Application of genipin-1-beta-D gentiobioside in medicine preparation

Abstract

The invention relates to an application of genipin-1-beta-D gentiobioside in preparation of medicines, in particular to an application of genipin-1-beta-D gentiobioside in preparation of medicines for treating respiratory tract inflammatory injury caused by coronavirus, respiratory syncytial virus or mycoplasma pneumoniae infection. The genipin-1-beta-D gentiobioside has the characteristics that the viral load, lung index and lung tissue inflammatory cytokines in lung tissues infected by coronavirus, respiratory syncytial virus or mycoplasma pneumoniae are reduced, and the genipin-1-beta-D gentiobioside has an obvious protective effect on death caused by coronavirus infection.

Description

Technical Field

[0001] The invention relates to an application of genipin-1-β-D gentiobioside, in particular to an application of genipin-1-β-D gentiobioside in preparing a medicine for treating coronavirus infection, respiratory syncytial virus infection or mycoplasma pneumoniae infection. Background Art

[0002] Coronavirus is the largest known RNA virus group. It is an enveloped virus with a positive-strand RNA genome. It has a wide range of natural hosts and is an important pathogen of humans and vertebrates. It can infect the human respiratory tract, gastrointestinal tract, liver, kidney and nervous system, and cause life-threatening pneumonia and bronchitis. In coronavirus (CoV), the envelope spike glycoprotein (S) is responsible for the entry of coronavirus into cells and its spread between cells. After coronavirus enters the cell through endosome engulfment, it releases viral RNA and nucleocapsid into the cytoplasm, replicates through the host, and completes the viral replication cycle. Coronavirus can cause acute respiratory infections in humans, especially the new coronavirus, which can cause acute pneumonia and death in severe cases.

[0003] At present, the drugs for treating coronavirus infection are mainly small molecule compounds that act on the viral proliferation cycle, such as Paxilovid, Monuprivir, Azvudine and VV116. Due to the single target and limited efficacy, these drugs are difficult to deal with virus mutation and drug resistance, especially for severe or fatal cases caused by factors such as inflammatory cytokine storm, disseminated intravascular coagulation, and low immune function.

[0004] Respiratory syncytial virus (RSV) is a circular negative-sense single-stranded RNA virus of the Parainfluenza virus family. Human respiratory syncytial virus is a common highly contagious virus that causes acute lower respiratory tract infection in infants and is the cause of high mortality in the elderly and children. RSV ranges from mild respiratory tract infection to severe respiratory tract infection, including pneumonia and bronchitis, and can cause serious complications such as respiratory failure. In the prior art, ribavirin is used as the preferred anti-RSV virus drug for respiratory syncytial virus pneumonia, but because ribavirin has multiple side effects, it is rarely used clinically. Therefore, for the treatment of respiratory syncytial virus pneumonia, comprehensive treatment (anti-infection, glucocorticoids, bronchoscope, anticoagulation, etc.) is currently mainly used, and effective drugs are still lacking in the clinic.

[0005] Mycoplasma pneumoniae (MP) is an important pathogen that causes respiratory infections in children. Mycoplasma pneumoniae pneumonia (MPP) caused by MP is currently the most common community-acquired pneumonia (CAP) in children aged 5 years and above in my country. Previous data showed that during epidemics, MPP accounted for 20% to 40% of CAP in the general population and up to 70% in closed populations. In the past, it was believed that the clinical manifestations of MPP were usually mild or even self-limiting. However, in recent years, more and more severe MPP (SMPP) have been reported.

[0006] In the prior art, macrolide antibiotics are the first choice for the treatment of Mycoplasma pneumonia, but drug resistance has continued to emerge in recent years, resulting in poor efficacy; while new tetracycline antibiotics, such as doxycycline and minocycline, are alternative drugs for the treatment of MPP, and have a definite effect on drug-resistant MPP, but may cause yellowing of the patient's teeth and enamel hypoplasia, so new tetracycline antibiotics are relatively limited in clinical application; quinolone antibiotics are alternative drugs for the treatment of MPP, and have a definite effect on macrolide-resistant MPP, and are used for suspected or confirmed MP-resistant MUMPP, RMMP, SMPP treatment, but due to the risk of quinolone antibiotics causing cartilage damage in young animals and tendon rupture in humans, their clinical application is limited. In summary, at present, comprehensive treatment (anti-infection, glucocorticoids, bronchoscopy, anticoagulation, etc.) is still the main treatment for Mycoplasma pneumonia, and there is still a lack of effective drugs in the clinic. Summary of the invention

[0007] The purpose of the present invention is to provide an application of genipin-1-β-D gentiobioside in the preparation of medicines. The present invention finds that genipin-1-β-D gentiobioside has the characteristics of reducing the viral load, lung index, and inflammatory cytokines in lung tissue after coronavirus infection, respiratory syncytial virus infection, or mycoplasma pneumoniae infection, and has a significant protective effect on death caused by coronavirus infection.

[0008] The technical solution of the present invention is the use of genipin-1-β-D gentiobioside in the preparation of a drug for treating respiratory tract damage caused by coronavirus, respiratory syncytial virus or mycoplasma pneumoniae infection.

[0009] The aforementioned application of genipin-1-β-D gentiobioside in the preparation of medicines, and the application of genipin-1-β-D gentiobioside in the preparation of medicines for treating inflammatory damage of pneumonia caused by coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

[0010] The aforementioned application of genipin-1-β-D gentiobioside in the preparation of medicines, and the application of genipin-1-β-D gentiobioside in the preparation of medicines with death protection effect for treating pneumonia caused by coronavirus infection.

[0011] In the use of the aforementioned genipin-1-β-D gentiobioside in the preparation of drugs, the coronavirus includes any one of the new coronavirus SARS-CoV-2, human coronavirus 229E, and human coronavirus OC43.

[0012] In the use of the aforementioned genipin-1-β-D gentiobioside in the preparation of drugs, the administration route of the genipin-1-β-D gentiobioside includes any one of atomization inhalation, oral administration, injection, sublingual administration, spray or anal administration; the dosage form of the drug includes any one of an inhalant, oral agent, injection, spray, film, and suppository.

[0013] An atomized inhalation agent containing genipin-1-β-D-gentiobioside is used to achieve the above application, and comprises the following raw materials: genipin-1-β-D-gentiobioside, a pH regulator for adjusting the pH value to 4.5-7.0, an osmotic pressure regulator accounting for 0-0.9% of the weight of genipin-1-β-D-gentiobioside, and a solvent.

[0014] In the aforementioned aerosol inhalation, the preparation method of the aerosol inhalation comprises the following steps:

[0015] (1) measuring 40% to 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0016] (2) When the temperature of the first solution is 20 to 80° C., the osmotic pressure regulator is added to the first solution and stirred evenly to obtain a second solution;

[0017] (3) adding a pH regulator and genipin-1-β-D-gentiobioside to the second solution to adjust the pH value of the second solution to a target value to obtain a third solution;

[0018] (4) Adding solvent to the third solution to make up to the total volume of the prepared solution, stirring evenly, and obtaining a nebulized inhalation preparation of genipin-1-β-D-gentiobioside.

[0019] An injection containing genipin-1-β-D gentiobioside, which realizes the above application, comprises the following raw materials: genipin-1-β-D gentiobioside, a pH regulator for adjusting the pH value to 4.5-7.0, an osmotic pressure regulator for adjusting the osmotic pressure to isotonic, and an injection solvent.

[0020] In the aforementioned injection, the preparation method of the injection comprises the following steps:

[0021] (1) measuring 40% to 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0022] (2) adding the osmotic pressure regulator to the first solution under the condition that the temperature of the first solution is 20 to 80° C. and stirring evenly to obtain a second solution;

[0023] (3) adding a pH regulator and genipin-1-β-D-gentiobioside to the second solution to adjust the pH value of the second solution to a target value to obtain a third solution;

[0024] (4) Adding solvent to the third solution to make up to the total volume of the prepared solution, stirring evenly, and obtaining an injection of genipin-1-β-D-gentiobioside.

[0025] A method for preparing genipin-1-β-D-gentiobioside, which is used to extract genipin-1-β-D-gentiobioside in the above application, comprises the following steps:

[0026] a. Take Gardenia jasminoides, extract with water, and concentrate the extract under reduced pressure to make the crude drug content in the extract 0.03 to 0.2 g / ml;

[0027] b. The extract was applied to a macroporous resin column, first eluted with 1 to 5 column volumes of deionized water, then eluted with 1 to 5 column volumes of ethanol at a volume concentration of 10 to 20%, the ethanol eluate was collected, the ethanol was recovered under reduced pressure, concentrated to 0.1 times the volume of the crude drug, ethanol was added to an alcohol volume concentration of 90%, allowed to stand, precipitated, filtered, the alcohol precipitate was passed through a neutral alumina column, successively eluted with 1 to 8 column volumes of ethanol at a volume concentration of 50 to 90%, the ethanol eluate at a volume concentration of 50 to 60% was collected, the ethanol was recovered under reduced pressure, and dried to obtain a crude product;

[0028] c. The crude product was purified 2 to 3 times by hot-melt recrystallization with ethanol. After the purified product was dried, the ethanol was removed to obtain high-purity genipin-1-β-D gentiobioside.

[0029] In the aforementioned method for preparing genipin-1-β-D gentiobioside, in step a, the specific steps of water extraction are: crushing the gardenia medicinal material, adding 12 times, 10 times, and 10 times the amount of water respectively, and decocting for 3 times, and the decocting time for each time is 1 to 1.5 hours; in step b, the weight ratio of the wet volume of the macroporous resin to the gardenia medicinal material is 3:2 to 3 ml / g, and the weight ratio of neutral alumina to the gardenia medicinal material is 1:3 to 3.5.

[0030] In the aforementioned method for preparing genipin-1-β-D gentiobioside, step b is specifically as follows: loading the gardenia extract onto an NKA-9 macroporous resin column, first eluting with 2 column volumes of deionized water, collecting the loading solution and the water eluate, combining and loading the X-5 macroporous resin, first eluting with 1 column volume of deionized water, then eluting with 5 column volumes of ethanol with a volume concentration of 10%, collecting the ethanol eluate, recovering the ethanol under reduced pressure, concentrating to a relative density of 1.08 to 1.15 at 60° C., adding ethanol to an alcohol volume concentration of 90%, placing, precipitating, filtering, passing the alcohol precipitate supernatant through a neutral alumina column, eluting with 6 column volumes of ethanol with a volume concentration of 90% and 4 column volumes of ethanol with a volume concentration of 60%, collecting the ethanol eluate with a volume concentration of 60%, recovering the ethanol under reduced pressure, and drying to obtain a crude product.

[0031] In the aforementioned method for preparing genipin-1-β-D gentiobioside, in step c, the method for recrystallizing the crude product after heat dissolving it in ethanol is specifically as follows: adding 0.5 to 1 times of anhydrous ethanol to the crude product, heating it under reflux to dissolve it, filtering it while hot, allowing it to precipitate, and filtering it to obtain a refined product.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention has discovered through animal experiments that genipin-1-β-D gentiobioside has a significant protective effect on the death of mice caused by the new coronavirus SARS-CoV-2 infection, and has a high lung index inhibition rate for mice infected with coronavirus, respiratory syncytial virus and Mycoplasma pneumoniae. It can reduce the viral load of mice infected with coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae, and reduce the content of inflammatory factors TNF-α, IL-6 and IL-10 in the lung tissue of mice infected with coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae.

[0034] Therefore, genipin-1-β-D gentiobioside has a definite and significant therapeutic effect on viral pneumonia caused by coronavirus, respiratory syncytial virus or mycoplasma pneumonia infection by reducing multiple inflammatory cytokines after coronavirus infection, respiratory syncytial virus infection or mycoplasma pneumonia infection, inhibiting inflammatory cytokine storm, and significantly reducing mortality, lung index, and lung tissue viral load. It shows that genipin-1-β-D gentiobioside has an anti-coronavirus, respiratory syncytial virus and mycoplasma pneumonia infection effect, and has a certain broad spectrum, can treat respiratory diseases, severe pneumonia and other diseases caused by coronavirus, respiratory syncytial virus and mycoplasma pneumonia infection, and has a death protection effect on inflammatory damage; it can be used in the preparation of drugs for treating respiratory diseases caused by coronavirus infection, respiratory syncytial virus infection or mycoplasma pneumonia infection, drugs for pneumonia inflammatory damage, and drugs with pneumonia death protection effect.

[0035] The administration routes of genipin-1-β-D gentiobioside include aerosol inhalation, oral administration, injection, sublingual administration, spray and rectal administration. Genipin-1-β-D gentiobioside can be made into a variety of preparations commonly used by medical students.

[0036] The present invention also provides a method for preparing genipin-1-β-D gentiobioside, wherein the crude product obtained by the method removes most of the pigments and iridoid impurities, and the content of genipin-1-β-D gentiobioside in the crude product is higher than 60%. The method can efficiently separate and obtain the effective part that meets the requirements of the new drug, wherein the content of the effective part reaches more than 50% of the extract. In the final obtained extract product, the purity of genipin-1-β-D gentiobioside reaches more than 96%.

[0037] In addition, in the preparation process of the present invention, toxic and harmful reagents are avoided and reusable macroporous resins are selected for purification. Therefore, the process route of the method is green, safe, relatively low in cost, and suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a graph showing the virus titer detection in the lung tissue of mice infected with the new coronavirus. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0040] The present invention discovers the application of genipin-1-β-D gentiobioside in preparing medicine for treating coronavirus, respiratory syncytial virus or mycoplasma pneumoniae infection.

[0041] Application of genipin-1-β-D gentiobioside in the preparation of medicines for treating respiratory tract damage caused by coronavirus, respiratory syncytial virus or mycoplasma pneumoniae infection.

[0042] Application of genipin-1-β-D gentiobioside in the preparation of medicines for treating inflammatory damage of pneumonia caused by coronavirus, respiratory syncytial virus or mycoplasma pneumoniae infection.

[0043] Application of genipin-1-β-D gentiobioside in the preparation of a drug with a death protection effect for treating pneumonia caused by coronavirus, respiratory syncytial virus or mycoplasma pneumoniae infection.

[0044] Furthermore, genipin-1-β-D gentiobioside is used in the preparation of drugs for treating increased levels of inflammatory factors TNF-α, IL-6, and IL-10 in lung tissue cells caused by coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

[0045] The coronavirus includes any one of the new coronavirus SARS-CoV-2, human coronavirus 229E, and human coronavirus OC43.

[0046] The genipin-1-β-D-gentiobioside described in the invention can be extracted from the traditional Chinese medicine Gardenia jasminoides, or prepared by artificial synthesis.

[0047] The method for extracting genipin-1-β-D-gentiobioside from the Chinese medicinal material Gardenia jasminoides can be carried out by the following steps:

[0048] a. Extraction of medicinal materials: Take the gardenia medicinal materials, crush the gardenia medicinal materials, add 12 times, 10 times, and 10 times the amount of water and boil three times, each boiling time is 1 to 1.5 hours, and the extract is concentrated under reduced pressure to make the crude drug content in the extract 0.03 to 0.2 g / ml;

[0049] b. Preparation of crude product: The extract is put on a macroporous resin column, the macroporous resin is preferably NKA-9 and X-5, and the weight ratio of wet volume to gardenia medicinal material is 3:2~3ml / g. First, elute with 1~5 times column volume of deionized water, then elute with 1~5 times column volume of ethanol with a volume concentration of 10~20%, collect the ethanol eluate, recover the ethanol under reduced pressure, concentrate to 0.1 times the volume of the crude drug, add ethanol to the alcohol volume concentration of 90%, place, precipitate, filter, and pass the alcohol precipitation supernatant through a neutral alumina column, the specification of neutral alumina is 100~200 mesh, and the weight ratio of wet volume to gardenia medicinal material is 1:3~3.5, and then elute with 1~8 times column volume of ethanol with a volume concentration of 50~90%, collect the ethanol eluate with a volume concentration of 50~60%, recover the ethanol under reduced pressure, and dry to obtain a crude product;

[0050] c. Preparation of genipin-1-β-D gentiobioside: The crude product is purified 2 to 3 times by ethanol hot dissolution recrystallization. The specific purification method is as follows: the crude product is added with 0.5 to 1 times of anhydrous ethanol, heated under reflux to dissolve it, filtered while hot, allowed to stand for precipitation, and filtered to obtain a purified product. After the purified product is dried, the ethanol is removed to obtain high-purity genipin-1-β-D gentiobioside.

[0051] The administration route of genipin-1-β-D gentiobioside includes any one of aerosol inhalation, oral administration, injection, sublingual administration, spray or anal administration.

[0052] The atomization inhalation and injection drug administration methods adopted by the present invention target the respiratory tract and lungs and directly administer the drugs. The drugs are transmitted to the respiratory tract and lungs in the form of atomization. It has the advantages of rapid onset of action, high local drug concentration in the inflammatory lesions of the respiratory tract and lungs, small dosage, convenient application and few systemic adverse reactions. It can be used as an important treatment for respiratory diseases.

[0053] The present invention also provides a medicine for treating coronavirus infection, comprising genipin-1-β-D gentiobioside and pharmaceutically acceptable excipients.

[0054] The dosage form of the drug includes any one of inhalation, oral preparation, injection, spray, film, and suppository.

[0055] Administration of the inhalation, injection, spray, suppository and film (non-oral film) can avoid the first-pass effect of the liver and intestine, and prevent the genipin-1-β-D gentiobioside containing glycosidic bonds in the structure from being degraded by gastric acid and intestinal bacteria and becoming ineffective.

[0056] A nebulizer inhaler containing genipin-1-β-D-gentiobioside for the above application comprises the following raw materials: 20-200 mg of genipin-1-β-D-gentiobioside, a pH regulator for adjusting the pH value to 4.5-7.0, an osmotic pressure regulator accounting for 0-0.9% of the weight of genipin-1-β-D-gentiobioside, and 1-5 ml of a solvent.

[0057] The preparation method of the aerosol inhalation containing genipin-1-β-D gentiobioside comprises the following steps:

[0058] (1) measuring 40% to 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0059] (2) When the temperature of the first solution is 20 to 80° C., the osmotic pressure regulator is added to the first solution and stirred evenly to obtain a second solution;

[0060] (3) adding a pH regulator and genipin-1-β-D-gentiobioside to the second solution to adjust the pH value of the second solution to a target value to obtain a third solution;

[0061] (4) Adding solvent to the third solution to make up to the total volume of the prepared solution, stirring evenly, and filtering with a 0.22 μm filter membrane or filter element to obtain a nebulized inhalation preparation of genipin-1-β-D-gentiobioside.

[0062] An injection containing genipin-1-β-D gentiobioside for the above application comprises the following raw materials: 20-200 mg of genipin-1-β-D gentiobioside, a pH regulator for adjusting the pH value to 4.5-7.0, an osmotic pressure regulator for adjusting the osmotic pressure to isotonicity, and 1-5 ml of an injection solvent.

[0063] The preparation method of the injection comprises the following steps:

[0064] (1) measuring 40% to 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0065] (2) adding the osmotic pressure regulator to the first solution under the condition that the temperature of the first solution is 20 to 80° C. and stirring evenly to obtain a second solution;

[0066] (3) adding a pH regulator and genipin-1-β-D-gentiobioside to the second solution to adjust the pH value of the second solution to a target value to obtain a third solution;

[0067] (4) Adding solvent to the third solution to make up to the total volume of the prepared solution, stirring evenly, and obtaining an injection of genipin-1-β-D-gentiobioside.

[0068] Extraction Example 1:

[0069] The preparation method of genipin-1-β-D gentiobioside comprises the following steps:

[0070] a. Extraction of medicinal materials: Take 2kg of Gardenia medicinal materials, crush the Gardenia medicinal materials, add 12 times, 10 times, and 10 times the amount of water and boil three times, each boiling time is 1 hour, combine and filter the extracts, and concentrate under reduced pressure to make the extract content of the crude drug 0.033g / ml;

[0071] b. Preparation of crude product: The Gardenia extract was loaded onto an NKA-9 macroporous resin column, first eluted with 2 column volumes of deionized water, the loading solution and the water eluate were collected, combined and loaded onto an X-5 macroporous resin, first eluted with 1 column volume of deionized water, then eluted with 5 column volumes of 10% ethanol by volume, the ethanol eluate was collected, ethanol was recovered under reduced pressure, concentrated to 200 ml, the relative density was 1.15 at 60 ° C, ethanol was added to an alcohol volume concentration of 90%, allowed to stand, precipitated, filtered, the alcohol precipitate was passed through a 100 mesh neutral alumina column, successively eluted with 6 column volumes of 90% ethanol by volume and 4 column volumes of 60% ethanol by volume, the ethanol eluate was collected with a volume concentration of 60%, ethanol was recovered under reduced pressure, and dried to obtain a crude product;

[0072] c. Preparation of genipin-1-β-D gentiobioside: The crude product was added with 1.2L of anhydrous ethanol, heated under reflux to dissolve it, filtered while hot, allowed to precipitate, and filtered to obtain a purified product-1; the purified product-1 was added with 1.4L of anhydrous ethanol, heated under reflux to dissolve it, filtered while hot, allowed to precipitate, filtered, and ethanol was removed to obtain a purified product-2. After the purified product-2 was dried (60°C, 0.7MPa), the ethanol was removed to obtain 11.7g of high-purity genipin-1-β-D gentiobioside with a mass content of 96.2%.

[0073] Extraction Example 2

[0074] The preparation method of genipin-1-β-D gentiobioside comprises the following steps:

[0075] a. Extraction of medicinal materials: Take 5 kg of gardenia medicinal materials, crush the gardenia medicinal materials, add 12 times the amount of water for the first time, extract for 1.5 hours, add 10 times the amount of water for the second time, extract for 1 hour, add 10 times the amount of water for the third time, extract for 1 hour, combine and filter the extracts, and concentrate under reduced pressure to make the crude drug content in the extract 0.2 g / ml;

[0076] b. Preparation of crude product: The Gardenia extract was loaded onto an NKA-9 macroporous resin column, first eluted with 3 column volumes of deionized water, the loading solution and the water eluate were collected, combined and loaded onto an X-5 macroporous resin, first eluted with 1 column volume of deionized water, then eluted with 3 column volumes of ethanol at a volume concentration of 20%, the ethanol eluate was collected, ethanol was recovered under reduced pressure, concentrated to 1L, the relative density was 1.08 at 60°C, ethanol was added to an alcohol volume concentration of 90%, allowed to stand, precipitated, filtered, the alcohol precipitate was passed through a 100 mesh neutral alumina column, eluted with 6 column volumes of 90% ethanol and 8 column volumes of 50% ethanol, the ethanol eluate was collected at a volume concentration of 50%, ethanol was recovered under reduced pressure, and dried to obtain a crude product;

[0077] c. Preparation of genipin-1-β-D gentiobioside: The crude product was added to 2.5L of anhydrous ethanol, heated to reflux to dissolve, filtered while hot, allowed to precipitate, and filtered to obtain a purified product-1; the purified product-1 was added to 2.5L of anhydrous ethanol, heated to reflux to dissolve, filtered while hot, allowed to precipitate, filtered, and ethanol was removed to obtain a purified product-2; the purified product-2 was added to 2.8L of anhydrous ethanol, heated to reflux to dissolve, filtered while hot, allowed to precipitate, filtered, and ethanol was removed to obtain a purified product-3. After the purified product-3 was dried (60°C, 0.7MPa), the ethanol was removed to obtain 20.7g of high-purity genipin-1-β-D gentiobioside with a mass content of 96.7%.

[0078] Preparation Example 1: Inhalation Administration - Solution for Inhalation

[0079] A nebulized inhalation solution containing genipin-1-β-D gentiobioside, the prescription composition is: genipin-1-β-D gentiobioside 20 mg, citric acid to adjust the pH value to 4.5, sodium chloride to adjust the osmotic pressure to isotonic, and solvent to 1 ml.

[0080] The preparation method of the atomized inhalation solution comprises the following steps:

[0081] (1) measuring 40% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0082] (2) Control the temperature of the first solution to 25°C, add sodium chloride and stir until dissolved to obtain the second solution

[0083] (3) adding citric acid to adjust the pH value of the solution to 4.5 to obtain a third solution;

[0084] (4) adding genipin-1-β-D-gentiobioside to the third solution, stirring evenly, and adding an appropriate amount of citric acid to maintain the pH of the solution at 4.5 to obtain a fourth solution;

[0085] (5) adding solvent to the fourth solution to make up the total volume of the solution required for preparation, and stirring evenly to obtain a fifth solution;

[0086] (6) filtering the fifth solution through a 0.22 μm filter membrane or filter element to obtain a sixth solution;

[0087] (7) Fill the sixth solution into an ampoule or vial of 1 ml and seal it to obtain a nebulized inhalation solution of genipin-1-β-D-gentiobioside.

[0088] Preparation Example 2: Inhalation Administration - Solution for Inhalation

[0089] A nebulized inhalation solution containing genipin-1-β-D gentiobioside, the prescription composition is: genipin-1-β-D gentiobioside 100 mg, citric acid 0.1%, sodium citrate to adjust pH value to 5.0, solvent to 3 ml.

[0090] The preparation method of the above-mentioned atomized inhalation solution comprises the following steps:

[0091] (1) measuring 60% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0092] (2) while controlling the temperature of the first solution to be 50° C., adding citric acid to the first solution and stirring evenly to obtain a second solution;

[0093] (3) adding sodium citrate to adjust the pH value of the solution to 5.0 to obtain a third solution;

[0094] (4) adding genipin-1-β-D-gentiobioside to the second solution, stirring evenly, and adding an appropriate amount of sodium citrate to maintain the pH value of the solution at 5.0 to obtain a fourth solution;

[0095] (5) adding solvent to the fourth solution to make up the total volume of the solution to be prepared, and stirring evenly to obtain a fifth solution;

[0096] (6) filtering the fifth solution through a 0.22 μm filter membrane or filter element to obtain a sixth solution;

[0097] (7) Fill the sixth solution into a 3 ml ampoule or vial and seal it to obtain a nebulized inhalation solution of genipin-1-β-D-gentiobioside.

[0098] Preparation Example 3: Inhalation Administration - Solution for Inhalation

[0099] A nebulized inhalation solution containing genipin-1-β-D gentiobioside, the prescription composition is: genipin-1-β-D gentiobioside 200 mg, sodium citrate 0.5%, sodium chloride to adjust the osmotic pressure to isotonic, sodium hydroxide to adjust the pH value to 7.0, and solvent to 5 ml.

[0100] The preparation method of the above-mentioned atomized inhalation solution comprises the following steps:

[0101] (1) measuring 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0102] (2) while controlling the temperature of the first solution to be 80° C., adding sodium citrate to the first solution and stirring evenly to obtain a second solution;

[0103] (3) adding sodium chloride and stirring until dissolved to obtain a third solution;

[0104] (4) adding sodium hydroxide to adjust the pH value of the solution to 7.0 to obtain a fourth solution;

[0105] (5) adding genipin-1-β-D-gentiobioside to the fourth solution, stirring evenly, and adding an appropriate amount of sodium hydroxide to maintain the pH value of the solution at 7.0 to obtain a fifth solution;

[0106] (6) adding solvent to the fifth solution to make up the total volume of the solution to be prepared, and stirring evenly to obtain a sixth solution;

[0107] (7) filtering the sixth solution through a 0.22 μm filter membrane or filter element to obtain a seventh solution;

[0108] (8) Fill the seventh solution into a 5 ml ampoule or vial and seal it to obtain a nebulized inhalation solution of genipin-1-β-D-gentiobioside.

[0109] Preparation Examples 1-3, when in use, place the nebulizer (the atomization principle can be air compression, vibrating mesh or ultrasound) on a flat surface, and keep the machine as far away from textiles as possible when working to prevent textile hair from clogging the machine's air inlet; after correctly installing the nebulizer cup according to the instruction manual. Open the medicine box, take out the ampoule or vial, transfer the liquid medicine therein to the nebulizer cup after aspirating it with a syringe, sit or stand in an upright position to ensure normal breathing, confirm that the nebulizer mask covers the mouth and nose or the nebulizer mouthpiece is placed in the mouth, turn on the nebulizer button, start nebulization, and continue inhaling until no more droplets are sprayed out. In order to reduce the risk of infection, disease or contamination, clean and disinfect the nebulizer according to the instructions after the treatment.

[0110] After stability testing, the quality and atomization characteristics of the three formulation examples after being placed at 25°C for 6 months all met the requirements.

[0111] Preparation Example 4: Inhalation Administration - Lyophilized Powder for Inhalation

[0112] A lyophilized powder for inhalation containing genipin-1-β-D gentiobioside, the prescription composition of which is: 20 mg of genipin-1-β-D gentiobioside, hydrochloric acid to adjust the pH value to 4.5, and solvent to 1 ml.

[0113] The preparation method of the above-mentioned freeze-dried powder for inhalation is:

[0114] (1) measuring 70% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0115] (2) controlling the temperature of the first solution to be 30° C., adding sodium hydroxide to the first solution, adjusting the pH value to 4.5, and stirring uniformly to obtain a second solution;

[0116] (3) adding genipin-1-β-D-gentiobioside to the second solution and stirring evenly to obtain a third solution;

[0117] (4) measuring the pH value. If the pH value is not between 4.4 and 4.6, an appropriate amount of sodium hydroxide is added again to adjust the pH value of the solution to 4.5 to obtain a fourth solution;

[0118] (5) adding solvent to the fourth solution to make up the total volume of the solution to be prepared, and stirring evenly to obtain a fifth solution;

[0119] (6) filtering the fifth solution through a 0.22 μm filter membrane or filter element to obtain a sixth solution;

[0120] (7) Fill the sixth solution into a 10 ml vial and half-stopper it.

[0121] (8) The above samples were transferred into a freeze dryer and freeze-dried according to the set freeze-drying curve: pre-freeze at -45°C for 6 hours, evacuate and heat to -15°C, perform sublimation drying, maintain for 10 hours, heat to 25°C, perform analytical drying, maintain for 4 hours. After the freeze-drying is completed, the stopper is fully pressed, the sample is taken out of the box, and the cap is rolled to obtain the freeze-dried powder of genipin-1-β-D-gentiobioside for inhalation.

[0122] Preparation Example 5: Inhalation Administration - Lyophilized Powder for Inhalation

[0123] A freeze-dried powder for inhalation containing genipin-1-β-D-gentiobioside, the prescription composition is: genipin-1-β-D-gentiobioside 80 mg, citric acid 0.1%, sodium citrate adjusted to pH 5.0, and solvent to 2 ml.

[0124] The preparation method of the above-mentioned freeze-dried powder for inhalation is:

[0125] (1) measuring 50% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0126] (2) while controlling the temperature of the first solution to be 40° C., adding citric acid and sodium citrate to the first solution, adjusting the pH value to 5.0, and stirring uniformly to obtain a second solution;

[0127] (3) adding genipin-1-β-D-gentiobioside to the second solution and stirring evenly to obtain a third solution;

[0128] (4) measuring the pH value. If the pH value is not between 4.9 and 5.1, an appropriate amount of sodium citrate is added again to adjust the pH value of the solution to 5.0 to obtain a fourth solution;

[0129] (5) adding solvent to the fourth solution to make up the total volume of the solution to be prepared, and stirring evenly to obtain a fifth solution;

[0130] (6) filtering the fifth solution through a 0.22 μm filter membrane or filter element to obtain a sixth solution;

[0131] (7) Fill the sixth solution into a 10 ml vial and half-stopper it.

[0132] (8) The above samples were transferred into a freeze dryer and freeze-dried according to the set freeze-drying curve: pre-freeze at -45°C for 6 hours, evacuate and heat to -15°C, perform sublimation drying, maintain for 15 hours, heat to 30°C, perform analytical drying, maintain for 6 hours. After the freeze-drying is completed, the stopper is fully pressed, the sample is taken out of the box, and the cap is rolled to obtain the freeze-dried powder of genipin-1-β-D-gentiobioside for inhalation.

[0133] Preparation Example 6: Inhalation Administration - Lyophilized Powder for Inhalation

[0134] A lyophilized powder for inhalation containing genipin-1-β-D gentiobioside, the prescription composition of which is: 200 mg of genipin-1-β-D gentiobioside, 0.1% of citric acid, disodium hydrogen phosphate to adjust the pH value to 7.0, and solvent to 5 ml.

[0135] The preparation method of the above-mentioned freeze-dried powder for inhalation is:

[0136] (1) measuring 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0137] (2) while controlling the temperature of the first solution to be 80° C., adding citric acid and disodium hydrogen phosphate to the first solution, adjusting the pH value to 7.0, and stirring uniformly to obtain a second solution;

[0138] (3) adding genipin-1-β-D-gentiobioside to the second solution and stirring evenly to obtain a third solution;

[0139] (4) measuring the pH value. If the pH value is not between 6.9 and 7.1, an appropriate amount of disodium hydrogen phosphate is added again to adjust the pH value of the solution to 7.0 to obtain a fourth solution;

[0140] (5) adding solvent to the fourth solution to make up the total volume of the solution to be prepared, and stirring evenly to obtain a fifth solution;

[0141] (6) filtering the fifth solution through a 0.22 μm filter membrane or filter element to obtain a sixth solution;

[0142] (7) Fill the sixth solution into a 10 ml vial and half-stopper it.

[0143] (8) The above samples were transferred into a freeze dryer and freeze-dried according to the set freeze-drying curve: pre-freeze at -45°C for 6 hours, evacuate and heat to -15°C, perform sublimation drying, maintain for 25 hours, heat to 20°C, perform analytical drying, maintain for 10 hours. After the freeze-drying is completed, the stopper is fully pressed, the sample is taken out of the box, and the cap is rolled to obtain the freeze-dried powder of genipin-1-β-D-gentiobioside for inhalation.

[0144] When using the samples prepared in formulation examples 4-6, place the nebulizer (the atomization principle can be air compression, vibrating mesh or ultrasound) on a flat surface, and keep the machine as far away from textiles as possible when working to avoid textile lint blocking the machine's air inlet; after correctly installing the nebulizer cup according to the instructions for use. Open the drug packaging box, take out the vial, use a syringe to draw 1 to 5 ml of water for injection, inject it into the vial, shake it to dissolve all the lyophilized powder, then draw the liquid medicine with a syringe and transfer it to the nebulizer cup, sit or stand in an upright position to ensure normal breathing, confirm that the nebulizer mask covers the mouth and nose or the nebulizer mouthpiece is placed in the mouth, turn on the nebulizer button, start nebulization, and continue inhaling until no more droplets are sprayed out. In order to reduce the risk of infection, disease or contamination, clean and disinfect the nebulizer according to the instructions after the treatment.

[0145] Through the stability test experiment, the contents and related substances of the freeze-dried powder obtained from the three formulation examples were relatively stable, and the atomization characteristics met the requirements.

[0146] Preparation Example 7: Injection Administration-Lyophilized Powder

[0147] A lyophilized powder for injection containing genipin-1-β-D gentiobioside, the prescription composition is: genipin-1-β-D gentiobioside 100 mg, sodium chloride to adjust the osmotic pressure to isotonic, sulfuric acid / sodium hydroxide to adjust the pH value to 6.0, water for injection to 3 ml.

[0148] The preparation method of the lyophilized powder for injection comprises the following steps:

[0149] (1) measuring 40% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0150] (2) adding sodium chloride to the first solution while controlling the temperature of the first solution to be 20° C. and stirring the solution to obtain a second solution;

[0151] (3) adding sodium hydroxide to the second solution, adjusting the pH value to 6.0, and stirring uniformly to obtain a third solution;

[0152] (4) adding genipin-1-β-D-gentiobioside to the third solution and stirring evenly to obtain a fourth solution;

[0153] (5) If the pH value is not between 5.9 and 6.1, an appropriate amount of sulfuric acid or sodium hydroxide is added again to adjust the pH value of the solution to 6.0 to obtain a fifth solution;

[0154] (6) adding water for injection to the fifth solution to make up the total volume of the solution required for preparation, and stirring evenly to obtain a sixth solution;

[0155] (7) filtering the sixth solution through a 0.22 μm filter membrane or filter element to obtain a seventh solution;

[0156] (8) Fill the seventh solution into a 10 ml vial and half-stopper it.

[0157] (9) The samples were transferred into a freeze dryer and freeze-dried according to the set freeze-drying curve: pre-freeze at -45°C for 6 hours, evacuate and heat to -20°C, perform sublimation drying, maintain for 14 hours, heat to -10°C, continue sublimation drying, maintain for 12 hours, after sublimation drying is completed, heat to 20°C, perform analytical drying, and keep warm for 4 hours. After freeze-drying is completed, fully press the stopper, take out of the box, and roll the cap.

[0158] Preparation Example 8: Injection Administration-Lyophilized Powder

[0159] A lyophilized powder for injection containing genipin-1-β-D gentiobioside, the prescription composition of which is: 200 mg of genipin-1-β-D gentiobioside, sodium chloride to adjust the osmotic pressure to isotonic, and water for injection to 5 ml.

[0160] The preparation method of the lyophilized powder for injection comprises the following steps:

[0161] (1) measuring 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0162] (2) controlling the temperature of the first solution to be 50° C., adding sodium chloride to the first solution and stirring the solution uniformly to obtain a second solution;

[0163] (3) adding genipin-1-β-D-gentiobioside to the second solution and stirring evenly to obtain a third solution;

[0164] (4) adding water for injection to the third solution to make up the total volume of the solution required for preparation, and stirring evenly to obtain a fourth solution;

[0165] (5) filtering the fourth solution through a 0.22 μm filter membrane or filter element to obtain a fifth solution;

[0166] (6) Fill the fifth solution into a 10 ml vial and half-stopper it.

[0167] (7) The samples were transferred into a freeze dryer and freeze-dried according to the set freeze drying curve: pre-freeze at -45 °C for 6 hours, evacuate and heat to -20 °C, perform sublimation drying, maintain for 20 hours, heat to -10 °C, continue sublimation drying, maintain for 16 hours, after sublimation drying is completed, heat to 30 °C, perform analytical drying, and keep warm for 8 hours. After freeze drying is completed, fully press the stopper, take out of the box, and roll the cap to obtain the product.

[0168] Preparation Example 9: Injection Administration-Lyophilized Powder

[0169] A lyophilized powder for injection containing genipin-1-β-D gentiobioside, the prescription composition of which is: 30 mg of genipin-1-β-D gentiobioside, appropriate amount of sodium dihydrogen phosphate and disodium hydrogen phosphate to adjust the pH value to 7.0, sodium chloride to adjust the osmotic pressure to isotonic, and water for injection to 5 ml.

[0170] The preparation method of the lyophilized powder for injection comprises the following steps:

[0171] (1) measuring 60% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0172] (2) adding sodium chloride to the first solution while controlling the temperature of the first solution to 80° C. and stirring the solution to obtain a second solution;

[0173] (3) adding sodium dihydrogen phosphate and disodium hydrogen phosphate to the second solution, adjusting the pH value to 7.0, and stirring uniformly to obtain a third solution;

[0174] (4) adding genipin-1-β-D-gentiobioside to the third solution and stirring evenly to obtain a fourth solution;

[0175] (5) Determine the pH value. If the pH value is not between 6.9 and 7.1, add an appropriate amount of sodium dihydrogen phosphate / sodium hydrogen phosphate to adjust the pH value of the solution to 7.0 to obtain a fifth solution;

[0176] (6) adding water for injection to the fifth solution to make up the total volume of the solution required for preparation, and stirring evenly to obtain a sixth solution;

[0177] (7) filtering the sixth solution through a 0.22 μm filter membrane or filter element to obtain a seventh solution;

[0178] (8) Fill the seventh solution into a 10 ml vial and half-stopper it.

[0179] (9) The above samples were transferred into a freeze dryer and freeze-dried according to the set freeze-drying curve: pre-freeze at -45°C for 6 hours, evacuate and heat to -20°C, perform sublimation drying, maintain for 18 hours, heat to -10°C, continue sublimation drying, maintain for 15 hours, after sublimation drying is completed, heat to 20-30°C, perform analytical drying, and keep warm for 7 hours. After freeze-drying is completed, the stopper is fully pressed, the sample is taken out of the box, and the cap is rolled to obtain the freeze-dried powder of genipin-1-β-D gentiobioside for injection.

[0180] When using the lyophilized powder prepared in Preparation Examples 7-9, open the medicine packaging box, take out the vial, use a syringe to draw 1-5 ml of water for injection, inject it into the vial, shake it to dissolve the lyophilized powder completely, and then use it by intramuscular injection, intravenous injection, etc. or mix it with other infusion solutions and then use it by infusion.

[0181] The stability test of the freeze-dried powders prepared in Preparation Examples 7-9 showed that the contents and related substances of the freeze-dried powders were relatively stable after being stored at 25°C for 6 months.

[0182] Pharmacological Experimental Example 1: The protective effect of intravenous injection of genipin-1-β-D gentiobioside on the death of the pneumonia model of mice infected with human novel coronavirus

[0183] 1 Test materials

[0184] 1.1 Test drug: Genipin-1-β-D-gentiobioside, properties: white powder, solubility: very soluble in water. The dosage for mice is 37.5 mg / kg and 75 mg / kg, intraperitoneal injection, once a day for 5 consecutive days.

[0185] 1.2 Experimental animals: hACE2 transgenic C57BL / 6 mice, 6-7 weeks old, 18-25 g, 32 in total, provided by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., license number SCXK(Su)2018-008.

[0186] 1.3 Experimental conditions: The experiment was carried out in the ABSL-3 laboratory of Guangzhou Institute of Respiratory Health.

[0187] 2 Test methods

[0188] 2.1 Grouping and administration: hACE2 transgenic C57BL / 6 mice were divided into 4 groups, namely, blank control group, SARS-CoV-2 infection group, genipin-1-β-D gentiobioside (37.5 mg / kg) administration group, and genipin-1-β-D gentiobioside (75 mg / kg) administration group, with 8 mice in each group. Except for the normal group, which was given PBS by intranasal drops, mice in other groups were infected with 104PFU of SARS-CoV-2 virus by intranasal drops. Two hours after infection, mice in the administration group were intraperitoneally injected, once a day, for 5 consecutive days. The death of mice was recorded daily after infection, and the 5-day mortality rate was calculated. After the experiment, lung tissue was dissected and homogenized to detect the virus titer.

[0189] 2.2 Detection of virus titer in mouse lung tissue homogenate:

[0190] After removing the mouse lung tissue, place it in a culture dish, cut it into pieces, transfer it to a homogenizer tube, dilute it with physiological saline 1:10 (w / v), and homogenize it at 8000rpm / min for 10 minutes. All the above operations are performed in an ice bath. Transfer the homogenate to a 1.5mL EP tube and centrifuge it at 4℃, 10000rpm for 10 minutes. Pipette the supernatant, aliquot it, and store it at -80℃ for later use.

[0191] VERO E6 cells with good growth status were inoculated in 96-well plates at 1×104 cells / well and cultured for 24 hours. After the cells attached to the wall and grew into a complete monolayer, the supernatant was discarded and the cells were washed twice with PBS. The frozen lung homogenate supernatant was thawed and diluted 10 times to 10 -1 ~10 -5 Five concentrations, add the diluted lung homogenate supernatant of each group of mice to the 96-well plate, add 100 μL of lung supernatant to each well, set up blank control wells at the same time, add cell culture medium to the blank control wells, make 4 replicates for each concentration in parallel, and culture in an incubator. Observe continuously for 4 days, observe the cytopathic effect (CPE) every day, record the number of lesion wells for each gradient concentration, and calculate its TCID50 value for VERO E6 cells.

[0192] 3 Test results

[0193] 3.1 Protective effect of genipin-1-β-D gentiobioside on death in mice infected with ARS-CoV-2-induced pneumonia

[0194] Table 1 Protective effect of genipin-1-β-D gentiobioside on death of mice caused by novel coronavirus ARS-CoV-2 infection

[0195]

[0196] The results in Table 1 show that no death occurred in the blank control group during the experiment; after SARS-CoV-2 virus infection, the mortality rate of mice in the model control group was 75%; the 37.5 mg / kg and 75 mg / kg dose groups of genipin-1-β-D gentiobioside were intraperitoneally injected once a day for 5 consecutive days, which can significantly reduce the number of deaths of mice after SARS-CoV-2 infection, with mortality rates of 37.5% and 25%, respectively. This shows that genipin-1-β-D gentiobioside has a significant protective effect on the death of mice caused by the new coronavirus SARS-CoV-2 infection, and has a good dose-effect correlation.

[0197] 3.2 Effect on virus titer in lung tissue of mice infected with SARS-CoV-2

[0198] like Figure 1 As shown, the results showed that after viral infection, a large amount of virus replication occurred in the lung tissue of mice in the model control group; after administration of 37.5 mg / kg and 75 mg / kg doses of genipin-1-β-D-gentiobioside, the virus titer in the lung tissue of mice was significantly reduced, which was significantly different from that of the model control group (P<0.05, P<0.01), and there was a good dose-effect correlation.

[0199] Pharmacological Experimental Example 2: Therapeutic Effect of Intravenous Injection of Genipin-1-β-D Gentianobioside on Pneumonia Model of Mouse Infected with Human Coronavirus 229E and OC43

[0200] 1 Test materials

[0201] 1.1 Test drug: Genipin-1-β-D-gentiobioside, properties: white powder, solubility: very soluble in water. The dosage for mice is 150 mg / kg, 75 mg / kg, and 37.5 mg / kg, administered by intravenous injection, once a day for 4 consecutive days.

[0202] 1.2 Positive drugs: Chloroquine phosphate tablets, Sichuan Shenghe Pharmaceutical Co., Ltd., batch number: 2002114, production date: February 26, 2020, expiration date: January 2022. Specification: 0.25g / tablet, dosage: 0.5g / 60kg / d, oral.

[0203] 1.3 Experimental animals

[0204] Table 2 Experimental animals

[0205]

[0206] 1.4 Virus strains and cells: Human coronavirus 229E (HCoV-229E) was provided by the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences; human coronavirus HCoV-OC43 was purchased from the ATCC bacterial strain storage center in the United States. It was passaged in this laboratory and stored in a -80℃ refrigerator for future use. Human embryonic lung fibroblasts MRC-5 were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, passaged in this laboratory, and stored in liquid nitrogen for future use.

[0207] 1.5 Test equipment

[0208] Table 3 Test instruments

[0209]

[0210] 1.6 Test site: ABSL-2 laboratory of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences

[0211] 2 Test methods

[0212] 2.1 Drug dosage design and preparation

[0213] Test drug: The dosage for mice was 150 mg / kg / d, 75 mg / kg / d, and 37.5 mg / kg / d;

[0214] Corresponding solutions were prepared with physiological saline and administered to mice by intravenous injection at a rate of 2 ml / 10 g / d, once a day for 4 consecutive days.

[0215] Chloroquine phosphate tablets: The clinical dose is: 0.5g / 60kg / d for humans, orally.

[0216] The mouse dose is: Convert the clinical dose to the mouse dose: 0.5g / 60kg / d×11=0.09g / kg / d

[0217] The dosage for mice is: 20 ml / kg / d, intragastric administration;

[0218] The preparation concentration is: 0.09g / kg / d÷20ml / kg / d=0.0045g / ml.

[0219] 2.2 Virus Passaging

[0220] Take a 25 cm2 culture flask with MRC-5 cells grown into a monolayer, discard the culture medium, rinse the cell surface with cell maintenance medium three times, add 5 ml of cell maintenance medium, and then add 200 μl of HCoV-229E or OC43 virus solution, and place it at 37°C with a volume concentration of 5% CO 2 The cells were cultured in an incubator for 72 to 96 h, and the cell lesions were observed under an inverted microscope every day until 80% of the cells showed obvious lesions (CPE). The cell culture flasks were then placed in a -80°C low-temperature refrigerator for storage. The virus solution was repeatedly frozen and thawed three times before being used for virus titer determination.

[0221] 2.3 Virus titer determination

[0222] Take a 96-well plate with MRC-5 cells grown into a monolayer, discard the culture medium, wash the cells three times with cell maintenance medium, and then resuspend them in 10-fold (10 -1 ~10 -8 ) Dilute and inoculate HCoV-229E or OC43 virus liquid of different titers, a total of 8 dilutions, 100 μl / well, 4 replicates for each dilution, and set up normal cell controls. The 96-well plate was placed at 37°C and 5% CO 2 The cells were cultured in an incubator for 72 to 96 hours, and the cytopathic effect was observed under an inverted microscope every day, and the cytopathic effect of each well was recorded. The 50% cytopathic effect concentration (TCID50) was calculated according to the Reed-Muench method.

[0223] 2.4 Construction and drug administration of human coronavirus mouse pneumonia model

[0224] BALB / c mice were randomly divided into three dose groups according to body weight: normal control group, model control group, chloroquine phosphate control group, and genipin-1-β-D gentiobioside, with 10 mice in each group, half male and half female. Except for the normal control group, mice in other groups were lightly anesthetized with ether and infected with 100TCID50 HCoV-229E or OC43 nasally, 50μl / mouse, once every other day, for a total of 2 infections. On the day of the first infection, each medication group began intravenous administration, once a day, for 4 consecutive days. After weighing the body weight on the 5th day, the lungs were dissected and weighed, and the mouse lung index and inhibition rate were calculated.

[0225] Lung index = [lung wet weight (g) / body weight (g)] × 100

[0226] Lung index inhibition rate = (lung index of model control group - lung index of drug administration group) / (lung index of model control group - lung index of normal control group) × 100%

[0227] 3 Test results

[0228] Table 4 The therapeutic effect of intravenous injection of genipin-1-β-D gentiobioside on the pneumonia model of mice infected with human coronavirus 229E

[0229]

[0230]

[0231] Note: Compared with the normal control group ## P<0.01, # P<0.05; compared with the model control group ** P<0.01, *P<0.05.

[0232] The results in Table 4 show that after the mice were infected with human coronavirus 229E virus, the lung index of the mice increased significantly, which was significantly different from the normal control group (P<0.01); after 4 days of intravenous injection of genipin-1-β-D gentiobioside from the day of infection, the three doses tested could significantly reduce the lung index of mice infected with 229E virus, which was significantly different from the model control group (P<0.01), and the lung index inhibition rates were 99.06%, 71.04%, and 75.12%, respectively. The drug efficacy is equivalent to that of chloroquine phosphate.

[0233] Table 5 The therapeutic effect of intravenous injection of genipin-1-β-D gentiobioside on the pneumonia model of mice infected with human coronavirus OC43

[0234]

[0235] Note: Compared with the normal control group ## P<0.01, # P<0.05; **P<0.01, *P<0.05 compared with the model control group.

[0236] The results in Table 5 show that after mice were infected with human coronavirus OC43, the lung index of mice increased significantly, which was significantly different from that of the normal control group (P<0.01); after 4 days of intravenous injection of genipin-1-β-D gentiobioside starting on the day of infection, the three doses tested could significantly reduce the lung index of mice infected with OC43 virus, which was significantly different from that of the model control group (P<0.05, P<0.01), and the lung index inhibition rates were 39.93%, 42.34%, and 41.59%, respectively; the efficacy was equivalent to that of chloroquine phosphate.

[0237] Pharmacological Experiment Example 3: Therapeutic effect of aerosol inhalation of genipin-1-β-D gentiobioside on the pneumonia model of mice infected with human coronavirus 229E and OC43

[0238] 1 Test materials

[0239] 1.1 Test drug: Genipin-1-β-D-gentiobioside, batch number: 20210106, properties: white powder, solubility: very soluble in water.

[0240] 1.2 Experimental animals: BALB / c mice, SPF grade, 13-15g, 140 mice, half male and half female, from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0241] 1.3 Virus strains and cells: Human coronavirus 229E (HCoV-229E) and human coronavirus OC43 (HCoV-OC43) were purchased from the ATCC bacterial strain collection in the United States, with a TCID50 of 10-4. They were propagated in this laboratory and stored in a -80℃ refrigerator for future use. Human embryonic lung fibroblasts MRC-5 were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, propagated in this laboratory, and stored in liquid nitrogen for future use.

[0242] 1.4 Test reagents:

[0243] Table 6 Test reagents

[0244]

[0245] 1.5 Test equipment:

[0246] Table 7 Test Instruments

[0247]

[0248]

[0249] 1.6 Test site: ABSL-2 laboratory of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences

[0250] 2 Test methods

[0251] 2.1 Drug dosage design and preparation

[0252] Drugs tested:

[0253] 1. In the 75 mg / ml group of genipin-1-β-D gentiobioside, the median particle size was 2.02±0.06μm; in the 37.5 mg / ml group of genipin-1-β-D gentiobioside, the median particle size was 2.12±0.08μm; in the 18.755 mg / ml group of genipin-1-β-D gentiobioside, the median particle size was 2.01±0.09μm; in the 9.375 mg / ml group of genipin-1-β-D gentiobioside, the median particle size was 2.07±0.06μm; the nebulization time for each group was 25 min.

[0254] 2. Chloroquine phosphate tablets, clinical dose is 0.5g / 60kg / d, oral; mouse dose is: 0.5g / 60kg / d×11=0.09g / kg / d; mouse dosage is 20ml / kg / d, gavage; preparation concentration is 0.09g / kg / d÷20ml / kg / d=0.0045g / ml.

[0255] 2.2 Grouping and Dosing

[0256] 70 ICR mice were randomly divided into 7 groups according to weight level, including normal control group, model control group, chloroquine phosphate control group, genipin-1-β-D gentiobioside 75mg / ml, 37.5mg / ml, 18.75mg / ml, 9.375mg / ml four dose groups, 10 mice in each group. Except for the normal control group, the remaining mice were lightly anesthetized with isoflurane and infected with 100TCID50 coronavirus liquid 229E strain or OC43 strain intranasally, 50μl per mouse, and reinfected once every other day. The first day of infection, the drug was administered by nebulization, with a flow rate of 7.5L / min. The four dose groups of genipin-1-β-D gentiobioside 75mg / ml, 37.5mg / ml, 18.75mg / ml, and 9.375mg / ml were nebulized for 25min respectively. The chloroquine phosphate control group was gavaged at 0.2ml / 10g body weight, once a day for 4 consecutive days. The normal control group and the model control group were nebulized with distilled water for 25min under the same conditions. The animals were dissected on the 5th day and the following indicators were tested:

[0257] ① The mice were weighed and dissected, the whole lungs were removed and weighed, and the lung index and inhibition rate were calculated;

[0258] ② The left lung lobe of the mouse was taken for viral nucleic acid detection, and the right lung lobe was taken for inflammatory factor content detection: IL-6, IL-10, TNF-α;

[0259] The results were analyzed statistically using t-test for comparison among groups.

[0260] Lung index = [lung wet weight (g) / body weight (g)] × 100

[0261] Lung index inhibition rate = (lung index of model control group - lung index of drug administration group) / (lung index of model control group - lung index of normal control group) × 100%

[0262] 2.3 Detection of viral load in lung tissue (RT-PCR method)

[0263] ① Nucleic acid lysis treatment

[0264] After the mouse was dissected, the lung tissue was stored in a -80℃ freezer. The mouse lung tissue was taken out of the -80℃ freezer, placed in a clean mortar, and a small amount of liquid nitrogen was poured into it and ground into powder using a pestle. The powder was collected in a 1.5ml centrifuge tube and 1ml TRIzol was immediately added. Reagent, flick the bottom of the tube and mix the sample as quickly as possible until it is resuspended; place the centrifuge tube horizontally at room temperature and incubate for 20 minutes; centrifuge at 4°C, 12000rpm for 10 minutes; transfer the clarified supernatant to a new 1.5ml centrifuge tube; add 0.2ml chloroform, cover the tube tightly, shake the centrifuge tube vigorously for 15s, and incubate at room temperature for 2-3min until the liquid separates; centrifuge at 4°C, 12000rpm for 15min; carefully transfer the transparent supernatant to a new 1.5ml centrifuge tube, add 0.5ml isopropanol, mix well, and incubate at room temperature for 30min; centrifuge at 4°C, 12000rpm for 10min; discard the supernatant and gently wash the precipitate with 1ml75% ethanol (so that the white precipitate floats lightly); centrifuge at 4°C, 7500rpm for 5min; aspirate the supernatant and briefly dry the RNA precipitate for 5-10min; dissolve the precipitate with 20μl DEPC water and store in a -80°C low-temperature refrigerator.

[0265] ② Nucleic acid determination

[0266] Nucleic acid treatment of control substances: DEPC-H2O is used as negative control. 10 7 , 10 6 , 10 5 , 10 4 The number of copies / ml was adjusted by gradient dilution to 4 concentrations.

[0267] Reagent preparation: Take n×18μl HCoV-229E nucleic acid fluorescence PCR detection mixture, n×1μl internal control, and n×1μl RT-PCR enzyme (n is the number of reaction tubes), shake and mix for a few seconds, and centrifuge at 3000rpm for a few seconds.

[0268] Add sample: Take 20μl of the above mixed solution and place it in a PCR tube, then add 5μl of sample nucleic acid extract, DEPC-H2O, and positive control into the PCR tube respectively, cover the tube tightly, centrifuge for a few seconds to allow all the liquid to settle at the bottom, and immediately carry out PCR amplification reaction.

[0269] PCR amplification: the reaction tube was placed on a quantitative fluorescence PCR instrument, and the cycle parameters were set as follows: 45°C × 10 min; 95°C × 15 min; then 95°C × 15 sec → 60°C × 60 sec, for 40 cycles; single-point fluorescence detection was at 60°C, and the reaction system was 25 μl.

[0270] Fluorescence channel detection selection: select FAM and HEX / VIC / JOE channels.

[0271] ③Calculation method: Draw a standard curve according to the ct value of the positive control with different concentrations, and calculate the viral nucleic acid concentration of the sample according to the ct value of the sample.

[0272] 3 Test results

[0273] Table 8 The therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside on the pneumonia model of HCOV-229E infected mice

[0274]

[0275]

[0276] Note: Compared with the normal control group ## P<0.01, # P<0.05; compared with the model control group ** P<0.01, * P<0.05.

[0277] The results in Table 8 show that after the mice were infected with human coronavirus 229E strain, the lung index of the mice in the model control group increased significantly, which was significantly different from that in the normal control group (P<0.01); after 4 days of aerosol inhalation treatment of genipin-1-β-D gentiobioside starting from the day of infection, the four dose groups of genipin-1-β-D gentiobioside (75mg / ml, 37.5mg / ml, 18.75mg / ml, and 9.375mg / ml) could significantly reduce the lung index, which was significantly different from that in the model control group (P<0.01), and the lung index inhibition rates were 109.19%, 64.22%, 65.59%, and 65.98%, respectively. The drug efficacy is better than that of chloroquine phosphate.

[0278] Table 9 Effect of nebulized inhalation of genipin-1-β-D gentiobioside on the viral load in lung tissue of mice infected with HCOV-229E

[0279]

[0280] Note: Compared with the normal group ## p<0.01, compared with the model group ** p<0.01.

[0281] The results in Table 9 show that there is no viral nucleic acid expression in the lung tissue of normal mice. After the mice were infected with the human coronavirus 229E strain virus, the coronavirus nucleic acid expression in the lung tissue of the mice was obvious; after 4 days of aerosol inhalation treatment of genipin-1-β-D gentiobioside starting on the day of infection, the expression of coronavirus 229E nucleic acid in the lung tissue of mice in the four dose groups of genipin-1-β-D gentiobioside of 75 mg / ml, 37.5 mg / ml, 18.75 mg / ml, and 9.375 mg / ml were significantly reduced, and there were significant differences compared with the model control group (P<0.01).

[0282] Table 10 The therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside on the pneumonia model of HCOV-OC43 infected mice

[0283]

[0284] Note: Compared with the normal control group ## P<0.01, # P<0.05; compared with the model control group ** P<0.01, * P<0.05.

[0285] The results in Table 10 show that after mice were infected with human coronavirus OC43 strain, the lung index of mice in the model control group was significantly increased, which was significantly different from that in the normal control group (P<0.01); after 4 days of aerosol inhalation treatment with genipin-1-β-D gentiobioside starting on the day of infection, the four dose groups of genipin-1-β-D gentiobioside (75 mg / ml, 37.5 mg / ml, 18.75 mg / ml, and 9.375 mg / ml) were able to significantly reduce the lung index of mice after infection, which was significantly different from that in the model control group (P<0.05, P<0.01), and the lung index inhibition rates were 102.88%, 90.30%, 75.73% and 49.42%, respectively.

[0286] Table 11 Effect of nebulized inhalation of genipin-1-β-D gentiobioside on the viral load in lung tissue of mice infected with HCOV-OC43

[0287]

[0288] Note: Compared with the normal group ## p<0.01, compared with the model group ** p<0.01.

[0289] The results in Table 11 show that there is no viral nucleic acid expression in the lung tissue of normal mice. After the mice were infected with the human coronavirus OC43 strain virus, the coronavirus nucleic acid expression in the lung tissue of the mice in the model control group was obvious; after 4 days of aerosol inhalation treatment of genipin-1-β-D gentiobioside starting on the day of infection, the coronavirus nucleic acid expression levels in the three dose groups of genipin-1-β-D gentiobioside of 75 mg / ml, 37.5 mg / ml, and 18.75 mg / ml were significantly reduced, and there were significant differences compared with the model control group (P<0.01).

[0290] Pharmacological Experiment Example 4: Effects of Genipin-1-β-D Gentianobioside on Inflammatory Cytokines in Lung Tissue of Mice Infected with HCOV-229E

[0291] The above-mentioned atomized inhalation administration sample was used. Weigh the weight of the lung tissue, add an appropriate amount of physiological saline to make a 10% tissue homogenate, use a high-throughput tissue grinder to homogenize the tissue, and use a low-temperature high-speed centrifuge to centrifuge at 4°C, 3000rpm for 10 minutes. After absorbing the supernatant, divide it into aliquots and store it in a -80°C refrigerator for future use. Avoid repeated freezing and thawing. During the detection, follow the instructions of the kit and use an enzyme reader to detect the absorbance at 450nm.

[0292] Table 12 Effects of genipin-1-β-D gentiobioside on cytokines in lung tissue of mice infected with human coronavirus 229E

[0293]

[0294] Compared with the normal control group ## P<0.01, # P<0.05; compared with the model group, ** P<0.01.

[0295] The results in Table 12 show that after the human coronavirus 229E infection model was established, the levels of TNF-α, IL-6, and IL-10 cytokines in the lung tissue of the mice in the model group were significantly increased, which were significantly different from those in the normal control group (P<0.01). The nebulized inhalation of genipin-1-β-D gentiobioside was given for 4 days starting from the day of infection. The four dose groups could reduce the levels of TNF-α, IL-6, and IL-10 in the lung tissue of the mice, which were significantly different from those in the model group (P<0.01).

[0296] Pharmacological Example 5: Therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside on the pneumonia model of mice infected with respiratory syncytial virus (RSV)

[0297] 1 Test materials

[0298] 1.1 Drugs under investigation

[0299] Genipin-1-β-D gentiobioside preparation, specification: 5ml; 375mg, batch number: 230608; properties: light yellow liquid; storage: store at room temperature.

[0300] 1.2 Experimental animals

[0301] Balb / c mice, SPF grade, weighing 9-11 g, 50 mice, half male and half female, license number: SCXK (Beijing) 2016-0006.

[0302] 1.3 Virus strains

[0303] Human respiratory syncytial virus (RSV), VR1580TM, was purchased from ATCC, propagated in this laboratory, and stored in a -80°C refrigerator for future use.

[0304] 1.4 Test equipment

[0305] Table 13 Test Instruments

[0306]

[0307] 2 Test methods and results

[0308] 2.1 Effects on mouse lung index and lung index inhibition rate

[0309] Sixty BALB / C mice weighing 10±1g were randomly divided into four groups: normal control group, model control group, high-dose group of genipin-1-β-D-gentiobioside 75mg / ml, and low-dose group of genipin-1-β-D-gentiobioside 37.5mg / ml, with 10 mice in each group, half male and half female. Except for the normal control group, mice in each group were lightly anesthetized with isoflurane and infected with respiratory syncytial virus RSV intranasally, 35μl per mouse. On the day of infection, each group began to receive nebulized inhalation medication, and each group nebulized for 25min each time, once a day, for 4 consecutive days. The normal control group and the model control group nebulized normal saline under the same conditions. After weighing on the 5th day, the mice were killed by cervical dislocation, and then the lung tissues were dissected and weighed to calculate the lung index and lung index inhibition rate. The retained lung tissues were used for viral load, pathological detection and inflammatory factor detection. The results were statistically processed by t-test for inter-group comparison.

[0310] Lung index = [lung wet weight (g) / body weight (g)] × 100

[0311] Lung index inhibition rate = (lung index of model control group - lung index of drug administration group) / (lung index of model control group - lung index of normal control group) × 100%

[0312] Table 14 The therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside on the pneumonia model of RSV-infected mice

[0313]

[0314]

[0315] Note: Compared with the normal control group ## p<0.01; compared with the model control group ** p<0.01.

[0316] The results in Table 14 show that the lung index of mice in the RSV model control group was significantly increased, which was significantly different from that in the normal control group (p<0.01); after the 75 mg / ml and 37.5 mg / ml dose groups of genipin-1-β-D gentiobioside were administered by aerosol for 25 minutes each time, once a day, for 4 consecutive days, the lung index of mice was significantly reduced, which was significantly different from that in the model control group (p<0.01), and the lung index inhibition rates were 88.35% and 68.79%, respectively.

[0317] 2.2 Effects on viral load in mouse lung tissue

[0318] Nucleic acid detection in lung tissue (RT-PCR method)

[0319] ① Nucleic acid lysis treatment

[0320] After the mouse was dissected, the lung tissue was stored in a -80℃ freezer. The mouse lung tissue was taken out of the -80℃ freezer, placed in a clean mortar, and a small amount of liquid nitrogen was poured into it and ground into powder using a pestle. The powder was collected in a 1.5ml centrifuge tube and 1ml TRIzol was immediately added. Reagent, flick the bottom of the tube and mix the sample as quickly as possible until it is resuspended; place the centrifuge tube horizontally at room temperature and incubate for 20 minutes; centrifuge at 4°C, 12000rpm for 10 minutes; transfer the clarified supernatant to a new 1.5ml centrifuge tube; add 0.2ml chloroform, cover the tube tightly, shake the centrifuge tube vigorously for 15s, and incubate at room temperature for 2-3min until the liquid separates; centrifuge at 4°C, 12000rpm for 15min; carefully transfer the transparent supernatant to a new 1.5ml centrifuge tube, add 0.5ml isopropanol, mix well, and incubate at room temperature for 30min; centrifuge at 4°C, 12000rpm for 10min; discard the supernatant and gently wash the precipitate with 1ml75% ethanol (so that the white precipitate floats lightly); centrifuge at 4°C, 7500rpm for 5min; aspirate the supernatant and briefly dry the RNA precipitate for 5-10min; dissolve the precipitate with 20μl DEPC water and store in a -80°C low-temperature refrigerator.

[0321] ② Nucleic acid determination

[0322] Nucleic acid treatment of control substances: DEPC-H2O was used as negative control. Positive control substances were diluted to 107, 106, 105, 104 copies / ml in a gradient dilution, with a total of 4 concentrations.

[0323] Reagent preparation: Take n×18 μl RSV nucleic acid fluorescence PCR detection mixture, n×1 μl internal control, and n×1 μl RT-PCR enzyme (n is the number of reaction tubes), shake and mix for a few seconds, and centrifuge at 3000 rpm for a few seconds.

[0324] Add sample: Take 20μl of the above mixed solution and place it in a PCR tube, then add 5μl of sample nucleic acid extract, DEPC-H2O, and positive control into the PCR tube respectively, cover the tube tightly, centrifuge for a few seconds to allow all the liquid to settle at the bottom, and immediately carry out PCR amplification reaction.

[0325] PCR amplification: the reaction tube was placed on a quantitative fluorescence PCR instrument, and the cycle parameters were set as follows: 45°C × 10 min; 95°C × 15 min; then 95°C × 15 sec → 60°C × 60 sec, for 40 cycles; single-point fluorescence detection was at 60°C, and the reaction system was 25 μl.

[0326] Fluorescence channel detection selection: select FAM and HEX / VIC / JOE channels.

[0327] Note: When using ABI series thermal cyclers, please be sure to select "none" for both passive reference and quencher.

[0328] Calculation method: Draw a standard curve according to the ct value of the positive control with different concentrations, and calculate the viral nucleic acid concentration of the sample according to the ct value of the sample.

[0329] Table 15 The therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside on the pneumonia model of RSV-infected mice

[0330]

[0331] The results in Table 15 show that the RSV viral load in the lung tissue of mice in the RSV model control group was significantly increased; after the 75 mg / ml and 37.5 mg / ml dose groups of genipin-1-β-D gentiobioside were administered by aerosol for 25 minutes each time, once a day, for 4 consecutive days, the RSV viral load in the lung tissue of mice was significantly reduced; the inhibition rates were 56.08% and 57.54%, respectively.

[0332] Pharmacological Example 6: Therapeutic effect of genipin-1-β-D gentiobioside aerosol inhalation on mice infected with Mycoplasma pneumoniae

[0333] 1.1 Test drug: Genipin-1-β-D-gentiobioside preparation, specification: 5ml; 375mg, batch number: 230608.

[0334] 1.2 Positive drug: Azithromycin capsules: Batch number: 23032004, production date: 2023.03.14, valid until: 2025.02. Produced by Sunflower Pharmaceutical Co., Ltd. Ingredients: The main ingredient of this product is azithromycin. Properties: This product is a capsule, and the content is white or off-white crystalline powder. Indications: Pneumonia caused by Mycoplasma pneumoniae. Specifications: 0.25g / capsule. Usage and dosage: Oral. Adults take 0.5g once a day, once a day, for 3 consecutive days. Children take 10mg / kg per day according to body weight. Storage conditions: sealed and stored in a dry place.

[0335] 1.3 Experimental animals

[0336] 100 Balb / c mice, SPF grade, weighing 13-15 g, half male and half female.

[0337] 1.4 Virus strain: Mycoplasma pneumoniae (MP), VR15531 TM .

[0338] 1.5 Test equipment

[0339] Table 16 Test Instruments

[0340] Instrument Name model Manufacturer use Type A2 Biological Safety Cabinet MSC1.2 Thermo Virus solution preparation Type A2 Biological Safety Cabinet MSC1.8 Thermo Animal infection Electronic balance BSA3202S-CW Sartorius Scientific Instruments Co., Ltd. Weighing Electronic balance AL204 Mettler-Toledo Instruments Ltd. Weigh lung weight Buxco Small Animal Snout Exposure Tower Buxco DSI Corporation Drug administration in mice

[0341] 1.6 Test reagents

[0342] Table 17 Test reagents

[0343]

[0344] 2 Test methods and results

[0345] 2.1 Effects on mouse lung index and lung index inhibition rate

[0346] Fifty BALB / C mice weighing 14±1g were randomly divided into five groups: normal control group, model control group, azithromycin control group, high-dose group of genipin-1-β-D gentiobioside (75mg / ml) nebulized for 15min, and low-dose group of genipin-1-β-D gentiobioside (37.5mg / ml) nebulized for 15min, with 10 mice in each group, half male and half female. Except for the normal control group, mice in each group were lightly anesthetized with isoflurane and infected with Mycoplasma pneumoniae intranasally, 50μl per mouse, for 3 consecutive days. On the day of infection, each drug-treated group was nebulized once a day for 4 consecutive days, and normal control group and model control group were nebulized with saline under the same conditions. After weighing on the 5th day, the mice were killed by cervical dislocation, and then the lung tissue was dissected and weighed to calculate the lung index and lung index inhibition rate; the content of inflammatory factors in lung tissue was determined; the content of CRP in serum was determined; and the lung tissue was taken for pathological examination. The results were statistically analyzed using t-test for comparison among groups.

[0347] Lung index = [lung wet weight (g) / body weight (g)] × 100

[0348] Lung index inhibition rate = (lung index of model control group - lung index of drug administration group) / (lung index of model control group - lung index of normal control group) × 100%

[0349] Table 18 The therapeutic effect of genipin-1-β-D gentiobioside on MP-infected mouse pneumonia model

[0350]

[0351] Note: Compared with the normal control group ## p<0.05; compared with the model control group ** p<0.01.

[0352] The results in Table 18 show that the lung index of the MP model control group was significantly increased, which was significantly different from that of the normal control group (p<0.01). After 4 days of aerosol administration of genipin-1-β-D gentiobioside, the lung index of mice in the high- and low-dose groups of genipin-1-β-D gentiobioside was significantly different from that of the model control group (p<0.01), and the two batches of experimental results had good repeatability.

[0353] 2.2 Detection of inflammatory factors in mouse lung tissue (ELISA method)

[0354] ①Sample collection and storage: Tissue homogenate sample: After weighing the lung tissue of mice, collect lung tissue from 8 mice in each group and store at -80℃. Use MPFastprep-245G rapid sample preparation instrument to homogenize the tissue, and use a low-temperature high-speed centrifuge at -4℃, 3000r / min for 10 minutes. After absorbing the supernatant, divide it into aliquots and store it in a -80℃ refrigerator for future use. Avoid repeated freezing and thawing.

[0355] ② Take out the microplate from the sealed bag that has been equilibrated to room temperature, and add different concentrations of standard to the corresponding wells, 50μL per well; add 10μL of experimental sample to each well, and then add 40μL of diluent. Except for the blank well, add 100μL of HRP to each well, seal the reaction well with sealing tape, and incubate at 37℃ for 1 hour. Wash the plate with washing solution and repeat the operation 4 times. After the last wash, turn the plate upside down and pat dry all residual liquid on absorbent paper; add 50μL of substrate A and B to each microwell, seal the reaction well with sealing tape, incubate at 37℃ for 15min, add 50μL of stop solution to each microwell, and measure the absorbance value at 450nm using an enzyme reader within 15min. Calculate the results.

[0356] Table 19 The therapeutic effect of genipin-1-β-D gentiobioside on MP-infected mouse pneumonia model

[0357]

[0358] Note: Compared with the normal control group ## p<0.01; compared with the model control group ** p<0.01.

[0359] The results in Table 19 show that the inflammatory factors IL-6, IL-1β, and TNF-α in the lung tissue of the MP model control group were significantly increased, which was significantly different from the normal control group (p<0.01); after 4 days of nebulization administration of high and low doses of genipin-1-β-D gentiobioside, the content of inflammatory factors in pneumonia mice was significantly reduced, which was significantly different from the model control group (p<0.01).

[0360] 2.3 Detection of CRP in mouse serum (ELISA method)

[0361] ① Sample collection and storage: Blood was collected from the mouse orbit and allowed to stand for 2 hours. The blood was then centrifuged at 2000g for 10 minutes. The supernatant was aspirated and stored at -80℃ for later use to avoid repeated freezing and thawing. When testing CRP, the blood was diluted 3000 times with normal saline.

[0362] ② Take out the microplate from the sealed bag that has been equilibrated to room temperature, add different concentrations of standards and samples to the corresponding wells, 100μL per well, and incubate at room temperature for 2 hours. Wash the plate with washing solution and repeat the operation 5 times. Add 100μL of biotinylated antibody and incubate at room temperature for 60min. Wash the plate 5 times and add 100μL of horseradish peroxidase, incubate in the dark for 20min. After washing the plate 5 times, add the color developer and incubate for 20min, and finally add 50μL of stop solution. Use an enzyme reader to measure the absorbance value at 450nm within 15min. Calculate the results.

[0363] Table 20 The therapeutic effect of genipin-1-β-D gentiobioside on MP-infected mouse pneumonia model

[0364]

[0365] Note: Compared with the normal control group ## p<0.01; compared with the model control group ** p<0.01.

[0366] The results in Table 20 show that the serum CRP content of mice in the MP model control group was significantly increased, which was significantly different from that in the normal control group (p<0.01); after 4 days of nebulization administration of high and low doses of genipin-1-β-D gentiobioside, the CRP level of inflammatory factors in pneumonia mice was significantly reduced, which was significantly different from that in the model control group (p<0.01).

[0367] 2.4 Pathological changes of mouse lung tissue (HE staining)

[0368] Normal control group: The alveolar surface was smooth without obvious deformation or rupture, the bronchiolar epithelial cells were arranged neatly without edema, degeneration or necrosis, there was no fibrous tissue hyperplasia and inflammatory cell infiltration in the pulmonary interstitium, and the tissue structure was normal.

[0369] Model control group: Microscopically, a large number of alveolar congestion, edema, and exudation were observed, and some alveolar walls were ruptured, forming alveolar fusion and consolidation. The alveolar septa were slightly or moderately thickened, accompanied by a large number of inflammatory cell infiltrations, mainly neutrophils, as well as a small number of lymphocytes, macrophages, and plasma cells. The bronchial mucosal epithelial cells were slightly or mildly edematous and degenerated, and a small number of necrotic and detached epithelial and inflammatory cells were observed in the lumen. There was a significant difference compared with the normal control group (P < 0.01).

[0370] BD-77 high-dose group: The mice had focal mild or mild congestion and edema of the alveoli, mild or mild thickening of the alveolar wall, accompanied by infiltration of a small number of inflammatory cells, mainly neutrophils, mild or mild edema of the bronchial epithelium of some animals, and the arrangement of bronchial epithelial cells was more regular, which was significantly alleviated compared with the model group (P < 0.1).

[0371] BD-77 low-dose group: Microscopically, the alveolar wall of mice was slightly or mildly thickened, accompanied by a small amount of inflammatory cell infiltration, which was significantly alleviated compared with the model group (P < 0.1); multifocal alveolar congestion or mild edema was alleviated compared with the model group, but no statistical difference was observed; bronchial mucosal epithelial cells were slightly to moderately edematous and degenerated, and some bronchial epithelial cells were necrotic, which was not significantly different from the model group.

[0372] Table 21 The therapeutic effect of BD-77 on MP-infected mouse pneumonia model

[0373]

[0374] Pharmacological Experiment Example 7: Therapeutic effect of intravenous injection of genipin-1-β-D gentiobioside on LPS-induced pneumonia model in mice

[0375] 1 Test materials

[0376] 1.1 Drug: Genipin-1-β-D-gentiobioside preparation, specification: 5ml; 375mg, batch number: 230608; properties: light yellow liquid; storage: store at room temperature.

[0377] 1.2 Animals: ICR mice, SPF grade, weighing 18-20 g, 60 mice, half male and half female, provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd., Animal Production License No.: SCXK(Beijing)2021-0006; kept in an ABSL-2 biosafety room.

[0378] 1.3 Reagents and instruments: Escherichia coli endotoxin (LPS) was produced by Sigma, lot number: lot#057M4013V. Isoflurane, Jiangsu Hengfengqiang Biotechnology Co., Ltd., lot number: 20211202. Production date: 20211224, expiration date: 20231223. MC 1.8 biological safety cabinet, Thermo Company. BSA3202S-CW, BSA323S-CW electronic balance, Sartorius Instrument Co., Ltd. AL-204 METTLER TOLEDO electronic balance, Mettler-Toledo Instrument (Shanghai) Co., Ltd.

[0379] 2 Test methods and results:

[0380] 2.1 Effects on mouse lung index and lung index inhibition rate

[0381] 60 healthy mice, 18-20g, half male and half female, were taken and stratified by body weight and then randomly divided into normal control group, model control group, three dose groups of 150mg / kg / d, 75mg / kg / d and 37.5mg / kg / d of genipin-1-β-D gentiobioside, and 50mg / kg / d of positive control drug sivelestat sodium, 10 mice in each group, half male and half female. After grouping, each drug group was intravenously injected with 20ml / kg, once a day for 2 consecutive days, and the normal control group and model control group were injected with normal saline; 1 hour after the second day, except for the normal control group, the other groups were lightly anesthetized with isoflurane, and the mice were dripped with LPS normal saline solution with a concentration of 20mg / ml, 0.05ml / mouse, to create a mouse pneumonia model. 6 hours after infection, the animals were killed and dissected, the whole lungs were removed and weighed, and the lung index was calculated (lung index = lung weight / body weight × 100%).

[0382] Table 22 Effects of genipin-1-β-D gentiobioside on LPS-induced pneumonia model in mice

[0383]

[0384] Note: Compared with the normal control group ## p<0.01; compared with the model control group ** p<0.01.

[0385] The results in Table 22 show that 6 hours after mice inhaled LPS, the lung index of mice in the model control group increased significantly, which was significantly different from that in the normal control group (p<0.01); the lung index of the high and medium dose groups of genipin-1-β-D gentiobioside administration was significantly reduced, which was significantly different from that in the model control group (p<0.01, p<0.05). This shows that the two dose groups of genipin-1-β-D gentiobioside 150mg / kg and 75mg / kg have a protective effect on pneumonia in mice caused by LPS.

[0386] 2.2 Detection of inflammatory factors in mouse lung tissue (ELISA method)

[0387] ① Sample collection and storage: Tissue homogenate sample: After weighing the lung tissue of mice, collect the lung tissue of mice No. 1, 2, 3, 6, 7, and 8 in each group and store them at -4°C. Use an ultrasonic cell disruptor to homogenize the tissue and use a low-temperature high-speed centrifuge at -4°C.

[0388] Centrifuge at 1000r / min for 10 minutes. Pipette out the supernatant and store in a -80℃ refrigerator for future use. Avoid repeated freezing and thawing.

[0389] ② Take out the microplate from the sealed bag that has been equilibrated to room temperature, and add different concentrations of standards, experimental samples or quality control products to the corresponding wells, 100μL per well. Seal the reaction wells with sealing tape and incubate at room temperature for 2 hours. Wash the plate with washing solution and repeat the operation 4 times. After the last wash, turn the plate upside down and pat dry all residual liquid on absorbent paper; add 100μL enzyme-labeled detection antibody to each microwell. Seal the reaction wells with sealing tape and incubate at room temperature for 2 hours. Repeat the washing operation in step 4, add 100μL of color substrate to each microwell, and incubate at room temperature for 30 minutes. Be careful to avoid light. Within 30 minutes after adding 100μL of stop solution to each microwell, use an enzyme reader to measure the absorbance value at 450nm. Calculate the results.

[0390] Table 23 Effect of genipin-1-β-D gentiobioside on LPS-induced pneumonia model in mice

[0391]

[0392] Note: Compared with the normal control group ## p<0.01; compared with the model control group * p<0.05, ** p<0.01.

[0393] The results in Table 23 show that 6 hours after mice inhaled LPS, the levels of IL-6, IL-10, and TNF-α in the lung tissue of mice in the model control group increased significantly, which was significantly different from that in the normal control group (p<0.01, p<0.05); the genipin-1-β-D gentiobioside administration group could significantly reduce the levels of IL-6, IL-10, and TNF-α, which was significantly different from that in the model control group (p<0.01, p<0.05). This indicates that the two dose groups of genipin-1-β-D gentiobioside 150 mg / kg and 75 mg / kg have a protective effect on pneumonia in mice caused by LPS.

Claims

1. The use of genipin-1-β-D gentiobioside in the preparation of medicines, characterized in that: Application of genipin-1-β-D gentiobioside in the preparation of medicines for treating respiratory tract damage caused by coronavirus, respiratory syncytial virus or mycoplasma pneumoniae infection.

2. The use of genipin-1-β-D-gentiobioside in the preparation of medicines according to claim 1, characterized in that: Application of genipin-1-β-D gentiobioside in the preparation of medicines for treating inflammatory damage of pneumonia caused by coronavirus, respiratory syncytial virus or mycoplasma pneumoniae infection.

3. The use of genipin-1-β-D gentiobioside in the preparation of medicines according to claim 3, characterized in that: Application of genipin-1-β-D gentiobioside in the preparation of a drug with a death protection effect for treating pneumonia caused by coronavirus infection.

4. The use of genipin-1-β-D-gentiobioside in the preparation of medicines according to claim 1, characterized in that: The coronavirus includes any one of the new coronavirus SARS-CoV-2, human coronavirus 229E, and human coronavirus OC43.

5. The use of genipin-1-β-D-gentiobioside in the preparation of medicines according to claim 1, characterized in that: The administration route of the genipin-1-β-D gentiobioside includes any one of atomization inhalation, oral administration, injection, sublingual administration, spray or anal administration; the dosage form of the drug includes any one of inhalation, oral administration, injection, spray, film and suppository.

6. A nebulizer inhalation agent containing genipin-1-β-D-gentiobioside, which realizes the use according to any one of claims 1 to 5, characterized in that: The atomized inhalation comprises the following raw materials: genipin-1-β-D gentiobioside, a pH regulator for adjusting the pH value to 4.5-7.0, an osmotic pressure regulator accounting for 0-0.9% of the weight of genipin-1-β-D gentiobioside, and a solvent.

7. The aerosol inhalation preparation containing genipin-1-β-D-gentiobioside according to claim 6, characterized in that: The preparation method of the atomized inhalation comprises the following steps: (1) measuring 40% to 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution; (2) When the temperature of the first solution is 20 to 80° C., the osmotic pressure regulator is added to the first solution and stirred evenly to obtain a second solution; (3) adding a pH regulator and genipin-1-β-D-gentiobioside to the second solution to adjust the pH value of the second solution to a target value to obtain a third solution; (4) Adding solvent to the third solution to make up to the total volume of the prepared solution, stirring evenly, and obtaining a nebulized inhalation preparation of genipin-1-β-D-gentiobioside.

8. An injection containing genipin-1-β-D-gentiobioside, which realizes the use according to any one of claims 1 to 5, characterized in that: The injection comprises the following raw materials: genipin-1-β-D gentiobioside, a pH regulator for adjusting the pH value to 4.5-7.0, an osmotic pressure regulator for adjusting the osmotic pressure to isotonicity, and an injection solvent.

9. The injection containing genipin-1-β-D-gentiobioside according to claim 8, characterized in that: The preparation method of the injection comprises the following steps: (1) measuring 40% to 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution; (2) adding the osmotic pressure regulator to the first solution under the condition that the temperature of the first solution is 20 to 80° C. and stirring evenly to obtain a second solution; (3) adding a pH regulator and genipin-1-β-D-gentiobioside to the second solution to adjust the pH value of the second solution to a target value to obtain a third solution; (4) Adding solvent to the third solution to make up to the total volume of the prepared solution, stirring evenly, and obtaining an injection of genipin-1-β-D-gentiobioside.

10. A method for preparing genipin-1-β-D-gentiobioside, for extracting genipin-1-β-D-gentiobioside for use in any one of claims 1 to 5, characterized in that: The steps include: a. Take Gardenia jasminoides, extract with water, and concentrate the extract under reduced pressure to make the crude drug content in the extract 0.03 to 0.2 g / ml; b. The extract was applied to a macroporous resin column, first eluted with 1 to 5 column volumes of deionized water, then eluted with 1 to 5 column volumes of ethanol at a volume concentration of 10 to 20%, the ethanol eluate was collected, the ethanol was recovered under reduced pressure, concentrated to 0.1 times the volume of the crude drug, ethanol was added to an alcohol volume concentration of 90%, allowed to stand, precipitated, filtered, the alcohol precipitate was passed through a neutral alumina column, successively eluted with 1 to 8 column volumes of ethanol at a volume concentration of 50 to 90%, the ethanol eluate at a volume concentration of 50 to 60% was collected, the ethanol was recovered under reduced pressure, and dried to obtain a crude product; c. The crude product was purified 2 to 3 times by hot-melt recrystallization with ethanol. After the purified product was dried, the ethanol was removed to obtain high-purity genipin-1-β-D gentiobioside.

11. The method for preparing genipin-1-β-D-gentiobioside according to claim 10, characterized in that: In step a, the specific steps of water extraction are: crushing the gardenia medicinal material, adding 12 times, 10 times and 10 times the amount of water respectively, and decocting for 3 times, and the decocting time for each time is 1 to 1.5 hours; in step b, the weight ratio of the wet volume of the macroporous resin to the gardenia medicinal material is 3:2 to 3 ml / g, and the weight ratio of the neutral alumina to the gardenia medicinal material is 1:3 to 3.

5.

12. The method for preparing genipin-1-β-D-gentiobioside according to claim 10, characterized in that: The specific step b is as follows: the gardenia extract is loaded onto an NKA-9 macroporous resin column, firstly eluted with 2 column volumes of deionized water, the loaded solution and the water eluate are collected, combined and loaded onto an X-5 macroporous resin, firstly eluted with 1 column volume of deionized water, then eluted with 5 column volumes of ethanol with a volume concentration of 10%, the ethanol eluate is collected, ethanol is recovered under reduced pressure, the relative density is 1.08-1.15 when concentrated to 60° C., ethanol is added to an alcohol volume concentration of 90%, the mixture is allowed to stand, a precipitate is precipitated, filtered, the supernatant of the alcohol precipitate is passed through a neutral alumina column, and eluted with 6 column volumes of ethanol with a volume concentration of 90% and 4 column volumes of ethanol with a volume concentration of 60%, the ethanol eluate with a volume concentration of 60% is collected, ethanol is recovered under reduced pressure, and dried to obtain a crude product.

13. The method for preparing genipin-1-β-D-gentiobioside according to claim 10, characterized in that: In step c, the method for recrystallizing the crude product after hot dissolution in ethanol is specifically as follows: adding 0.5 to 1 times of anhydrous ethanol to the crude product, heating to reflux to dissolve it, filtering it while hot, allowing it to precipitate, and filtering it to obtain a refined product.

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