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions: The present invention provides a drug combination for anti-influenza virus, and the drug combination includes andrographolide and tetrandrine.

[0007] Preferably, the active ingredients of the drug combination are composed of andrographolide and tetrandrine.

[0008] Preferably, the drug combination further includes pharmaceutically acceptable excipients.

[0009] Preferably, the mass ratio of andrographolide to tetrandrine is 15:5 to 25.

[0010] Preferably, the mass ratio of andrographolide to tetrandrine is 15:6, 15:12, 15:18 or 15:24.

[0011] The present invention provides a solid or liquid preparation for anti-influenza virus, comprising the above drug combination.

[0012] Preferably, the solid or liquid preparation further comprises a sodium carboxymethylcellulose solution.

[0013] Preferably, the mass-volume percentage of the sodium carboxymethylcellulose solution is 3 to 7%; the mass-volume ratio of andrographolide, tetrandrine and the sodium carboxymethylcellulose solution is 15 mg: (5 - 25) mg: (18 - 22) mL.

[0014] The present invention provides an application of the above drug combination or solid or liquid preparation in the preparation of anti-influenza virus drugs.

[0015] Preferably, the influenza virus includes influenza A virus.

[0016] The present invention provides an application of the above drug combination or solid or liquid preparation in the preparation of drugs for inflammation caused by influenza virus.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a drug combination, preparation and use for anti-influenza virus. The drug combination has dual effects, can both inhibit the replication of influenza virus in vivo and in vitro, and can relieve the inflammation caused by influenza virus, showing a synergistic effect. And the andrographolide and tetrandrine in the drug combination of the present invention have high safety, rich raw materials, are easy to obtain and prepare, and are suitable for industrial production. The present invention provides a new choice for anti-influenza virus drugs in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shows the effects of different groups on the cytopathic effect of H1N1 influenza A virus infection. A shows the results of the combined drugs 1 - 6 inhibiting the cytopathic effect of H1N1 influenza A virus infection, B shows the results of the combined drugs 7 - 10 inhibiting the cytopathic effect of H1N1 influenza A virus infection, and C shows the results of the combined drug 11 inhibiting the cytopathic effect of H1N1 influenza A virus infection; Figure 2 Shows the results of andrographolide combined with tetrandrine inhibiting the replication of influenza virus in vitro; Figure 3 Shows the results of andrographolide combined with tetrandrine inhibiting the protein expression of influenza virus in vitro; Figure 4 Results of the changes in the mRNA levels of inflammatory factors caused by inhibiting influenza virus in vitro by tetrandrine combined with andrographolide; Figure 5 Results of the effects of tetrandrine, andrographolide, and combined oral suspension 1 on the survival rate of mice infected with influenza virus; Figure 6 Results of the effects of combined oral suspensions 1 - 5 on the survival rate of mice infected with influenza virus; Figure 7 Results of the effects of combined oral suspensions 1 - 2 and 6 - 7 on the survival rate of mice infected with influenza virus; Figure 8 Results of the effects of administering combined oral suspension 7 at different dosing times on the survival rate of mice infected with influenza virus; Figure 9 Results of the inhibition of influenza virus replication in vivo by combined oral suspension 7. A shows the effect of combined oral suspension 7 on the expression of the M1 gene in the lung tissue of a mouse model infected with influenza virus, and B shows the effect of combined oral suspension 7 on the expression of the NS1 gene in the lung tissue of a mouse model infected with influenza virus; Figure 10 Results of the inhibition of influenza virus protein expression in vivo by combined oral suspension 7; Figure 11 Results of the effect of combined oral suspension 7 on the lung index of a mouse model infected with influenza virus; Figure 12 Results of the effect of combined oral suspension 7 on the pathological damage of the lung tissue of a mouse model infected with influenza virus; Figure 13 Results of the effect of combined oral suspension 7 on the release of inflammatory factors in vivo caused by influenza virus. A shows the effect of combined oral suspension 7 on the release of IL - 6 caused by influenza virus, B shows the effect of combined oral suspension 7 on the release of IL - 1β caused by influenza virus, and C shows the effect of combined oral suspension 7 on the release of TNF - α caused by influenza virus. Detailed implementation mode

[0019] The present invention provides a drug combination for anti - influenza virus, and the drug combination includes andrographolide and tetrandrine.

[0020] In the present invention, tetrandrine, andrographolide, dihydroartemisinin, and baicalin are combined in different ways. It is found that the combination of tetrandrine and andrographolide has a better effect on treating influenza virus compared with other drug combinations, and has a synergistic effect compared with tetrandrine and andrographolide alone. In addition, it is found that the combination of tetrandrine and dihydroartemisinin, the combination of tetrandrine and baicalin, the combination of andrographolide and dihydroartemisinin, the combination of andrographolide and baicalin, the combination of dihydroartemisinin and baicalin, and the combination of three or more of tetrandrine, andrographolide, dihydroartemisinin, and baicalin cannot significantly inhibit the cytopathic effect caused by influenza virus. It can be seen that the selection of components in 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 consist of andrographolide and tetrandrine. In the present invention, the drug combination further includes pharmaceutically acceptable excipients. The excipients include one or more of solvents, binders, diluents, disintegrants, antioxidants, dispersants, wetting agents, solubilizers, buffers, and surfactants. The dosage forms of the drug preferably include suspensions, tablets, pills, solutions, injections, granules, capsules, or sprays. The drugs described in the present invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously, or nasally. The present invention has no special limitation on the sources of andrographolide and tetrandrine, and the known preparation methods in the art or commercially available products can be used.

[0021] The structure of andrographolide is as follows: 。

[0022] The structure of tetrandrine is as follows: 。

[0023] In the present invention, the mass ratio of andrographolide to tetrandrine is preferably 15:5 to 25. Further preferably, the mass ratio of andrographolide to tetrandrine is preferably 15:6 to 24, such as the mass ratio of andrographolide to tetrandrine being 15:6, 15:12, 15:18, or 15:24. When the present invention uses andrographolide and tetrandrine in different mass ratios to prevent and / or treat influenza virus, it is found that the combination of andrographolide and tetrandrine in the above mass ratios has a more significant anti-influenza virus effect and has a synergistic effect.

[0024] The present invention provides a solid or liquid preparation against influenza virus, comprising the above-mentioned drug combination. The solid or liquid preparation further comprises a sodium carboxymethylcellulose solution, and the mass-volume percentage of the sodium carboxymethylcellulose solution is preferably 3-7%, more preferably 4-6%, and still more preferably 5%. The solvent of the sodium carboxymethylcellulose solution is water. As an embodiment, the preparation method of the 5% sodium carboxymethylcellulose solution is to weigh 1 g of CMCNa and mix it evenly with 200 mL of deionized water. The mass-volume ratio of andrographolide, tetrandrine and the sodium carboxymethylcellulose solution is preferably 15 mg:(5-25) mg:(18-22) mL, more preferably 15 mg:(6-24) mg:(19-21) mL, and still more preferably 15 mg:(6-24) mg:20 mL, such as 15 mg:6 mg:20 mL, 15 mg:12 mg:20 mL, 15 mg:18 mg:20 mL or 15 mg:24 mg:20 mL. The solid or liquid preparation includes 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 carboxymethylcellulose can disperse andrographolide and tetrandrine evenly in the liquid, ensuring the uniformity of the administered dose.

[0025] The present invention studies and finds that after infection with influenza A virus H1N1, after treatment with the drug combination of tetrandrine and andrographolide or the solid or liquid preparation, the expression levels of viral NS1, M1 genes and NP, HA proteins are detected, and it is 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 can also significantly inhibit the increase in the levels of inflammatory factors such as IL-6, TNF-α and IL-1β caused by influenza virus. Therefore, the drug combination of tetrandrine and andrographolide or the solid or liquid preparation provided by the present invention can be applied to the treatment of influenza virus infection and the inflammation caused by influenza virus infection.

[0026] The present invention provides an application of the above-mentioned drug combination or solid or liquid preparation in the preparation of an anti-influenza virus drug.

[0027] In the present invention, the influenza virus includes influenza A virus, such as influenza A virus H1N1.

[0028] The present invention provides an application of the above-mentioned drug combination or solid or liquid preparation in the preparation of a drug for inflammation caused by influenza virus.

[0029] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art.

[0030] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] In the following embodiments, the preparation of the stock solutions of tetrandrine, andrographolide, dihydroartemisinin and baicalin (for cell experiments): Weigh 20 mg of tetrandrine precisely with an electronic balance and dissolve it in 6.4 mL of DMSO to prepare a 10 mM tetrandrine stock solution. Weigh 10 mg of andrographolide precisely with an electronic balance and dissolve it in 2.8 mL of DMSO to prepare a 10 mM andrographolide stock solution. Weigh 10 mg of dihydroartemisinin precisely with an electronic balance and dissolve it in 3.5 mL of DMSO to prepare a 10 mM dihydroartemisinin stock solution. Weigh 10 mg of baicalin precisely with an electronic balance and dissolve it in 2.2 mL of DMSO to prepare a 10 mM baicalin stock solution.

[0032] Example 1 The combination of andrographolide, tetrandrine, dihydroartemisinin and baicalin inhibits the cytopathic effect of influenza virus infection (1) A549 cell culture A549 cells were cultured in F12K medium containing 10% fetal bovine serum and 1% double antibody in an incubator at 37 °C and 5% CO 2 2. After observing that the cell growth state was good and culturing until the logarithmic growth phase, the A549 cells were seeded into a 96-well plate at 1×10 5 cells / well and continued to be cultured.

[0033] (2) Influenza virus inoculation The optimal concentrations of tetrandrine, andrographolide, dihydroartemisinin and baicalin for single drug administration were 6, 25, 10, and 25 μM respectively. The concentrations of pairwise combinations were halved, the concentrations of three-three combinations were reduced to 0.33 of the original, and the concentrations of four-drug combinations were changed to 0.25 of the original, as follows: Combined drug 1 was prepared by mixing 25 μM andrographolide solution and 25 μM baicalin solution in a volume ratio of 1:1 (denoted as And+Bai group); Combined drug 2 was prepared by mixing 25 μM andrographolide solution and 6 μM tetrandrine solution in a volume ratio of 1:1 (denoted as And+Tet group); Combined drug 3 was prepared by mixing 25 μM andrographolide solution and 10 μM dihydroartemisinin solution in a volume ratio of 1:1 (denoted as And+Dih group); Combined drug 4 was prepared by mixing 25 μM baicalin solution and 6 μM tetrandrine solution in a volume ratio of 1:1 (denoted as Bai+Tet group); The combined drug 5 was prepared by mixing a 25 μM baicalin solution and a 10 μM dihydroartemisinin solution in a volume ratio of 1:1 (denoted as the Bai+Dih group); The combined drug 6 was prepared by mixing a 6 μM tetrandrine solution and a 10 μM dihydroartemisinin solution in a volume ratio of 1:1 (denoted as the Tet+Dih group); The combined drug 7 was prepared by mixing a 25 μM andrographolide solution, a 25 μM baicalin solution and a 6 μM tetrandrine solution in a volume ratio of 1:1:1 (denoted as the And+Bai+Tet group); The combined drug 8 was prepared by mixing a 25 μM andrographolide solution, a 6 μM tetrandrine solution and a 10 μM dihydroartemisinin solution in a volume ratio of 1:1:1 (denoted as the And+Tet+Dih group); The combined drug 9 was prepared by mixing a 25 μM baicalin solution, a 6 μM tetrandrine solution and a 10 μM dihydroartemisinin solution in a volume ratio of 1:1:1 (denoted as the Bai+Tet+Dih group); The combined drug 10 was prepared by mixing a 25 μM andrographolide solution, a 25 μM baicalin solution and a 10 μM dihydroartemisinin solution in a volume ratio of 1:1:1 (denoted as the And+Bai+Dih group); The combined drug 11 was prepared by mixing a 25 μM andrographolide solution, a 25 μM baicalin solution, a 6 μM tetrandrine solution and a 10 μM dihydroartemisinin solution in a volume ratio of 1:1:1:1 (denoted as the And+Bai+Tet+Dih group).

[0034] A549 cells inoculated into a 96-well plate were cultured for 24 h. After growing to 40 - 60% cell confluence, under the conditions of 0.3% BSA (volume percentage), 0.2 μg / mL trypsin-TPCK, 1% double antibody (volume percentage) and F12K medium, 200 TCID 50 H1N1 influenza A virus (H1N1, A / Puerto Rico / 8 / 34) was used to infect A549 cells. After 1 h, the virus solution was discarded. Cell culture media containing combined drugs 1 - 11 (the cell culture media was F12K medium containing 0.3% BSA (volume percentage), 0.2 μg / mL trypsin-TPCK, 1% double antibody (volume percentage)) were added to the cells infected with influenza virus respectively. The Control group was A549 cells not infected with H1N1 influenza A virus and not treated with any drug, and the H1N1 group was A549 cells infected with H1N1 influenza A virus but not treated with any drug. They were cultured at 37 °C and 5% CO 2 Continue the culture.

[0035] (3) CCK8 assay for cell viability 37 °C, 5% CO 2 After culturing for 48 h, wash twice with PBS, add 100 μL of medium and 10 μL of CCK8 reagent to each well, and culture at 37 °C for 10 - 30 min. Record the absorbance OD value at 450 nm.

[0036] (4)Results Figure 1 The results showed that the combination of andrographolide and tetrandrine (i.e., combined drug 2) had a significant effect on inhibiting the cytopathic effect of influenza virus-infected cells, and the results were better than those of the triple, quadruple, and other pairwise drug combinations (i.e., combined drugs 1, 3 - 11). In addition, the Bai + Dih group, Tet + Dih group, And + Bai + Tet group, And + Tet + Dih group, Bai + Tet + Dih group, And + Bai + Dih group, and And + Bai + Tet + Dih group (i.e., combined drugs 5 - 11) could not inhibit the cytopathic effect of influenza virus-infected cells. It can be seen that not any drug combination can achieve the effect of inhibiting the cytopathic effect of influenza virus-infected cells.

[0037] Example 2 Inhibition of influenza virus replication in vitro by the combination of andrographolide and tetrandrine (1)A549 cell culture A549 cells were cultured in F12K medium containing 10% fetal bovine serum and 1% double antibiotics in an incubator at 37 °C, 5% CO 2 Observe that the cell growth state is good. After culturing to the logarithmic growth phase, inoculate A549 cells into a 12-well plate at 1×10 5 cells / well and continue to culture.

[0038] (2)Influenza virus inoculation The A549 cells inoculated into the 12-well plate were cultured for 24 h. After growing to 40 - 60% cell confluence, under the conditions of F12K medium containing 0.3% BSA (by volume), 0.2 μg / mL trypsin-TPCK, 1% double antibiotics (by volume), infect A549 cells with 200 TCID 50 H1N1 influenza A virus. After 1 h, discard the virus solution, and add the cell culture medium containing combined drug 2 of Example 1 (the cell culture medium is F12K medium containing 0.3% BSA (by volume), 0.2 μg / mL trypsin-TPCK, 1% double antibiotics (by volume)) to the cells infected with influenza virus, and continue to culture at 37 °C, 5% CO 2 Continue to culture.

[0039] (3)Q-PCR detection of virus gene changes Total cellular RNA was extracted using a kit, and the operation was carried out according to the instruction manual on the cells infected with influenza virus after culturing at 37 °C and 5% CO 2 for 2 days in step (2). First-strand cDNA synthesis premix kit was used to reverse-transcribe the extracted RNA according to the instruction manual, and Talent fluorescence quantitative detection kit (SYBR Green) was used to amplify the target genes of M1 and HA.

[0040] (4)Western blot detection of viral protein changes Cells were lysed with RIPA cell lysate, and phosphatase and protease inhibitors were added simultaneously. Proteins were separated from the total cell lysates by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk in TPBS for 2 h 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 4 times for 5 min with TPBS and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000) for 1 h at room temperature. After washing 5 times for 5 min in PBS-Tween buffer, the chemiluminescence of the labeled proteins was visualized using HRP substrate, and the chemiluminescence of the labeled proteins was captured under the instrument.

[0041] (5)Results The results were as Figure 2 shown. Andrographolide and tetrandrine combination (Combined drug 2 in Example 1) inhibited the mRNA levels of viral M1 and HA genes. The results were as Figure 3 shown. Andrographolide and tetrandrine combination (Combined drug 2 in Example 1) inhibited the expression of viral HA and NP proteins.

[0042] Example 3 Andrographolide and tetrandrine inhibit influenza virus-induced inflammation in vitro (1)A549 cell culture A549 cells were cultured in F12K medium containing 10% fetal bovine serum and 1% double antibody in an incubator at 37 °C and 5% CO 2 . After observing that the cell growth state was good and culturing to the logarithmic growth phase, A549 cells were seeded into 12-well plates at 1×10 5 cells / well and continued to be cultured.

[0043] (2)Influenza virus inoculation The cells seeded in the 12-well plates were cultured for 24 h. After growing to 40 - 60% cell confluence, under the conditions of 0.3% BSA by volume, 0.2 μg / mL trypsin-TPCK, 1% double antibody by volume, and F12K medium, 200 TCID 50The A549 cells were infected with influenza virus. After 1 h, the virus solution was discarded, and the cell culture medium containing the combined drug 2 of Example 1 (the cell culture medium was F12K medium containing 0.3% BSA, 0.2 μg / mL trypsin-TPCK, and 1% double antibody by volume) was added to the cells infected with influenza virus, and the cells were cultured at 37 °C and 5% CO 2 The culture was continued.

[0044] (3)Detection of virus gene changes by Q-PCR The total RNA extraction kit for cells was used and the operation was carried out with reference to the instruction manual to extract RNA from the cells infected with influenza virus after culturing at 37 °C and 5% CO 2 for 2 days in step (2). The cDNA first-strand synthesis premix kit was used and the operation was carried out with reference to the instruction manual to reverse-transcribe the extracted RNA, and the Talent fluorescence quantitative detection kit (SYBR Green) was used to amplify the target gene.

[0045] (4)Results The results were as Figure 4 shown. Andrographolide combined with tetrandrine (the combined drug 2 of Example 1) could significantly inhibit the increase in the mRNA levels of inflammatory factors such as IL-6 and TNF-α caused by influenza virus in vitro.

[0046] Example 4 At the animal level, the protective effect of the andrographolide and tetrandrine combined oral suspension on influenza model mice, the inhibitory effect on influenza virus in vivo, and the alleviation of inflammation caused by influenza virus were evaluated.

[0047] I. Protective effect of andrographolide, tetrandrine administered alone and in combination on mice infected with influenza virus (1)Preparation of andrographolide and tetrandrine combined oral suspension 1 g of sodium carboxymethylcellulose (CMCNa) was precisely weighed with an electronic balance and added to 200 mL of deionized water, and the mixture was shaken overnight on a shaker to prepare a 5% CMCNa solution. Then, 150 mg of andrographolide and 60 mg of tetrandrine were precisely weighed with an electronic balance respectively, and were suspended in 10 mL of 5% CMCNa respectively to prepare 6 mg / mL tetrandrine suspension and 15 mg / mL andrographolide suspension; the combined oral suspension 1 was prepared by suspending 150 mg of andrographolide and 60 mg of tetrandrine in 20 mL of 5% CMCNa. In the positive drug group, 20 mg of oseltamivir phosphate was weighed with an electronic balance and dissolved in 10 mL of physiological saline to prepare a 2 mg / mL oseltamivir phosphate solution.

[0048] (2)Establishment of influenza virus-infected mouse model: 6- to 8-week-old Balb / c mice were used in the experiment. They were randomly grouped and after 3 days of adaptive feeding, the H1N1 influenza A virus was diluted with PBS to 4LD 50 , and after the mice were anesthetized, 20 μL of the influenza virus was instilled into their noses. The normal group was instilled with normal saline into their noses.

[0049] (3)Grouping and administration methods: The mice were divided into a normal control group (Control), a positive drug group (H1N1+Ose), an influenza virus-infected group (H1N1), an andrographolide group (H1N1+And), a tetrandrine group (H1N1+Tet), and a combination group of andrographolide and tetrandrine (H1N1+And+Tet), with 10 mice in each group. Among them, the Control group was given 5% CMCNa by gavage to normal mice, 0.2 mL each time, once a day; H1N1+Ose was given oseltamivir phosphate solution at 2 mg / mL by gavage to influenza virus-infected mice, 0.2 mL each time, once a day; H1N1 (model group) was given 5% CMCNa by gavage to influenza virus-infected mice, 0.2 mL each time, once a day; H1N1+And was given andrographolide suspension at 15 mg / mL by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 150 mg / kg / d; H1N1+Tet was given tetrandrine suspension at 6 mg / mL by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 60 mg / kg / d; H1N1+Tet+And was given combined oral suspension 1 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 75 mg / kg / d And + 30 mg / kg / d Tet.

[0050] (4)Effects of tetrandrine, andrographolide, and combined oral suspension 1 on the survival and body weight of model mice The drugs were administered once a day for 7 consecutive days after virus challenge, and the observation lasted until 15 days. The survival of the mice was observed and recorded daily.

[0051] (5)Results As Figure 5The results showed that when using the combined oral suspension of andrographolide and tetrandrine, the death of mice infected with influenza A virus (IAV) could be delayed. The oral suspension of tetrandrine (60 mg / kg) and the oral suspension of andrographolide (150 mg / kg) alone could also improve the survival rate of influenza-infected mice. However, even when the dosage of the combined group of andrographolide and tetrandrine was reduced to 1 / 2 of the single-drug dosage, the survival rate was 66.7% higher than that of the model group. According to the equivalent half-dose addition method (see the evaluation of research methods for measuring the effects of combined drugs, Guo Jianyou et al., Pharmacology and Clinics of Chinese Materia Medica, 2005), q = 1.8 > 1 was calculated, and it was determined to be a synergistic effect. When the oral suspension of tetrandrine and the oral suspension of andrographolide were given alone, the survival rates were 37.5% and 37.5% higher than that of the model group, respectively. Therefore, the administration of andrographolide combined with tetrandrine could synergistically treat influenza A virus.

[0052] II. Protective effects of oral suspensions of andrographolide and tetrandrine with different proportion dosages on influenza-infected model mice S1. Preparation of combined oral suspensions of andrographolide and tetrandrine in different proportions: Precisely weigh 1 g of CMCNa using an electronic balance, add it to 200 mL of deionized water, and shake overnight in a shaker to prepare a 5% CMCNa solution. Then, precisely weigh 60 mg, 180 mg, and 360 mg of tetrandrine and 150 mg, 450 mg, and 900 mg of andrographolide using an electronic balance, respectively suspend them in 10 mL of 5% CMCNa, and prepare tetrandrine suspensions of 6 mg / mL, 18 mg / mL, and 36 mg / mL, and andrographolide suspensions of 15 mg / mL, 45 mg / mL, and 90 mg / mL, respectively; configure different ratios of andrographolide and tetrandrine (1:3, 1:6, 1:1, 3:1, 6:1, and this ratio value is calculated by comparing the dosages of andrographolide and tetrandrine with those in combined suspension 1), among which the combination of andrographolide and tetrandrine with a ratio of 1:1 is the above-mentioned combined oral suspension 1; the combination of andrographolide and tetrandrine with a ratio of 1:3 (denoted as combined oral suspension 2) is prepared by suspending 150 mg of andrographolide and 180 mg of tetrandrine in 20 mL of 5% CMCNa; the combination of andrographolide and tetrandrine with a ratio of 1:6 (denoted as combined oral suspension 3) is prepared by suspending 150 mg of andrographolide and 360 mg of tetrandrine in 20 mL of 5% CMCNa; the combination of andrographolide and tetrandrine with a ratio of 3:1 (denoted as combined oral suspension 4) is prepared by suspending 450 mg of andrographolide and 60 mg of tetrandrine in 20 mL of 5% CMCNa; the combination of andrographolide and tetrandrine with a ratio of 6:1 (denoted as combined oral suspension 5) is prepared by suspending 900 mg of andrographolide and 60 mg of tetrandrine in 20 mL of 5% CMCNa. For the positive drug group, weigh 20 mg of oseltamivir phosphate using an electronic balance, dissolve it in 10 mL of physiological saline, and prepare an oseltamivir phosphate solution of 2 mg / kg.

[0053] The method for establishing a mouse model infected with influenza virus is the same as step (2) of this example.

[0054] S3. Grouping and Administration Method: The mice were divided into a normal control group (Control), a positive drug group (H1N1+Ose), an influenza virus infection group (H1N1), a combination group of andrographolide and tetrandrine with a 1:1 ratio (1:1 H1N1+And+Tet), a combination group of andrographolide and tetrandrine with a 1:3 ratio (1:3 H1N1+And+Tet), a combination group of andrographolide and tetrandrine with a 1:6 ratio (1:6 H1N1+And+Tet), a combination group of andrographolide and tetrandrine with a 3:1 ratio (3:1H1N1+And+Tet), and a combination group of andrographolide and tetrandrine with a 6:1 ratio (6:1 H1N1+And+Tet), with 10 mice in each group. Among them, the Control group was given 5% CMCNa by gavage to normal mice, 0.2 mL each time, once a day; H1N1+Ose was given oseltamivir phosphate solution at 2 mg / mL by gavage to influenza virus-infected mice, 0.2 mL each time, once a day; H1N1 (model group) was given 5% CMCNa by gavage to influenza virus-infected mice, 0.2 mL each time, once a day; 1:1H1N1+Tet+And was given combined oral suspension 1 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 75 mg / kg / d And+30 mg / kg / d Tet; 1:3 H1N1+Tet+And was given combined oral suspension 2 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 75 mg / kg / d And+90 mg / kg / d Tet; 1:6 H1N1+Tet+And was given combined oral suspension 3 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 75 mg / kg / d And+180 mg / kg / d Tet; 3:1 H1N1+Tet+And was given combined oral suspension 4 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 225 mg / kg / d And+30 mg / kg / d Tet; 6:1 H1N1+Tet+And was given combined oral suspension 5 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 450 mg / kg / dAnd+30 mg / kg / d Tet.

[0055] S4. Effects of Combined Oral Suspensions 1-5 on the Survival of Model Mice.

[0056] Once a day after virus challenge for 7 consecutive days, and observed until 15 days. The survival of mice was observed and recorded daily.

[0057] S5. Results Figure 6The results showed that when using the combined oral suspension of andrographolide and tetrandrine in a ratio of 1:3, the death of mice infected with influenza virus could be significantly postponed, and the survival rate increased by about 50%, which was the highest. The 3:1 ratio had the lowest increase in the survival rate of mice, and the increase rates of the survival rates of other combinations with different ratios were relatively low.

[0058] III. Protective effects of combined oral suspensions of andrographolide and tetrandrine in different ratios on influenza model mice A1. Preparation of combined oral suspensions of andrographolide and tetrandrine in different ratios: Precisely weigh 1 g of CMCNa with an electronic balance, add it to 200 mL of deionized water, and shake it overnight on a shaker to prepare a 5% CMCNa solution. Then, precisely weigh 60 mg, 120 mg, 180 mg, and 240 mg of tetrandrine and 150 mg of andrographolide with an electronic balance respectively, and suspend them in 10 mL of 5% CMCNa respectively to prepare tetrandrine suspensions of 6 mg / mL, 12 mg / mL, 18 mg / mL, and 24 mg / mL, and an andrographolide suspension of 15 mg / mL; configure different ratios of andrographolide and tetrandrine (1:1, 1:2, 1:3, 1:4, and this ratio value is calculated by comparing the dosages of andrographolide and tetrandrine with those in combined suspension 1). Among them, the combination of andrographolide and tetrandrine with a ratio of 1:1 is the above-mentioned combined oral suspension 1; the combination of andrographolide and tetrandrine with a ratio of 1:2 (denoted as combined oral suspension 6) is prepared by suspending 150 mg of andrographolide and 120 mg of tetrandrine in 20 mL of 5% CMCNa; the combination of andrographolide and tetrandrine with a ratio of 1:3 (denoted as combined oral suspension 2) is prepared by suspending 150 mg of andrographolide and 180 mg of tetrandrine in 20 mL of 5% CMCNa; the combination of andrographolide and tetrandrine with a ratio of 1:4 (denoted as combined oral suspension 7) is prepared by suspending 150 mg of andrographolide and 240 mg of tetrandrine in 20 mL of 5% CMCNa. For the positive drug group, precisely weigh 20 mg of oseltamivir phosphate with an electronic balance, dissolve it in 10 mL of physiological saline, and prepare an oseltamivir phosphate solution of 2 mg / kg.

[0059] A2. The method for establishing the influenza virus-infected mouse model is the same as step (2) of this example.

[0060] A3. Grouping and administration method: The mice were divided into a normal control group (Control), a positive drug group (H1N1 + Ose), an influenza virus infection group (H1N1), a combination group of andrographolide and tetrandrine at a ratio of 1:1 (1:1 H1N1 + And + Tet), a combination group of andrographolide and tetrandrine at a ratio of 1:2 (1:2 H1N1 + And + Tet), a combination group of andrographolide and tetrandrine at a ratio of 1:3 (1:3 H1N1 + And + Tet), and a combination group of andrographolide and tetrandrine at a ratio of 1:4 (1:4 H1N1 + And + Tet), with 10 mice in each group. Among them, the Control group was given 5% CMCNa by gavage to normal mice, 0.2 mL each time, once a day; H1N1 + Ose was given oseltamivir phosphate solution at 2 mg / mL by gavage to influenza virus-infected mice, 0.2 mL each time, once a day; H1N1 (model group) was given 5% CMCNa by gavage to influenza virus-infected mice, 0.2 mL each time, once a day; 1:1 H1N1 + Tet + And was given combined oral suspension 1 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 75 mg / kg / d And + 30 mg / kg / d Tet; 1:2 H1N1 + Tet + And was given combined oral suspension 6 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 75 mg / kg / d And + 60 mg / kg / d Tet; 1:3 H1N1 + Tet + And was given combined oral suspension 2 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 75 mg / kg / d And + 90 mg / kg / d Tet; 1:4 H1N1 + Tet + And was given combined oral suspension 7 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 75 mg / kg / d And + 120 mg / kg / d Tet.

[0061] A4. Effects of combined oral suspensions 1 - 2 and 6 - 7 on the survival and body weight of influenza virus-infected model mice The drug was administered once a day for 7 consecutive days after virus challenge, and the observation lasted for 15 days. The survival of the mice was observed and recorded daily.

[0062] A5. Results Figure 7 The results showed that when using the combined oral suspension of andrographolide and tetrandrine at a ratio of 1:4, the death time of influenza virus-infected mice could be postponed, and the survival rate was the highest, increasing by about 60%, while the survival rate increase ratios of other combinations with different ratios were relatively low.

[0063] IV. Protective effects of andrographolide and tetrandrine oral suspensions administered at different times on influenza virus-infected model mice B1. Preparation of andrographolide and tetrandrine combination oral suspension: Precisely weigh 1 g of CMCNa using an electronic balance, add it to 200 mL of deionized water, and shake overnight on a shaker to prepare a 5% CMCNa solution. Then, precisely weigh 150 mg of andrographolide and 240 mg of tetrandrine using an electronic balance, and suspend them in 10 mL of 5% CMCNa to prepare 24 mg / mL tetrandrine suspension and 15 mg / mL andrographolide suspension respectively. The 1:4 ratio combination of andrographolide and tetrandrine (denoted as combination oral suspension 7) is prepared by suspending 150 mg of andrographolide and 240 mg of tetrandrine in 20 mL of 5% CMCNa. For the positive drug group, weigh 2 mg of oseltamivir phosphate using an electronic balance, dissolve it in 10 mL of physiological saline to prepare a 2 mg / kg oseltamivir phosphate solution.

[0064] B2. The method for establishing an influenza virus-infected mouse model (administering drugs on the first day after virus challenge) is the same as step (2) of this example.

[0065] B3. Grouping and administration methods: The mice are divided into a normal control group (Control), a positive drug group (H1N1+Ose), an influenza virus-infected group (H1N1), and a 1:4 ratio combination group of andrographolide and tetrandrine (1:4 H1N1+And+Tet), with 10 mice in each group. Among them, the Control group is given 5% CMCNa by gavage to normal mice, 0.2 mL each time, once a day; H1N1+Ose is given a 2 mg / mL oseltamivir phosphate solution by gavage to influenza virus-infected mice, 0.2 mL each time, once a day; H1N1 (model group) is given 5% CMCNa by gavage to influenza virus-infected mice, 0.2 mL each time, once a day; 1:4H1N1+Tet+And is given combination oral suspension 7 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 75 mg / kg / d And+120 mg / kg / d Tet.

[0066] B4. Effects of combination oral suspension 7 at different administration times on the survival of model mice Administer drugs at 0 (control group), 1, 3, and 5 days after virus challenge, once a day for 6 consecutive days, and observe until 15 days. Record the survival of mice every day.

[0067] B5. Results Figure 8The results showed that when using the combined oral suspension of andrographolide and tetrandrine in a ratio of 1:4, and administering it one day after infection, the death time of mice infected with influenza virus could be significantly postponed, the survival rate increased by 80%, and the survival rate was the highest, which was close to the protection rate of oseltamivir. The survival rate increase ratios at other different administration times were relatively low. After one day of infection, the later the combined drug intervention was carried out, the worse the treatment effect was.

[0068] Example 5 The combined oral suspension of andrographolide and tetrandrine inhibits the replication of influenza virus in vivo (1) Preparation of the combined oral suspension of andrographolide and tetrandrine Refer to step B1 of Example 4.

[0069] (2) Establishment of a mouse model infected with influenza virus In the experiment, 6 - 8 - week - old Balb / c mice were randomly grouped. After 3 days of adaptive feeding, the H1N1 influenza A virus was diluted with PBS to 4LD 50 , and after the mice were anesthetized, 20 μL of influenza virus was instilled into the nasal cavity. The normal group was instilled with normal saline into the nasal cavity.

[0070] (3) Grouping and administration methods: The mice were divided into a normal control group (Control), a positive drug group (H1N1 + Ose), an influenza virus - infected group (H1N1), and a combined group of andrographolide and tetrandrine with a ratio of 1:4 (1:4 H1N1 + And + Tet), with 10 mice in each group. Among them, the Control group was given 5% CMCNa by gavage to normal mice, 0.2 mL each time, once a day; H1N1 + Ose was given a 2 mg / mL oseltamivir phosphate solution by gavage to influenza - virus - infected mice, 0.2 mL each time, once a day; H1N1 (model group) was given 5% CMCNa by gavage to influenza - virus - infected mice, 0.2 mL each time, once a day; 1:4H1N1 + Tet + And was given the combined oral suspension 7 by gavage to influenza - virus - infected mice, 0.2 mL each time, once a day, that is, 75 mg / kg / d And + 120 mg / kg / d Tet.

[0071] (4) After virus challenge, the drug was administered on the first day for 4 consecutive days, once a day.

[0072] (5) Detection of virus gene changes in the lung tissue of influenza - infected mice by Q - PCR On the 4th day after taking the medicine for detoxification, the lung tissues of mice in each group were taken. The total RNA extraction kit for animal tissues was used to extract RNA from the lung tissues of mice in each group according to the instructions. The first-strand cDNA synthesis premix kit was used to reverse-transcribe the extracted RNA according to the instructions. The Talent fluorescence quantitative detection kit (SYBR Green) was used to amplify the target genes of M1 and NS1.

[0073] (6) Detection of changes in viral protein expression in the lung tissues of influenza-infected mice by Western blot Lung tissues were lysed with RIPA cell lysate, and phosphatase and protease inhibitors were added simultaneously. Proteins were separated from the total tissue lysate by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk in TPBS for 2 h 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 4 times for 5 min with TPBS and incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) (1:3000) for 1 h at room temperature. After washing 5 times for 5 min in PBS-Tween buffer, the chemiluminescence of the labeled protein was visualized using an HRP substrate, and the chemiluminescence of the labeled protein was captured using an instrument.

[0074] (7) Results Figure 9 The results showed that the combination oral suspension of andrographolide and tetrandrine (Combination oral suspension 7) could significantly inhibit the expression of M1 and NS1 genes in the lung tissues of the influenza virus-infected mouse model on the 4th day.

[0075] In addition, Figure 10 The results showed that the combination oral suspension of andrographolide and tetrandrine (Combination oral suspension 7) could also inhibit the expression of viral HA and NP proteins in the lung tissues of the influenza virus-infected mouse model on the 4th day.

[0076] Example 6 Andrographolide and tetrandrine oral suspension inhibits in vivo inflammation caused by influenza virus The preparation of the andrographolide and tetrandrine oral suspension refers to step B1 of Example 4.

[0077] (2) Establishment of an influenza virus-infected mouse model In the experiment, 6-8-week-old Balb / c mice were randomly grouped. After 3 days of adaptive feeding, the H1N1 influenza virus was diluted to 4LD with PBS 50 , and the mice were anesthetized and infected with 20 μL of influenza virus by nasal drip. The normal group was given normal saline by nasal drip.

[0078] (3)Grouping and administration method: The mice were divided into a normal control group (Control), a positive drug group (H1N1+Ose), an influenza virus infection group (H1N1), and a combination group of andrographolide and tetrandrine at a ratio of 1:4 (1:4 H1N1+And+Tet), with 10 mice in each group. Among them, the Control group was given 5% CMCNa by gavage to normal mice, 0.2 mL each time, once a day; H1N1+Ose was given oseltamivir phosphate solution at 2 mg / mL by gavage to influenza virus-infected mice, 0.2 mL each time, once a day; H1N1 (model group) was given 5% CMCNa by gavage to influenza virus-infected mice, 0.2 mL each time, once a day; 1:4H1N1+Tet+And was given the combined oral suspension 7 by gavage to influenza virus-infected mice, 0.2 mL each time, once a day, that is, 75 mg / kg / d And+120 mg / kg / d Tet.

[0079] (4)After virus challenge, drugs were administered on the first day for 4 consecutive days, once a day.

[0080] (5)Lung index determination Lung tissues of mice in each group were taken after 4 days, and the wet weight of the lungs was weighed with an electronic balance to calculate the lung index. Lung index = wet weight of lungs / body weight.

[0081] (6)HE pathological analysis Lung tissues of mice in each group were taken after 4 days, fixed with 4% paraformaldehyde, embedded in paraffin, cut into 5 µm sections, stained with hematoxylin-eosin staining (HE), and scanned with an electron microscope to analyze pathological changes.

[0082] (7)ELISA detection of changes in inflammatory factors in the lung tissues of influenza mice Alveolar lavage fluid of mice in each group was taken after 4 days, and experiments were carried out according to the ELISA instruction manual to detect inflammatory factors such as IL-6, TNF-α, and IL-1β.

[0083] (8)Results Figure 11 The results showed that the combined oral suspension of andrographolide and tetrandrine (combined oral suspension 7) could significantly reduce the lung index of model mice on the 4th day. Figure 12 The results showed that the treatment with the combined oral suspension of andrographolide and tetrandrine (combined oral suspension 7) could reduce the infiltration of inflammatory cells in mice and decrease the thickness of the alveolar space. Figure 13 The results showed that the oral suspension of andrographolide and tetrandrine could significantly inhibit the increase in the levels of inflammatory factors such as IL-6, TNF-α, and IL-1β caused by influenza virus.

[0084] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A drug combination for resisting influenza virus, characterized in that: The drug combination includes andrographolide and tetrandrine.

2. The drug combination according to claim 1, characterized in that The active ingredients of the drug combination consist of andrographolide and tetrandrine.

3. The drug combination according to claim 1 or 2, characterized in that: The mass ratio of the andrographolide to the tetrandrine is 15:5-25.

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

24.

5. A solid or liquid preparation for resisting influenza virus, characterized in that: Comprising the drug combination according to any one of claims 1 to 4.

6. The solid or liquid preparation according to claim 5, characterized in that The solid or liquid preparations also include sodium carboxymethylcellulose solutions.

7. The solid or liquid preparation according to claim 6, 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.

8. Use of the pharmaceutical combination according to any one of claims 1 to 4 or the solid or liquid preparation according to any one of claims 5 to 7 in the preparation of anti-influenza virus drugs.

9. The use according to claim 8, characterized in that: The influenza virus includes influenza A virus.

10. Use of the drug combination according to any one of claims 1 to 4 or the solid or liquid preparation according to any one of claims 5 to 7 in the preparation of drugs for treating inflammation caused by influenza virus.

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