Use of Genipin-1-β-D Gentiobioside in Preparation of Medicine

Abstract

The present invention discloses the use of genipin-1-β-D gentiobioside in the preparation of a medicament for treating airway damage caused by coronavirus, respiratory syncytial virus, or Mycoplasma pneumoniae infection. The present invention has discovered that genipin-1-β-D gentiobioside reduces the viral burden, lung index, and pulmonary tissue inflammatory cytokines in lung tissue after coronavirus, respiratory syncytial virus, or Mycoplasma pneumoniae infection, and has a significant protective effect against death from coronavirus infection.

Description

[Technical Field]

[0001] The present invention relates to the use of genipin-1-β-D gentiobioside, in particular to the use of genipin-1-β-D gentiobioside for preparing a medicament for treating coronavirus infection, respiratory syncytial virus infection or Mycoplasma pneumoniae infection. [Background technology]

[0002] Coronaviruses are the largest known RNA virus family, enveloped viruses with a positive-strand RNA genome. They have a wide range of natural hosts and are important pathogens of humans and vertebrates, infecting the human respiratory tract, gastrointestinal tract, liver, kidneys, and nervous system, and causing life-threatening outbreaks of pneumonia and bronchitis. In coronaviruses (CoVs), the envelope spike glycoprotein (S) is responsible for coronavirus entry into cells and cell-to-cell transmission. After phagocytosing the intracellular body, coronaviruses release viral RNA and nuclear envelopes into the cytoplasm, where they are replicated by the host, completing the viral replication cycle. Coronaviruses can cause acute respiratory infections in humans, with the novel coronavirus in particular causing acute pneumonia and death in severe cases.

[0003] Currently, drugs used to treat coronavirus infections are mainly small molecule compounds that act on the viral replication cycle, such as Paxilovid, Monupivir, Azfidine, and VV116. These drugs have a single target and limited therapeutic effect, making it difficult to respond to viral mutations and drug resistance, and are particularly ineffective in severe or fatal cases accompanied by factors such as inflammatory factor storms, diffuse intravascular coagulation, and impaired immune function.

[0004] Respiratory syncytial virus (RSV) is a circular, negative-sense, single-stranded RNA virus belonging to the influenza virus family. Human RSV is a highly contagious virus that commonly causes acute lower respiratory tract infections in infants and is responsible for high mortality rates among the elderly and children. RSV can cause mild to severe respiratory tract infections, including pneumonia and bronchitis, and can lead to serious complications such as respiratory failure. Ribavirin has traditionally been used as the preferred antiviral drug for RSV pneumonia, but due to its numerous side effects, it has rarely been used in clinical practice. Therefore, comprehensive therapy (combining anti-infectives, glucocorticoids, bronchoscopy, and anticoagulation) is currently the primary treatment for RSV pneumonia, and there is still a lack of effective treatments in clinical practice.

[0005] Mycoplasma pneumoniae (MP) is a significant pathogen causing respiratory infections in children, and its associated disease, Mycoplasma pneumoniae pneumonia (MPP), is currently the most common community-acquired pneumonia (CAP) among children aged 5 years and older in China. Past data suggest that during epidemics, MPP accounts for 20% to 40% of CAP cases in the general population and up to 70% in the closed population. While clinical symptoms of MPP were typically mild and considered a self-limiting disease, an increasing number of severe cases of MPP (SMPP) have been reported in recent years.

[0006] Previously, macrolide antibiotics were the first-line treatment for Mycoplasma pneumoniae pneumoniae pneumoniae, but in recent years, drug resistance has been steadily emerging, resulting in poor therapeutic efficacy. However, new tetracycline antibiotics, such as polycycycline and minocycline, are alternatives for treating MPP. While they are effective against drug-resistant MPP, their clinical application is relatively limited due to the risk of tooth yellowing and poor enamel development in patients. Quinolone antibiotics are alternatives for treating MPP. They are effective against macrocyclic lactone-resistant MPPs, allowing for the diagnosis and confirmation of MPP-resistant MUMPP, RMMP, and SMPP. However, their clinical application is limited due to the risk of cartilage damage in young animals and tendon rupture in humans. Therefore, currently, comprehensive treatment (combined anti-infective, glucocorticoid, bronchoscopy, anticoagulation, etc.) is primarily used for treating M. pneumoniae pneumoniae pneumoniae pneumoniae, and there is still a lack of effective drugs in clinical practice. Summary of the Invention

[0007] The present invention provides a use of genipin-1-β-D gentiobioside in the preparation of medicine. The present invention has discovered that genipin-1-β-D gentiobioside can reduce the viral burden, lung index, and pulmonary tissue inflammatory cytokines in lung tissue after infection with coronavirus, respiratory syncytial virus, or Mycoplasma pneumoniae, and has significant protective effects against death from coronavirus infection.

[0008] In the technical solution of the present invention, genipin-1-β-D gentiobioside is used to prepare a drug for treating airway damage caused by coronavirus, respiratory syncytial virus, or Mycoplasma pneumoniae infection.

[0009] In the aforementioned use of genipin-1-β-D gentiobioside in a drug preparation, genipin-1-β-D gentiobioside is used to prepare a drug for treating pneumonia inflammatory damage caused by coronavirus, respiratory syncytial virus, or Mycoplasma pneumoniae infection.

[0010] In the aforementioned use of genipin-1-β-D gentiobioside in a drug preparation, genipin-1-β-D gentiobioside is used to prepare a drug that treats pneumonia caused by coronavirus infection and has a death-protective effect.

[0011] In the above-mentioned use of genipin-1-β-D gentiobioside in the preparation of a drug, the coronavirus includes any one of the novel coronavirus SARS-CoV-2, human coronavirus 229E, and human coronavirus OC43.

[0012] In the use of genipin-1-β-D gentiobioside in the preparation of a drug, the administration route of the genipin-1-β-D gentiobioside includes any one of atomized inhalation, oral administration, injection, sublingual, spray, and anal administration routes, and the dosage form of the drug includes any one of inhalation, oral administration, injection, spray, film, and suppository.

[0013] The atomized inhalant containing genipin-1-β-D gentiobioside that can be used as described above comprises the following raw materials: genipin-1-β-D gentiobioside; a pH adjuster that adjusts the pH to 4.5 to 7.0; an osmotic pressure adjuster that accounts for 0 to 0.9% by weight of genipin-1-β-D gentiobioside; and a solvent.

[0014] The atomized inhalant described above is prepared by the method of: Measure out 40% to 90% (v / v) of the total amount of water for injection of the solution prepared as needed to obtain a first solution; adding an osmolality adjuster to the first solution under the condition that the temperature of the first solution is 20 to 80°C, and stirring the mixture uniformly to obtain a second solution; adding a pH adjuster 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; and replenishing the third solution with a solvent as needed to adjust the volume to the total amount of the prepared solution, and stirring uniformly to obtain an atomized inhalant of genipin-1-β-D gentiobioside.

[0015] The above-mentioned use is realized by an injection containing genipin-1-β-D gentiobioside, which contains the following raw materials: genipin-1-β-D gentiobioside, a pH adjuster for adjusting the pH to 4.5-7.0, an osmotic adjuster for adjusting the osmotic pressure to isotonicity, and an injection solvent.

[0016] In the above-mentioned injection, the method for preparing the injection comprises: Measure out 40% to 90% (v / v) of the total amount of water for injection of the solution prepared as needed to obtain a first solution; adding an osmolality adjuster to the first solution under a condition where the temperature of the first solution is 20 to 80°C, and stirring the mixture uniformly to obtain a second solution; adding a pH adjuster 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; and replenishing the third solution with a solvent to adjust the volume to the total volume of the prepared solution as needed, and stirring the mixture uniformly to obtain an injection of genipin-1-β-D gentiobioside.

[0017] The method for preparing genipin-1-β-D gentiobioside for the above-mentioned uses includes: a. Collecting gardenia herbal medicine, extracting it with water, concentrating the extract under reduced pressure, and adjusting the herbal medicine content in the extract to 0.03-0.2g / ml; b. The extract is passed through a large-pore resin column, first eluted with 1-5 column volumes of deionized water, then eluted with 1-5 column volumes of 10-20% ethanol, the ethanol eluate is collected, the ethanol is recovered under reduced pressure, concentrated to 0.1 times the volume of the crude drug, and ethanol is added to an alcohol concentration of 90%, allowed to stand, precipitated, filtered, and the alcohol precipitate supernatant is passed through a neutral alumina column, and then eluted with 1-8 column volumes of 50-90% ethanol, the ethanol eluate with a volume concentration of 50-60% is collected, the ethanol is recovered under reduced pressure, and dried, thus obtaining a crude product; c. The crude product is purified by hot dissolving in ethanol and recrystallizing it two or three times, and the purified product is dried and then the ethanol is removed to obtain high-purity genipin-1-β-D gentiobioside.

[0018] The herbal content refers to the amount of medicinal material (g) divided by the volume of medicinal liquid (mL). For example, if 2 kg of gardenia herb is boiled, filtered, and concentrated to 60 L, the concentration of the herbal material is 2 kg / 60 L = 0.033 g / mL.

[0019] The term "concentrating the herb volume to 0.1 times" means reducing the volume of the extract by removing the solvent so that the final volume is 10% (i.e., 0.1 times) of the original herb volume.

[0020] In the above-mentioned method for preparing genipin-1-β-D gentiobioside, the specific steps of extracting with water in step a are to crush the gardenia herb and add 12 times, 10 times, and 10 times the amount of water to decoctate three times, each decocting for 1-1.5 hours; in step b, the weight ratio of the wet volume of the macroporous resin to the gardenia herb is 3:2-3 ml / g, and the weight ratio of the neutral alumina to the gardenia herb is 1:3-3.5.

[0021] In the aforementioned method for preparing genipin-1-β-D-gentiobioside, step b specifically involves loading the gardenia extract onto an NKA-9 macroporous resin column, first eluting with 2 column volumes of deionized water, and collecting the sample solution and the effluent. This is then combined with the sample X-5 macroporous resin, first eluting with 1 column volume of deionized water, and then eluting with 5 column volumes of 10% ethanol. The ethanol eluate is collected, the ethanol is recovered under reduced pressure, and the concentrate is concentrated to a relative density of 1.08-1.15 at 60°C. Ethanol is added to a volumetric concentration of 90% ethanol, allowed to stand, precipitated, and filtered. The supernatant of the alcohol precipitate is passed through a neutral alumina column, and then eluted with 6 column volumes of 90% ethanol and 4 column volumes of 60% ethanol, collecting the 60% ethanol eluate, recovering the ethanol under reduced pressure, and drying to obtain the crude product.

[0022] In the aforementioned preparation method of genipin-1-β-D-gentiobioside, in step c, the crude product is dissolved in ethanol and recrystallized. Specifically, the crude product is added to 0.5 to 1 times absolute ethanol, heated to reflux to dissolve, and then filtered by hot filtration. The precipitate is then allowed to stand and extracted to obtain a purified product.

[0023] The beneficial effects of the present invention compared to the prior art are as follows:

[0024] Through animal experiments, the present invention has discovered that genipin-1-β-D gentiobioside has a significant protective effect against mouse death caused by infection with the novel coronavirus SARS-CoV-2, has a high inhibitory rate on the lung index of mice infected with coronavirus, respiratory syncytial virus, and Mycoplasma pneumoniae, reduces the viral burden in mice infected with coronavirus, respiratory syncytial virus, or Mycoplasma pneumoniae, and can 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.

[0025] Genipin-1-β-D gentiobioside reduces various inflammatory cytokines and inhibits the inflammatory cytokine storm after coronavirus, respiratory syncytial virus, or Mycoplasma pneumoniae infection, significantly reducing mortality rates, lung index, and lung tissue viral burden, demonstrating a definite and significant therapeutic effect against viral pneumonia caused by coronavirus, respiratory syncytial virus, or Mycoplasma pneumoniae infection. Genipin-1-β-D gentiobioside exhibits anti-coronavirus, respiratory syncytial virus, and Mycoplasma pneumoniae infection effects, and has a broad spectrum of activity, allowing it to treat respiratory tract diseases caused by coronavirus, respiratory syncytial virus, and Mycoplasma pneumoniae infections, severe pneumonia, and other diseases, and has a protective effect against inflammatory damage. It can be used to manufacture drugs for treating respiratory diseases caused by coronavirus, respiratory syncytial virus, or Mycoplasma pneumoniae infection, drugs for preventing pneumonia inflammation damage, and drugs with a protective effect against pneumonia death.

[0026] Genipin-1-β-D gentiobioside can be administered via atomized inhalation, oral administration, injection, sublingual administration, spraying, and anal administration, and can be formulated into a variety of commonly used pharmaceutical formulations.

[0027] The present invention also provides a method for preparing genipin-1-β-D gentiobioside, which removes most of the pigments and iridoid impurities from the crude product, resulting in a genipin-1-β-D gentiobioside content of more than 60%. This method can efficiently isolate the active ingredient, achieving the requirement for a new drug with an active ingredient content of more than 50% in the extract. The purity of genipin-1-β-D gentiobioside in the final extract product reaches more than 96%.

[0028] In the preparation process of the present invention, the use of toxic and harmful reagents is avoided and reusable macroporous resins are selected for purification, so the process of this method is green, safe, relatively low cost, and suitable for large-scale production. [Brief explanation of the drawings]

[0029] [Figure 1] This is a graph showing the measurement of virus titration in lung tissue of a mouse infected with the new coronavirus. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will be further explained below with reference to examples, but the examples are not intended to limit the scope of the present invention.

[0031] The present invention has discovered that genipin-1-β-D gentiobioside can be used to prepare a medicament for treating coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

[0032] Genipin-1-β-D gentiobioside is used to prepare a drug for treating airway damage caused by coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

[0033] Genipin-1-β-D gentiobioside is used to prepare a drug for treating pneumonia inflammatory damage caused by coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

[0034] Genipin-1-β-D gentiobioside is used to prepare drugs for treating pneumonia caused by coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection and has a death-protective effect.

[0035] Furthermore, genipin-1-β-D gentiobioside can be used to prepare 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.

[0036] The coronavirus includes any one of the novel coronavirus SARS-CoV-2, human coronavirus 229E, and human coronavirus OC43.

[0037] The genipin-1-β-D gentiobioside of the present invention can be extracted from the traditional Chinese medicine Gardenia jasmine, or can be prepared by artificial synthesis.

[0038] The method for extracting genipin-1-β-D gentiobioside from the traditional Chinese medicine Gardenia can be carried out using the following procedure: a. Extraction of medicinal materials: Collect the gardenia crude drug, crush the gardenia crude drug, add 12 times, 10 times, and 10 times the amount of water, and decoctate three times, each time for 1 to 1.5 hours. Concentrate the extract under reduced pressure, and the medicinal herb content in the extract should be 0.03 to 0.2 g / ml. b. Preparation of crude product: The extract is applied to a large-pore resin column, the large-pore resin is preferably NKA-9 or X-5, and the ratio of wet volume to weight of Gardenia herb is 3:2~3ml / g. First, elution is performed with 1 to 5 column volumes of deionized water, and then elution is performed with 1 to 5 column volumes of 10 to 20% ethanol by volume. The ethanol eluate is collected, the ethanol is recovered under reduced pressure, and the mixture is concentrated to 0.1 times the volume of the herb. Ethanol is added to the mixture until the alcohol volume concentration reaches 90%. The mixture is left to stand for precipitation, and then filtered. The supernatant of the alcohol precipitate is passed through a neutral alumina column. The neutral alumina specification is 100 to 200 mesh, and the weight ratio to the gardenia medicinal material is 1:3 to 3.5. Sequential elution is performed with 1 to 8 column volumes of 50 to 90% ethanol by volume. The ethanol eluate with a volume concentration of 50 to 60% is collected, the ethanol is recovered under reduced pressure, and the mixture is dried to obtain the crude product. c. Preparation of genipin-1-β-D gentiobioside: The crude product is purified two to three times by hot dissolution in ethanol and recrystallization. Specifically, the crude product is added to 0.5 to 1 times absolute ethanol, heated to reflux to dissolve, and then filtered hot to precipitate. The purified product is then extracted and dried, after which the ethanol is removed to obtain high-purity genipin-1-β-D gentiobioside.

[0039] The administration routes of genipin-1-β-D gentiobioside include any one of atomized inhalation, oral administration, injection, sublingual, spray or anal administration routes.

[0040] The atomized inhalation and injection administration methods adopted in the present invention are directly administered to the airways and lungs as target organs, and the drug is delivered to the airways and lungs in the form of a mist, which has the advantages of rapid effectiveness, high local drug concentration in the respiratory tract and pulmonary inflammatory lesions, low drug consumption, convenient application, and few systemic side effects, making it an important means of treating respiratory diseases.

[0041] The present invention further provides a medicament for treating coronavirus infection, comprising genipin-1-β-D gentiobioside and a pharmaceutically acceptable excipient.

[0042] The dosage form of the drug includes any one of an inhalant, an oral administration agent, an injection agent, a spray agent, a film agent, and a suppository agent.

[0043] Administration of inhalants, injections, sprays, suppositories, and parenteral film formulations can avoid liver and intestinal overdose and prevent the inactivation of the genipin-1-β-D gentiobioside of the present invention, which contains glycosidic bonds in its structure, due to degradation by gastric acid and intestinal bacteria.

[0044] The atomized inhalant containing genipin-1-β-D gentiobioside is used for the above-mentioned purposes and contains the following raw materials: 20 to 200 mg of genipin-1-β-D gentiobioside, a pH adjuster that adjusts the pH to 4.5 to 7.0, an osmotic pressure adjuster that accounts for 0 to 0.9% by weight of genipin-1-β-D gentiobioside, and 1 to 5 ml of solvent.

[0045] The method for preparing an atomized inhalant containing genipin-1-β-D gentiobioside is as follows:

[0046] Measure out 40% to 90% (v / v) of the total amount of water for injection of the solution prepared as needed to obtain a first solution;

[0047] adding an osmolality adjuster to the first solution under the condition that the temperature of the first solution is 20 to 80°C, and stirring the mixture uniformly to obtain a second solution;

[0048] adding a pH adjuster 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;

[0049] The third solution is supplemented with a solvent as needed to adjust the volume to the total volume of the prepared solution, stirred uniformly, and filtered through a 0.22 μm filter membrane or filter wick to obtain an atomized inhalation agent of genipin-1-β-D gentiobioside.

[0050] The genipin-1-β-D gentiobioside-containing injection is used for the above-mentioned purposes and contains the following raw materials: 20 to 200 mg of genipin-1-β-D gentiobioside, a pH adjuster for adjusting the pH to 4.5 to 7.0, an osmotic adjuster for adjusting the osmotic pressure to isotonicity, and 1 to 5 ml of injection solvent.

[0051] a. The method for preparing the injection comprises: Measure out 40% to 90% (v / v) of the total amount of water for injection of the solution prepared as needed to obtain a first solution; adding an osmolality adjuster to the first solution under the condition that the temperature of the first solution is 20 to 80°C, and stirring the mixture uniformly to obtain a second solution; adding a pH adjuster 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; and replenishing the third solution with a solvent as needed to adjust the volume to the total amount of the prepared solution, and stirring uniformly to obtain an atomized inhalant of genipin-1-β-D gentiobioside.

[0052] Extraction Example 1: The preparation method of genipin-1-β-D gentiobioside is A. Extraction of medicinal materials: Collect 2 kg of gardenia herb, crush the gardenia herb, add 12 times, 10 times, and 10 times the amount of water and brew three times, each time for 1 hour, filter the extract, combine, and concentrate under reduced pressure until the content of the herb in the extract is 0.033 g / ml; B. Preparation of crude product: The gardenia extract is loaded onto an NKA-9 macroporous resin column, and first eluted with 2 column volumes of deionized water. The sample solution and the effluent are collected. The sample is then loaded onto an X-5 macroporous resin, and first eluted with 1 column volume of deionized water. Then, eluted with 5 column volumes of 10% ethanol. The ethanol eluate is collected, and the ethanol is recovered under reduced pressure. The concentrate is then concentrated to 200 ml, and the relative density at 60°C is 1.15. Add ethanol to the column until the alcohol concentration reaches 90%. Allow to stand, precipitate, and filter. The supernatant of the alcohol precipitate is passed through a 100-mesh neutral alumina column. Then, eluted with 6 column volumes of 90% ethanol and 4 column volumes of 60% ethanol. The 60% ethanol eluate is collected, and the ethanol is recovered under reduced pressure. Drying is performed to obtain the crude product. C. Preparation of genipin-1-β-D gentiobioside: The crude product was added to 1.2 L of absolute ethanol, heated to reflux to dissolve, and then filtered by hot standing to precipitate. Purified Product-1 was obtained by extraction. Purified Product-1 was added to 1.4 L of absolute ethanol, heated to reflux to dissolve, and then filtered by hot standing to precipitate. Purified Product-2 was obtained by removing the ethanol. Purified Product-2 was then dried (60°C, 0.7 MPa) and the ethanol was removed to obtain 11.7 g of high-purity genipin-1-β-D gentiobioside, with a mass content of 96.2%.

[0053] Extraction Example 2 The preparation method of genipin-1-β-D gentiobioside is A. Extraction of medicinal materials: Collect 5 kg of gardenia crude drug, crush the gardenia crude drug, 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, filter the extract, combine, and concentrate under reduced pressure until the content of the medicinal herb in the extract is 0.2 g / ml. B. Preparation of crude product: The gardenia extract is loaded onto an NKA-9 macroporous resin column, and first eluted with 3 column volumes of deionized water. The sample solution and the effluent are collected. The sample is then loaded onto an X-5 macroporous resin, and first eluted with 1 column volume of deionized water. Then, eluted with 3 column volumes of 20% ethanol. The ethanol eluate is collected, and the ethanol is recovered under reduced pressure. The mixture is concentrated to a relative density of 1.08 at 60°C (1L). Add ethanol to an alcohol concentration of 90%. Allow to stand, precipitate, and filter. The supernatant of the alcohol precipitate is passed through a 100-mesh neutral alumina column. Then, eluted with 6 column volumes of 90% ethanol and 8 column volumes of 50% ethanol, and the 50% ethanol eluate is collected, and the ethanol is recovered under reduced pressure. Drying is performed to obtain the crude product. C. Preparation of genipin-1-β-D gentiobioside: The crude product was added to 2.5 L of absolute ethanol, heated to reflux to dissolve, and then filtered with hot water to precipitate. Purified Product-1 was obtained by extraction. Purified Product-1 was added to 2.5 L of absolute ethanol, heated to reflux to dissolve, and then filtered with hot water to precipitate. The extraction was then filtered, and the ethanol was removed to obtain Purified Product-2. Purified Product-2 was added to 2.8 L of absolute ethanol, heated to reflux to dissolve, and then filtered with hot water to precipitate. The extraction was then filtered, and the ethanol was removed to obtain Purified Product-3. Purified Product-3 was dried (60°C, 0.7 MPa), and the ethanol was removed to obtain 20.7 g of high-purity genipin-1-β-D gentiobioside, with a mass content of 96.7%.

[0054] Formulation Example 1: Inhalation Administration - Inhalation Solution The formulation of the atomized inhalation solution containing genipin-1-β-D gentiobioside is as follows: 20 mg of genipin-1-β-D gentiobioside, citric acid to adjust the pH to 4.5, sodium chloride to adjust the osmotic pressure to isotonicity, and the solvent to 1 ml.

[0055] The method for preparing the atomized inhalation solution includes: Measure out 40% (v / v) of the total volume of the solution prepared as needed with water for injection to obtain a first solution; adding sodium chloride to the first solution and stirring until dissolved under conditions of controlling the temperature of the first solution at 25°C to obtain a second solution; adjusting the pH value of the solution to 4.5 by adding citric acid to obtain a third solution; adding genipin-1-β-D gentiobioside to the third solution, stirring evenly, and adding an appropriate amount of citric acid to maintain the solution pH at 4.5 to obtain a fourth solution; Adding a solvent to the fourth solution to a constant volume equal to the total volume of the solution prepared as needed, and stirring the mixture uniformly to obtain a fifth solution; filtering the fifth solution through a 0.22 μm filter membrane or filter core to obtain a sixth solution; The sixth solution is poured into a 1 ml standard ampoule or syringe bottle and sealed, thereby obtaining an atomized inhalation solution of genipin-1-β-D gentiobioside.

[0056] Formulation Example 2: Inhalation Administration - Inhalation Solution The formulation of the atomized inhalation solution containing genipin-1-β-D gentiobioside is 100 mg of genipin-1-β-D gentiobioside, 0.1% citric acid, sodium citrate to adjust the pH to 5.0, and 3 ml of solvent.

[0057] The method for preparing the atomized inhalation solution described above includes the steps of: Measure out 60% (v / v) of the total volume of the solution prepared as needed with water for injection to obtain a first solution; Adding citric acid to the first solution and stirring uniformly under the condition of controlling the temperature of the first solution at 50°C to obtain a second solution; adjusting the solution pH value to 5.0 by adding sodium citrate to obtain a third solution; Add genipin-1-β-D gentiobioside to the third solution, stir evenly, and add sodium citrate in an appropriate amount to maintain the solution pH at 5.0 to obtain a fourth solution. supplementing the fourth solution with a solvent to a constant volume equal to the total volume of the solution prepared as needed, and stirring uniformly to obtain a fifth solution; filtering the fifth solution through a 0.22 μm filter membrane or filter core to obtain a sixth solution; The sixth solution is poured into a 3 ml standard ampoule or syringe bottle and sealed, thereby obtaining an atomized inhalation solution of genipin-1-β-D gentiobioside.

[0058] Formulation Example 3: Inhalation Administration - Inhalation Solution The formulation of the atomized inhalation solution containing genipin-1-β-D gentiobioside is 200 mg of genipin-1-β-D gentiobioside, 0.5% sodium citrate, sodium chloride to adjust the osmotic pressure to isotonicity, sodium hydroxide to adjust the pH to 7.0, and 5 ml of solvent.

[0059] The method for preparing the atomized inhalation solution described above includes the steps of: Measure out 90% (v / v) of the total volume of the solution prepared as needed with water for injection to obtain a first solution; adding sodium citrate to the first solution and stirring uniformly under the condition of controlling the temperature of the first solution at 80°C to obtain a second solution; adding sodium chloride and stirring until dissolved to obtain a third solution; adjusting the pH value of the solution to 7.0 by adding sodium hydroxide to obtain a fourth solution; adding genipin-1-β-D gentiobioside to the fourth solution, stirring evenly, and adding an appropriate amount of sodium hydroxide to maintain the solution pH at 7.0 to obtain a fifth solution; supplementing the fifth solution with a solvent to a constant volume equal to the total volume of the solution prepared as needed, and stirring uniformly to obtain a sixth solution; filtering the sixth solution through a 0.22 μm filter membrane or filter core to obtain a seventh solution; The seventh solution is poured into a 5 ml standard ampoule or syringe bottle and sealed, thereby obtaining an atomized inhalation solution of genipin-1-β-D gentiobioside.

[0060] In the formulation examples 1-3, when using, the atomizer (atomization principle is air compression, vibrating mesh or ultrasonic) should be placed on a flat surface, the device should be as far away from the fabric as possible during operation to avoid fabric fluff blocking the device's air intake, and the atomization cup should be correctly installed according to the instruction manual. Then, open the medicine packaging box, take out the ampoule or syringe bottle, absorb the herbal liquid in it with a syringe, and transfer it to the atomization cup. Sit or stand upright, ensure normal breathing, ensure that the atomization mask covers the mouth and nose or the atomization nozzle is placed in the mouth, open the atomization button, start atomization, and continue inhaling until no more mist droplets are ejected. In order to reduce the risk of infection, disease or contamination, after treatment, the spray should be cleaned and disinfected according to the instruction manual.

[0061] After the stability test, the three formulation examples were left at 25°C for 6 months, and the quality and atomization properties all met the requirements.

[0062] Formulation Example 4: Inhalation Administration - Lyophilized Powder for Inhalation

[0063] The formulation of the freeze-dried powder for inhalation containing genipin-1-β-D gentiobioside is 20 mg of genipin-1-β-D gentiobioside, hydrochloric acid to adjust the pH value to 4.5, and solvent to 1 ml.

[0064] The method for preparing the freeze-dried powder for inhalation described above comprises the steps of: Measure out 70% (v / v) of the total volume of the solution prepared as needed with water for injection to obtain a first solution; Add sodium hydroxide to the first solution to adjust the pH value to 4.5 under the condition of controlling the temperature of the first solution at 30°C, and stir uniformly to obtain a second solution; adding genipin-1-β-D gentiobioside to the second solution and stirring uniformly to obtain a third solution; Measure the pH value. If the pH value is not between 4.4 and 4.6, add an appropriate amount of sodium hydroxide to adjust the pH value of the solution to 4.5, to obtain the fourth solution. supplementing the fourth solution with a solvent to a constant volume equal to the total volume of the solution prepared as needed, and stirring uniformly to obtain a fifth solution; filtering the fifth solution through a 0.22 μm filter membrane or filter core to obtain a sixth solution; Pour the sixth solution into a 10 ml syringe bottle and seal it. The sample is transferred to a freeze-dryer and freeze-dried according to a preset freeze-drying curve, pre-freezing at -45°C for 6 hours, evacuating and heating to -15°C, sublimation drying, maintaining this temperature for 10 hours, heating to 25°C, analytical drying, maintaining this temperature for 4 hours, and after freeze-drying is complete, the container is fully capped, removed, and rolled to obtain freeze-dried powder of genipin-1-β-D gentiobioside for inhalation.

[0065] Formulation Example 5: Inhalation Administration - Lyophilized Powder for Inhalation The formulation of the freeze-dried powder for inhalation containing genipin-1-β-D gentiobioside is 80 mg of genipin-1-β-D gentiobioside, 0.1% citric acid, an appropriate amount of sodium citrate to adjust the pH to 5.0, and 2 ml of solvent.

[0066] Method for preparing the above freeze-dried powder for inhalation: Measure out water for injection in an amount of 50% (v / v) of the total volume of the solution prepared as needed to obtain a first solution; While controlling the temperature of the first solution at 40°C, add citric acid and sodium citrate to the first solution, adjust the pH value to 5.0, and stir uniformly to obtain a second solution; adding genipin-1-β-D gentiobioside to the second solution and stirring uniformly to obtain a third solution; Measure the pH value, if the pH value is not between 4.9 and 5.1, add an appropriate amount of sodium citrate again to adjust the solution pH value to 5.0, to obtain the fourth solution; supplementing the fourth solution with a solvent to a constant volume equal to the total volume of the solution prepared as needed, and stirring uniformly to obtain a fifth solution; filtering the fifth solution through a 0.22 μm filter membrane or filter core to obtain a sixth solution; Pour the sixth solution into a 10 ml syringe bottle and seal it. The sample is transferred to a freeze-dryer and freeze-dried according to a preset freeze-drying curve, pre-freezing at -45°C for 6 hours, evacuating and heating to -15°C, sublimation drying, maintaining the temperature for 15 hours, heating to 30°C, analytical drying, maintaining the temperature for 6 hours, and after freeze-drying is complete, the container is fully capped, removed, and rolled to obtain freeze-dried powder of genipin-1-β-D gentiobioside for inhalation.

[0067] Formulation Example 6: Inhalation Administration - Lyophilized Powder for Inhalation

[0068] The formulation of the freeze-dried powder for inhalation containing genipin-1-β-D gentiobioside is 200 mg of genipin-1-β-D gentiobioside, 0.1% citric acid, disodium hydrogen phosphate to adjust the pH to 7.0, and 5 ml of solvent.

[0069] The method for preparing the freeze-dried powder for inhalation described above comprises the steps of: Measure out 90% (v / v) of the total volume of the solution prepared as needed with water for injection to obtain a first solution; While controlling the temperature of the first solution at 80°C, add citric acid and disodium hydrogen phosphate to the first solution, adjust the pH value to 7.0, and stir uniformly to obtain a second solution; adding genipin-1-β-D gentiobioside to the second solution and stirring uniformly to obtain a third solution; Measure the pH value. If the pH value is not between 6.9 and 7.1, add an appropriate amount of disodium hydrogen phosphate to adjust the pH value to 7.0 to obtain the fourth solution. supplementing the fourth solution with a solvent to a constant volume equal to the total volume of the solution prepared as needed, and stirring uniformly to obtain a fifth solution; filtering the fifth solution through a 0.22 μm filter membrane or filter core to obtain a sixth solution; Pour the sixth solution into a 10 ml syringe bottle and seal it. The sample is transferred to a freeze-dryer and freeze-dried according to a preset freeze-drying curve, pre-freezing at -45°C for 6 hours, evacuating and heating to -15°C, sublimation drying, maintaining the temperature for 25 hours, heating to 20°C, analytical drying, maintaining the temperature for 10 hours, and after freeze-drying is complete, the container is fully capped, removed, and rolled to obtain freeze-dried powder of genipin-1-β-D gentiobioside for inhalation.

[0070] For the samples prepared in Examples 4-6 of the formulation, when using the atomizer (using compressed air, vibrating mesh, or ultrasonic atomization) on a flat surface, keep the device as far away from the fabric as possible during operation to prevent fabric fluff from blocking the device's air intake, and install the atomization cup correctly according to the instruction manual. After opening the medicine box, take out the syringe, draw up 1-5 ml of water for injection with the syringe, inject it into the syringe, shake it, and dissolve the freeze-dried powder. After absorbing the herbal liquid with the syringe, transfer it to the atomization cup. While sitting or standing upright, ensure normal breathing, ensure the atomization mask covers the mouth and nose or the atomization nozzle is placed in the mouth, open the atomization button, start atomization, and continue inhaling until no more mist is emitted. To reduce the risk of infection, disease, or contamination, clean and disinfect the sprayer according to the instruction manual after treatment is completed.

[0071] After stability testing, the contents and related substances of the freeze-dried powders obtained in the three formulation examples were all stable, and the atomization properties all met the requirements.

[0072] Formulation Example 7: Injectable Administration - Lyophilized Powder

[0073] The formulation of the freeze-dried powder for injection containing genipin-1-β-D gentiobioside is 100 mg of genipin-1-β-D gentiobioside, sodium chloride to adjust the osmotic pressure to isotonicity, sulfuric acid / sodium hydroxide to adjust the pH to 6.0, and 3 ml of water for injection.

[0074] The method for preparing the lyophilized powder for injection is as follows: Measure out 40% (v / v) of the total volume of the solution prepared as needed with water for injection to obtain a first solution; adding sodium chloride to the first solution under the condition of controlling the temperature of the first solution at 20°C, and stirring uniformly to obtain a second solution; Add sodium hydroxide to the second solution to adjust the pH value to 6.0, and stir evenly to obtain a third solution; adding genipin-1-β-D gentiobioside to the third solution and stirring uniformly to obtain a fourth solution; If the pH value is not between 5.9 and 6.1, add an appropriate amount of sulfuric acid or sodium hydroxide to adjust the pH value of the solution to 6.0, to obtain a fifth solution. supplementing the fifth solution with water for injection to the total volume of the solution prepared as needed, and stirring uniformly to obtain a sixth solution; filtering the sixth solution through a 0.22 μm filter membrane or filter core to obtain a seventh solution; Pour the seventh solution into a 10 ml syringe bottle and seal it. The sample is transferred to a freeze-dryer and freeze-dried according to the set freeze-drying curve. The sample is pre-freezed at -45°C for 6 hours, vacuumed and heated to -20°C, sublimation-dried, maintained for 14 hours, heated to -10°C, continued sublimation-drying, maintained for 12 hours, and after the sublimation-drying is completed, the sample is heated to 20°C, analytical drying is performed, kept warm for 4 hours, and after the freeze-drying is completed, the sample is fully sealed, removed from the box, and rolled up.

[0075] Formulation Example 8: Injectable Administration - Lyophilized Powder

[0076] The formulation of the freeze-dried powder for injection containing genipin-1-β-D gentiobioside is 200 mg of genipin-1-β-D gentiobioside, sodium chloride to adjust the osmotic pressure to isotonicity, and 5 ml of water for injection.

[0077] The method for preparing the lyophilized powder for injection is as follows:

[0078] Measure out 90% (v / v) of the total volume of the solution prepared as needed with water for injection to obtain a first solution; adding sodium chloride to the first solution and stirring uniformly under the condition of controlling the temperature of the first solution at 50°C to obtain a second solution; adding genipin-1-β-D gentiobioside to the second solution and stirring uniformly to obtain a third solution; Add water for injection to the third solution to make the total volume of the solution prepared as needed, and stir uniformly to obtain a fourth solution; filtering the fourth solution through a 0.22 μm filter membrane or filter core to obtain a fifth solution; Pour the fifth solution into a 10 ml syringe bottle and seal it. The sample is transferred to a freeze-dryer and freeze-dried according to the set freeze-drying curve. The sample is pre-freezed at -45°C for 6 hours, vacuumed and heated to -20°C, sublimation drying is carried out, maintained for 20 hours, heated to -10°C, sublimation drying is continued, maintained for 16 hours, after sublimation drying is completed, the sample is heated to 30°C, analytical drying is carried out, the sample is kept warm for 8 hours, and after freeze-drying is completed, the sample is fully sealed, removed from the box, and rolled up.

[0079] Formulation Example 9: Injectable Administration - Lyophilized Powder

[0080] The formulation of the freeze-dried powder for injection containing genipin-1-β-D gentiobioside is 30 mg of genipin-1-β-D gentiobioside, sodium dihydrogen phosphate and disodium hydrogen phosphate to adjust the pH to 7.0, sodium chloride to adjust the osmotic pressure to isotonicity, and 5 ml of water for injection.

[0081] The method for preparing the lyophilized powder for injection is as follows: Measure out 60% (v / v) of the total volume of the solution prepared as needed with water for injection to obtain a first solution; adding sodium chloride to the first solution and stirring uniformly under the condition of controlling the temperature of the first solution at 80°C to obtain a second solution; 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; adding genipin-1-β-D gentiobioside to the third solution and stirring uniformly to obtain a fourth solution; Measure the pH value. If the pH value is not between 6.9 and 7.1, add an appropriate amount of sodium dihydrogen phosphate / disodium hydrogen phosphate to adjust the solution pH to 7.0, to obtain the fifth solution. supplementing the fifth solution with water for injection to the total volume of the solution prepared as needed, and stirring uniformly to obtain a sixth solution; filtering the sixth solution through a 0.22 μm filter membrane or filter core to obtain a seventh solution; Pour the seventh solution into a 10 ml syringe bottle and seal it. The sample is transferred to a freeze-dryer and freeze-dried according to a preset freeze-drying curve. The sample is pre-freezed at -45°C for 6 hours, vacuumed and heated to -20°C, sublimation-dried, maintained for 18 hours, heated to -10°C, continued sublimation-dried, maintained for 15 hours, and after the sublimation-drying is completed, the sample is heated to 20-30°C, analytical drying is performed, and the temperature is maintained for 7 hours. After the freeze-drying is completed, the sample is fully capped, removed from the box, and rolled up to obtain freeze-dried powder of genipin-1-β-D gentiobioside for injection.

[0082] When using the lyophilized powders prepared in Examples 7-9 of the formulations, the packaging box is opened, the syringe bottle is taken out, 1-5 ml of water for injection is drawn up with a syringe, and the syringe bottle is then shaken to completely dissolve the lyophilized powder, which can then be injected by intramuscular injection, intravenous injection, or mixed with other infusion solutions for infusion.

[0083] The freeze-dried powders prepared in formulation examples 7-9 underwent stability testing, and it was found that the contents and related substances in the freeze-dried powders were all stable after being left at 25°C for 6 months.

[0084] Pharmacological test example 1: Protective effect of intravenous administration of genipin-1-β-D gentiobioside on death in a mouse pneumonia model infected with the human novel coronavirus

[0085] 1. Test materials

[0086] 1.1 Test drug: genipin-1-β-D gentiobioside, physical state: white powder, solubility: highly soluble in water. Mice were given 37.5 mg / kg and 75 mg / kg doses via intraperitoneal injection once daily for 5 consecutive days.

[0087] 1.2 Test animals: hACE2 transgenic C57BL / 6 mice, 6-7 weeks old, 18-25 g, 32 mice in total, provided by Jiangsu Jishu Yaokang Bioscience and Technology Co., Ltd., license number SCXK(Su)2018-008.

[0088] 1.3 Experimental conditions: The ABSL-3 laboratory of Guangzhou Respiratory Health Research Institute was completed.

[0089] 2. Test Method

[0090] 2.1 Grouping and administration: The hACE2 transgenic C57BL / 6 mice were divided into four groups: a blank control group, a SARS-CoV-2 infected group, a genipin-1-β-D gentiobioside (37.5 mg / kg) administration group, and a genipin-1-β-D gentiobioside (75 mg / kg) administration group. Each group consisted of eight mice. Except for the normal group, which received PBS intranasally, each group consisted of 10 mice. 4 Mice were intranasally infected with PFU of SARS-CoV-2 virus. Two hours after infection, the treatment group received intraperitoneal injections of the virus once daily for five consecutive days. After infection, mouse mortality was recorded daily and the 5-day mortality rate was calculated. After the experiment, lung tissue was dissected and homogenized to measure viral infiltration.

[0091] 2.2 Mouse lung tissue homogenous virus instillation assay: Mouse lung tissue was excised, placed in a petri dish, sheared, and transferred to a homogenizing tube. It was diluted 1:10 (w / v) with saline and homogenized at 8,000 rpm / min for 10 min. All of these procedures were performed in an ice bath. The homogenized slurry was transferred to a 1.5 mL EP tube and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was aspirated, aliquoted, and stored at -80°C.

[0092] VERO E6 cells in good growth condition were seeded in a 96-well plate at 1 × 10 4 The cells were cultured at 10 cells / well for 24 hours. After the cells had grown into a complete monolayer, the supernatant was discarded and the cells were washed twice with PBS. After thawing the frozen lung homogenate supernatant, 10 cells / well were cultured at 10 cells / well for 24 hours. -1 ~10 -5 The diluted lung homogenate from each group of mice was diluted 10-fold into five concentrations and then added to a 96-well plate. 100 μL of lung supernatant was added to each well, and blank control wells were also set up. Cell culture medium was added to the blank control wells. Four parallel wells were created for each concentration, and the wells were then cultured in a culture box. Cell lesions (CPE) were observed daily for 4 days, and the number of lesions at each concentration was recorded. The TCID50 values ​​for VERO E6 cells were calculated.

[0093] 3. Test results 3.1 Genipin-1-β-D-gentiobioside protects against pneumonia caused by the novel coronavirus ARS-CoV-2 in mice

[0094] Table 1. Protective effect of genipin-1-β-D gentiobioside against death in mice infected with the novel coronavirus ARS-CoV-2 JPEG2026502403000002.jpg39155The results in Table 1 show that no deaths occurred in the blank control group of mice during the test period, while the mortality rate in the model control group of mice after SARS-CoV-2 infection was 75%. Genipin-1-β-D gentiobioside administered intraperitoneally at 37.5mg / kg and 75mg / kg doses, once daily for 5 consecutive days, significantly reduced the number of mice dying after SARS-CoV-2 infection, with mortality rates of 37.5% and 25%, respectively. This demonstrates that genipin-1-β-D gentiobioside has a significant protective effect against death in mice caused by SARS-CoV-2 infection, with a good dose-effect relationship.

[0095] 3.2 Effect of viral drip in lung tissue of mice infected with the novel coronavirus ARS-CoV-2

[0096] As shown in Figure 1, after viral infection, there was a large amount of viral replication in the lung tissue of the model and control mice. After administration of genipin-1-β-D gentiobioside at 37.5 mg / kg and 75 mg / kg doses, the viral drip in the lung tissue of the mice could be significantly reduced, with significant differences compared with the model and control groups (P<0.05, P<0.01), demonstrating a good dose-effect correlation.

[0097] Pharmacological test example 2: Therapeutic effect of intravenous administration of genipin-1-β-D gentiobioside in a mouse pneumonia model infected with human coronavirus 229E and OC43

[0098] 1. Test materials

[0099] 1.1 Test drug: Genipin-1-β-D gentiobioside, physical properties: white powder, solubility: highly soluble in water. Mice were administered intravenously at doses of 150 mg / kg, 75 mg / kg, and 37.5 mg / kg once daily for four consecutive days.

[0100] 1.2 Positive drug: Chloroquine phosphate tablets, Sichuan Shenghe Pharmaceutical Co., Ltd., Lot number: 2002114, Date of production: February 26, 2020, Expiration date: January 2022. Specification: 0.25g / tablet, Dosage: 0.5g / 60kg / day, Oral administration.

[0101] 1.3 Experimental animals Table 2 Experimental Animal List JPEG2026502403000003.jpg411521.4 Virulent strains and cells: Human coronavirus 229E (HCoV-229E) was provided by the Institute of Pharmaceutical and Biological Technology, Chinese Academy of Medical Sciences. Human coronavirus HCoV-OC43 was purchased from the American Transgenic Corynebacterium CCTV (ATCC) Virus Stock Collection. These cells were passaged in our laboratory and stored in a -80°C refrigerator. Human embryonic lung fibroblast MRC-5 cells were purchased from Beijing Beina Chuanglian Biotechnology Research Institute. These cells were passaged in our laboratory and stored in liquid nitrogen for backup.

[0102] 1.5 Test equipment Table 3 Test equipment list JPEG2026502403000004.jpg551521.6 Test location: ABSL-2 Laboratory, Institute of Traditional Chinese Medicine, Chinese Academy of Traditional Chinese Medicine

[0103] 2. Test Method

[0104] 2.1 Drug Dose Design and Preparation Test drug: The drug doses for mice were 150 mg / kg / d, 75 mg / kg / d, and 37.5 mg / kg / d. The corresponding solutions were prepared using physiological saline and administered intravenously to mice at 2ml / 10g / day once daily for 4 consecutive days. Chloroquine phosphate tablets: Clinical dose: 0.5g / 60kg / d for humans, orally administered. Mouse dose: The clinical dose was converted to a mouse dose of 0.5 g / 60 kg / d x 11 = 0.09 g / kg / d. Mouse dose: 20ml / kg / d, perfusion Preparation concentration: 0.09g / kg / d÷20ml / kg / d=0.0045g / ml.

[0105] 2.2 Virus passaging 25cm of MRC-5 cells grown as a monolayer 2 The culture bottles were collected, the culture medium discarded, and the cell surface washed three times with cell maintenance medium. 5 ml of cell maintenance medium was then added, followed by 200 ml of HCoV-229E or OC43 virus liquid. The bottles were then placed in a 5% CO2 culture chamber at 37°C for 72 to 96 hours of culture. The cell lesions were observed daily under an inverted microscope until 80% of the cells showed obvious lesions (CPE). The cell culture bottles were then frozen and stored in a -80°C low-temperature refrigerator. The virus liquid was frozen and thawed three times before being used for virus titration measurements.

[0106] 2.3 Virus drop measurement The 96-well plate containing monolayer MRC-5 cells was harvested, the culture medium was discarded, and the cells were washed three times with cell maintenance solution. Then, different drops of HCoV-229E or OC43 virus solution were added to the cells in a 10-fold (10 -1 ~10 -8 ) for a total of eight dilutions, with 100 ml per well, with four duplicate wells per dilution, and normal control cells were also included. The 96-well plates were cultured at 37°C in a 5% CO2 incubator for 72-96 hours, and the cytopathic status was observed daily under an inverted microscope, and the cytopathic status of each well was recorded. The 50% cytopathic concentration (TCID) was determined by the Reed-Muench method. 50 ) was calculated.

[0107] 2.4 Construction and administration of a human coronavirus mouse pneumonia model BALB / c mice were randomly divided into three groups according to body weight: normal control group, model control group, chloroquine phosphate control group, and genipin-1-β-D gentiobioside dose group. Each group consisted of 10 mice, half male and half female. Except for the normal control group, all mice in each group were lightly anesthetized with ethyl ether and then administered 100 TCID 50 Mice were intranasally infected with HCoV-229E or OC43 at 50 μl per mouse, once every other day, for a total of two infections. On the day of the initial infection, each treatment group received intravenous administration once daily for four consecutive days. On the fifth day, mice were weighed, dissected, and their lungs were collected. The lung weights were then used to calculate the lung index and inhibition rate.

[0108] Lung index = [lung wet weight (g) / body weight (g)] × 100

[0109] Lung index inhibition rate = (Lung index of model control group - Lung index of treatment group) / (Lung index of model control group - Lung index of normal control group) x 100%

[0110] 3. Test results Table 4. Therapeutic effect of intravenous administration of genipin-1-β-D gentiobioside in a mouse pneumonia model infected with human coronavirus 229E JPEG2026502403000005.jpg80128Note:Comparison with normal control group ## P<0.01, # P<0.05, compared with the model control group ** P<0.01, *P<0.05.

[0111] According to the results in Table 4, after mice were infected with human coronavirus 229E virus, the lung index of the mice was significantly increased, with a significant difference compared with the normal control group (P<0.01). Genipin-1-β-D gentiobioside was administered intravenously on the day of infection, and after 4 days of treatment, all three tested doses were able to significantly reduce the lung index of mice infected with 229E virus, with a significant difference compared with the model control group (P<0.01). The lung index inhibition rates were 99.06%, 71.04%, and 75.12%, respectively. The efficacy was comparable to that of chloroquine phosphate.

[0112] Table 5. Therapeutic effect of intravenous administration of genipin-1-β-D gentiobioside in a mouse pneumonia model infected with human coronavirus OC43 JPEG2026502403000006.jpg75134Note: Comparison with normal control group ## P<0.01, # P<0.05, **P<0.01, *P<0.05 compared with the model control group.

[0113] According to the results in Table 5, after mice were infected with human coronavirus OC43, the lung index of the mice increased significantly, with a significant difference compared with the normal control group (P<0.01). Genipin-1-β-D gentiobioside was administered intravenously on the day of infection, and after 4 days of treatment, the three tested doses were able to significantly reduce the lung index of OC43 virus-infected mice, with significant differences compared with the model control group (P<0.05, P<0.01). The lung index inhibition rates were 39.93%, 42.34%, and 41.59%, respectively, and the efficacy was comparable to that of chloroquine phosphate.

[0114] Pharmacological test example 3: Therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside in a mouse pneumonia model infected with human coronavirus 229E and OC43

[0115] 1. Test materials 1.1 Test drug: genipin-1-β-D gentiobioside, lot number: 20210106, physical state: white powder, solubility: very soluble in water.

[0116] 1.2 Experimental animals: BALB / c mice, SPF grade, 13-15g, 140 mice, half male and half female. Source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

[0117] 1.3 Toxic strains and cells: Human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), purchased from the American Toxic Strain Collection Center (ATCC), TCID 50 is 10 -4 The cells were passaged in this laboratory and stored in a refrigerator at -80°C. Human embryonic lung fibroblasts MRC-5 were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, and were passaged in this laboratory and stored in liquid nitrogen as a backup.

[0118] 1.4 Test Reagents: Table 6 Test Reagent Table JPEG2026502403000007.jpg1331561.5 Test equipment:

[0119] Table 7 Test equipment list JPEG2026502403000008.jpg1681561.6 Test location: ABSL-2 Laboratory, Institute of Traditional Chinese Medicine, Chinese Academy of Traditional Chinese Medicine

[0120] 2. Test Method 2.1 Drug Dose Design and Preparation Test drug: 1. Genipin-1-β-D gentiobioside 75mg / ml set, median particle size 2.02±0.06μm, genipin-1-β-D gentiobioside 37.5mg / ml set, median particle size 2.12±0.08μm, genipin-1-β-D gentiobioside 18.755mg / ml set, median particle size 2.01±0.09μm, genipin-1-β-D gentiobioside 9.375mg / ml set, median particle size 2.07±0.06μm, nebulization time for each set: 25min.

[0121] 2. Chloroquine phosphate tablets, clinical dose: 0.5g / 60kg / d, oral administration, mouse dose: 0.5g / 60kg / d × 11 = 0.09g / kg / d, mouse dose: 20ml / kg / d, gastric perfusion, preparation concentration: 0.09g / kg / d ÷ 20ml / kg / d = 0.0045g / ml.

[0122] 2.2 Grouping and Administration Seventy ICR mice were collected and randomly divided into seven groups according to weight grade: normal control group, model control group, chloroquine phosphate control group, and 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), each group consisting of 10 mice. Except for the normal control group, the remaining mice were lightly anesthetized with isofluoroalkane and administered 100 TCID 50Mice were intranasally infected with either the 229E or OC43 strain of coronavirus, with 50μl per mouse, and retested once the following day. Nebulization began on the day of the first infection, with a flow rate of 7.5L / min. Four doses of genipin-1-β-D gentiobioside (75mg / ml, 37.5mg / ml, 18.75mg / ml, and 9.375mg / ml) were nebulized for 25 minutes each. A chloroquine phosphate control group was perfused intragastrically at 0.2ml / 10g body weight once daily for four consecutive days. A normal control group and a model control group were nebulized with distilled water for 25 minutes under the same conditions. On the fifth day, mice were dissected and the following parameters were measured:

[0123] (1) After weighing the mice, they are dissected, and the whole lung weight is collected to calculate the lung index and inhibition rate.

[0124] (2) Measurement of viral nucleic acid in the left lung lobe of mice, and measurement of inflammatory factor content in the right lung lobe: IL-6, IL-10, and TNF-α.

[0125] The results were statistically analyzed using pairwise comparison t-tests.

[0126] Lung index = [lung wet weight (g) / body weight (g)] × 100

[0127] Lung index inhibition rate = (Lung index of model control group - Lung index of treatment group) / (Lung index of model control group - Lung index of normal control group) x 100%

[0128] 2.3 Measurement of virus load in lung tissue (RT-PCR method) (1) Nucleic acid decomposition treatment

[0129] After dissection, the lung tissues were separated and stored in a -80°C refrigerator. The mouse lung tissues were removed from the -80°C refrigerator, placed in a clean mortar, and crushed into powder using a pestle after adding a small amount of liquid nitrogen. The powder was collected in a 1.5 ml centrifuge tube, and 1 ml of TRIzol Reagent was immediately added. The tube was gently tapped to mix the sample as quickly as possible to form a heavy suspension. Place the tube level at room temperature, incubate for 20 min, centrifuge at 4°C, 12000 rpm for 10 min, transfer the clear supernatant to a new 1.5 ml centrifuge tube, add 0.2 ml of chloroform, close the tube cap tightly, shake the tube vigorously for 15 s, incubate at room temperature for 2-3 min to allow the liquid to stratify, centrifuge at 4°C, 12000 rpm for 10 min, carefully transfer the clear supernatant to a new 1.5 ml centrifuge tube, add 0.5 ml of isopropanol, mix evenly, incubate at room temperature for 30 min, centrifuge at 4°C, 12000 rpm for 10 min, discard the supernatant, gently wash the precipitate with 1 ml of 75% ethanol (allowing the white precipitate to float), centrifuge at 4°C, 7500 rpm for 5 min, siphon off the supernatant, briefly dry the RNA, and precipitate for 5-10 min. 20 μl of the precipitate was added. The solution was dissolved in DEPC water and stored in a low-temperature refrigerator at -80°C.

[0130] (2) Nucleic acid measurement

[0131] Control nucleic acid treatment: DEPC-H2O is the negative control. The positive control is 10 7 , 10 6 , 10 5 , 10 4 Four concentrations were diluted in copies / ml on the gradient scale.

[0132] Reagent preparation: n × 18 μl HCoV-229E nucleic acid fluorescent PCR assay mixture and n × 1 μl internal control were taken and uniformly mixed with n × 1 μl RT-PCR enzyme (n is the number of reaction tubes) for a few seconds, and then centrifuged at 3000 rpm for a few seconds.

[0133] Addition: 20 μl of the above mixture was taken and placed in a PCR tube, then 5 μl each of the sample nucleic acid extract, DEPC-H2O, and positive control was added to the PCR tube, the tube lid was closed, and the tube was centrifuged for a few seconds to settle all the liquid to the bottom, and the PCR amplification reaction was immediately carried out.

[0134] PCR amplification: The reaction tube was placed in a quantitative fluorescence PCR meter, and the cycling parameters were set to 45°C for 10 minutes, 95°C for 15 minutes, and then 95°C for 15 seconds, followed by 60°C for 60 seconds, for a total of 40 cycles. Single-point fluorescence measurement was performed at 60°C, and the reaction mixture was 25 μl.

[0135] Fluorescence channel measurement selection: Select FAM and HEX / VIC / JOE channels.

[0136] (3) Calculation method: A standard curve was drawn based on the ct values ​​of the positive control products at different concentrations, and the sample viral nucleic acid concentration was calculated based on the ct values ​​of the samples.

[0137] 3. Test results

[0138] Table 8. Therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside in a mouse model of pneumonia infected with HCV-229E JPEG2026502403000009.jpg85149Note:Comparison with normal control group ## P<0.01, # P<0.05, compared with the model control group ** P<0.01, * P<0.05.

[0139] According to the results in Table 8, after mice were infected with the human coronavirus 229E strain, the lung index of the model control group mice was significantly higher, with a significant difference compared with the normal control group (P<0.01). Genipin-1-β-D gentiobioside nebulization inhalation was administered on the day of infection, and after four days of treatment, the four tested doses of genipin-1-β-D gentiobioside (75mg / ml, 37.5mg / ml, 18.75mg / ml, and 9.375mg / ml) all significantly reduced the lung index, with a significant difference compared with the model control group (P<0.01). The lung index inhibition rates were 109.19%, 64.22%, 65.59%, and 65.98%, respectively. The efficacy is superior to that of chloroquine phosphate.

[0140] Table 9. Effect of nebulized inhalation administration of genipin-1-β-D gentiobioside on viral load in lung tissue of HCV-229E-infected mice JPEG2026502403000010.jpg90130Note: Comparison between normal and comparison groups ## p<0.01, compared with the model group ** p<0.01.

[0141] According to the results in Table 9, there was no viral nucleic acid expression in the lung tissue of normal mice. After mice were infected with the human coronavirus 229E strain virus, there was obvious coronavirus nucleic acid expression in the lung tissue of the mice. On the day of infection, nebulized genipin-1-β-D gentiobioside was administered, and after four days of treatment, the levels of coronavirus 229E nucleic acid expression in the lung tissue of mice in 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 all significantly reduced, with significant differences (P<0.01) compared with the model control group.

[0142] Table 10. Therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside in a mouse pneumonia model infected with HCV-OC43 JPEG2026502403000011.jpg81159Note: Comparison with normal control group ## P<0.01, #P<0.05, compared with the model control group ** P<0.01, * P<0.05.

[0143] According to the results in Table 10, after mice were infected with the human coronavirus OC43 strain, the lung index of the model control group mice was significantly higher, with a significant difference compared with the normal control group (P<0.01). Genipin-1-β-D gentiobioside nebulization inhalation was administered starting on the day of infection, and after four days of treatment, the four tested doses of genipin-1-β-D gentiobioside, 75mg / ml, 37.5mg / ml, 18.75mg / ml, and 9.375mg / ml, all significantly reduced the lung index of the mice after infection, with a significant difference compared with the model control group (P<0.05, P<0.01). The lung index inhibition rates were 102.88%, 90.30%, 75.73%, and 49.42%, respectively.

[0144] Table 11. Effect of nebulized inhalation administration of genipin-1-β-D gentiobioside on viral load in lung tissue of mice infected with HCV-OC43 JPEG2026502403000012.jpg129128Note: Comparison between normal and comparison groups ## p<0.01, compared with the model group ** p<0.01.

[0145] According to the results in Table 11, there was no viral nucleic acid expression in the lung tissue of normal mice. After mice were infected with the human coronavirus OC43 strain virus, there was obvious coronavirus nucleic acid expression in the lung tissue of the model control group of mice. On the day of infection, nebulized genipin-1-β-D gentiobioside was administered, and after four days of treatment, the coronavirus nucleic acid expression levels of the three tested genipin-1-β-D gentiobioside dose groups, 75 mg / ml, 37.5 mg / ml, and 18.75 mg / ml, all showed significant decreases compared to the model control group (P<0.01).

[0146] Pharmacological Test Example 4: Effect of genipin-1-β-D gentiobioside on inflammatory cytokines in lung tissue of HCV-229E-infected mice

[0147] The above-mentioned nebulized inhalation administration sample was used. The lung tissue was weighed, and an appropriate amount of saline was added to prepare a 10% homogenous tissue slurry. The tissue was homogenized using a high-flux tissue grinder and then centrifuged at 4°C and 3000 rpm for 10 minutes using a low-temperature high-speed centrifuge. The supernatant was aspirated, aliquoted, and stored in a -80°C refrigerator as a reserve. Repeated freezing and thawing was avoided. The absorbance at 450 nm was measured using an enzyme meter according to the kit's instructions.

[0148] Table 12. Effect of genipin-1-β-D-gentiobioside on cytokines in lung tissue of mice infected with human coronavirus 229E JPEG2026502403000013.jpg122160Comparison with normal control group ## P<0.01, # P<0.05, compared with the model group; ** P<0.01.

[0149] According to the results in Table 12, after modeling infection with human coronavirus 229E, the contents of cytokines TNF-α, IL-6, and IL-10 in the lung tissue of the model mice were significantly higher, with significant differences compared with the normal control group (P<0.01). On the day of infection, nebulized inhalation administration of genipin-1-β-D gentiobioside was started and treated for 4 days. All four dose groups reduced the contents of TNF-α, IL-6, and IL-10 in the lung tissue of mice, with significant differences compared with the model group (P<0.01).

[0150] Pharmacological Example 5: Therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside on respiratory syncytial virus (RSV)-infected mouse pneumonia model

[0151] 1. Test materials 1.1 Test drug Genipin-1-β-D gentiobioside preparation, specifications: 5ml, 375mg, lot number: 230608, properties: pale yellow liquid, storage: store at room temperature.

[0152] 1.2 Experimental animals Balb / c mice, SPF grade, weight 9-11g, 50 mice, half male and half female, permit number: SCXK(Kyoto)2016-0006.

[0153] 1.3 virulent virus strains Respiratory syncytial virus (RSV), VR1580™, was purchased from ATCC and passaged in this laboratory and stored in a −80°C refrigerator.

[0154] 1.4 Test equipment Table 13 Test equipment list JPEG2026502403000014.jpg851382 Test method and results

[0155] 2.1 Effects of lung index and lung index suppression rate on mice

[0156] Sixty BALB / C mice, weighing 10±1g, were randomly divided into four groups: normal control group, model control group, high-dose genipin-1-β-D gentiobioside group (75mg / ml), and low-dose genipin-1-β-D gentiobioside group (37.5mg / ml). Each group consisted of 10 mice, half male and half female. Except for the normal control group, mice in each group were lightly anesthetized with isofluoroalkane and intranasally infected with respiratory syncytial virus (RSV) at 35μl per mouse. On the day of infection, each treatment group began nebulized inhalation therapy. Each group was nebulized for 25 minutes, once daily for four consecutive days. The normal control and model control groups were nebulized with normal saline under the same conditions. On the fifth day, the mice were weighed and sacrificed by cervical dislocation. Lung tissue was then collected and weighed, and the lung index and lung index inhibition rate were calculated. Lung tissue was retained for viral load, pathological examination, and inflammatory factor measurements. Results were statistically analyzed using a pairwise t-test.

[0157] Lung index = [lung wet weight (g) / body weight (g)] × 100

[0158] Lung index inhibition rate = (Lung index of model control group - Lung index of treatment group) / (Lung index of model control group - Lung index of normal control group) x 100% Table 14. Therapeutic effect of nebulized inhaled genipin-1-β-D gentiobioside in a mouse model of RSV-infected pneumonia. JPEG2026502403000015.jpg65163Note: Comparison with normal control group ## p<0.01, compared with the model control group ** p<0.01.

[0159] According to the results in Table 14, the lung index of the mice in the RSV model control group was significantly increased, with a significant difference (p<0.01) compared with the normal control group. After nebulizing 75mg / ml and 37.5mg / ml doses of genipin-1-β-D gentiobioside for 25 minutes each time, once a day for 4 consecutive days, the lung index of the mice was significantly reduced, with a significant difference (p<0.01) compared with the model control group, with the lung index inhibition rates of 88.35% and 68.79%, respectively.

[0160] 2.2 Effect on virus load in mouse lung tissue Nucleic acid measurement in lung tissue (RT-PCR method) (1) Nucleic acid decomposition treatment After dissection, the lung tissue was stored separately in a -80°C refrigerator. The mouse lung tissue was removed from the -80°C refrigerator, placed in a clean mortar, and crushed into powder using a pestle. The powder was then transferred to a 1.5ml centrifuge tube. 1ml of TRIzol Reagent was immediately added, and the tube was gently tapped to mix the sample as quickly as possible to form a heavy suspension. The tube was placed level at room temperature and incubated for 20 min. Then, the tube was centrifuged at 4°C for 10 min at 12,000 rpm. The clear supernatant was transferred to a new 1.5ml centrifuge tube, 0.2ml of chloroform was added, the tube cap was tightly closed, and the tube was vigorously rocked for 15 s. The tube was incubated at room temperature for 2-3 min to allow the liquid to stratify. Then, the tube was centrifuged at 4°C for 10 min at 12,000 rpm. The clear supernatant was transferred to a new 1.5ml centrifuge tube and diluted with isopropyl alcohol. 0.2 ml of isopropanol was added, the tube cap was tightly closed, the tube was vigorously agitated for 15 seconds, and incubated at room temperature for 2-3 minutes to allow the liquid to stratify. The tube was then centrifuged at 12,000 rpm at 4°C for 15 minutes. The clear supernatant was carefully transferred to a new 1.5 ml centrifuge tube, 0.5 ml of isopropanol was added, the mixture was mixed evenly, and the tube was incubated at room temperature for 30 minutes. The tube was then centrifuged at 12,000 rpm at 4°C for 10 minutes. The supernatant was discarded, the precipitate was gently washed with 1 ml of 75% ethanol (allowing the white precipitate to float), and the tube was centrifuged at 7,500 rpm at 4°C for 5 minutes. The supernatant was then aspirated, the RNA was briefly dried, and the precipitate was allowed to settle for 5-10 minutes. The precipitate was then dissolved in 20 μl of DEPC water and stored in a -80°C refrigerator.

[0161] (2) Nucleic acid measurement Control nucleic acid treatment: DEPC-H2O is the negative control. 10 7 , 10 6 , 10 5 , 10 4 The solution was diluted to a total of four concentrations using a copies / ml gradient. Reagent preparation: n x 18 μl of RSV nucleic acid fluorescent PCR assay mixture was taken, and n x 1 μl of internal control and n x 1 μl of RT-PCR enzyme (n is the number of reaction tubes) were mixed evenly for a few seconds, followed by centrifugation at 3000 rpm for a few seconds. Addition: 20 μl of the above mixture was taken and placed in a PCR tube, then 5 μl each of the sample nucleic acid extract, DEPC-H2O, and positive control was added to the PCR tube, the tube lid was closed, and the tube was centrifuged for a few seconds to settle all the liquid to the bottom, and the PCR amplification reaction was immediately carried out. PCR amplification: The reaction tube was placed in a quantitative fluorescence PCR meter, and the cycling parameters were set to 45°C for 10 minutes, 95°C for 15 minutes, and then 95°C for 15 seconds, followed by 60°C for 60 seconds, for a total of 40 cycles. Single-point fluorescence measurement was performed at 60°C, and the reaction mixture was 25 μl. Fluorescence channel measurement selection: Select FAM and HEX / VIC / JOE channels. Note: To use an ABI series PCR meter, select "none" for both the passive reference and quencher. Calculation method: A standard curve was drawn based on the ct values ​​of different concentrations of positive control products, and the sample viral nucleic acid concentration was calculated based on the ct values ​​of the samples.

[0162] Table 15. Therapeutic effect of nebulized inhaled genipin-1-β-D gentiobioside in a mouse model of RSV-infected pneumonia. JPEG2026502403000016.jpg70162 According to the results in Table 15, the RSV virus burden in the lung tissue of mice in the RSV model control group was significantly increased. After nebulizing 75 mg / ml and 37.5 mg / ml doses of genipin-1-β-D gentiobioside for 25 minutes each time, once a day for 4 consecutive days, the RSV virus burden in the lung tissue of mice was significantly reduced, with the inhibition rates being 56.08% and 57.54%, respectively.

[0163] Pharmacological Example 6: Therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside in mice infected with Mycoplasma pneumoniae

[0164] 1.1 Test drug: Genipin-1-β-D gentiobioside preparation, specification: 5 ml, 375 mg, lot number: 230608. 1.2 Positive drug: Azismycin capsules: Lot number: 23032004, production date: March 14, 2023, expiration date: February 2025. Produced by Sunflower Pharmaceutical Co., Ltd. Ingredients: The main ingredient of this product is azismycin. Properties: This product is in capsule form, and the contents are a white or white crystalline powder. Indications: Pneumonia caused by Mycoplasma pneumoniae. Specifications: 0.25g / capsule. Dosage and administration: Oral administration. Adults: 0.5g once a day for three consecutive days. Children: 10mg / kg of body weight daily. Storage conditions: Sealed and stored in a dry place. 1.3 Experimental animals Balb / c mice, SPF grade, weight 13-15g, several hundred mice, half male and half female. 1.4 Viral strains: Mycoplasma pneumoniae (MP), VR15531 TM . 1.5 Test equipment Table 16 Test Equipment List JPEG2026502403000017.jpg621491.6 Test Reagent Table 17 Test Reagent Table JPEG2026502403000018.jpg70138

[0165] 2 Test method and results 2.1 Effects on lung index and lung index inhibition rate in mice Fifty BALB / C mice, weighing 14±1g, were randomly divided into five groups: a normal control group, a model control group, an azithromycin control group, a high-dose genipin-1-β-D gentiobioside group (75mg / ml) nebulized for 15 minutes, and a low-dose genipin-1-β-D gentiobioside group (37.5mg / ml) nebulized for 15 minutes. Each group consisted of 10 mice, half male and half female. Except for the normal control group, each group was lightly anesthetized with isofluoroalkane and intranasally infected with Mycoplasma pneumoniae at 50μl per mouse for three consecutive days. On the day of infection, each treatment group was administered nebulized once daily for four consecutive days. The normal control group and model control group were administered saline nebulization under the same conditions. On the fifth day, the mice were weighed and then sacrificed by cervical dislocation. Lung tissue was then collected and weighed, and the lung index and lung index inhibition rate were calculated. Inflammatory factor content in lung tissue was measured, and serum CRP content was measured. Lung tissue was collected for pathological examination. Results were statistically analyzed using a pairwise t-test.

[0166] Lung index = [lung wet weight (g) / body weight (g)] × 100

[0167] Lung index inhibition rate = (Lung index of model control group - Lung index of treatment group) / (Lung index of model control group - Lung index of normal control group) x 100%

[0168] Table 18. Therapeutic effect of genipin-1-β-D gentiobioside in MP-infected mouse pneumonia model JPEG2026502403000019.jpg109170Note: Comparison with normal control group ## p<0.05, compared with the model control group ** p<0.01.

[0169] According to the results in Table 18, the lung index of the MP model control group was significantly elevated and significantly different from the normal control group (p<0.01). Four days after nebulized administration of genipin-1-β-D gentiobioside, the lung index of the low-dose mice was significantly higher than that of the model control group (p<0.01). The results of the two experiments showed good overlap.

[0170] 2.2 Measurement of inflammatory factors in mouse lung tissue (ELISA method) (1) Sample collection and storage: Tissue homogenized slurry sample: After weighing the lung tissues of the mice, the lung tissues of eight mice in each group were collected and stored at -80°C. The tissues were homogenized using an MP Fastprep-24 5G high-speed sample preparation device and centrifuged at -4°C and 3000 r / min for 10 minutes using a low-temperature high-speed centrifuge. The supernatant was aspirated, aliquoted, and stored in a -80°C refrigerator as a backup. Repeated freezing and thawing was avoided.

[0171] (2) After equilibrating to room temperature, the micropore plate was removed from the sealed bag and different concentrations of standard solution were added to the corresponding wells. 50 μL per well, 10 μL of experimental sample was added to each well, and 40 μL of dilution solution was then added. After removing the cavity, 100 μL of HRP was added to each well. The reaction wells were sealed with a sealing sheet and incubated at 37°C for 1 hour. The plate was then washed with washing solution, this was repeated four times. After the last wash, the plate was turned upside down and patted dry with absorbent paper to remove any remaining liquid. 50 μL each of substrates A and B was added to each well, the reaction wells were sealed with a sealing sheet, and the plate was incubated at 37°C for 15 minutes. After this, 50 μL of stop solution was added to each well, and the absorbance at 450 nm was measured within 15 minutes using an enzyme meter. The results were then calculated.

[0172] Table 19. Therapeutic effect of genipin-1-β-D gentiobioside in MP-infected mouse pneumonia model JPEG2026502403000020.jpg94166Note: Comparison with normal control group ## p<0.01, compared with the model control group ** p<0.01.

[0173] According to the results in Table 19, the inflammatory factors IL-6, IL-1β, and TNF-α in the lung tissue of the MP model control group were significantly elevated, with a significant difference compared with the normal control group (p<0.01). After 4 days of nebulized administration of high and low doses of genipin-1-β-D gentiobioside, the inflammatory factor content of the pneumonia mice was significantly reduced, with a significant difference compared with the model control group (p<0.01).

[0174] 2.3 Measurement of CRP in mouse serum (ELISA method) (1) Sample collection and storage: Blood was collected from the mouse orbit and left to stand for 2 hours, then centrifuged at 2000 g for 10 minutes. The supernatant was absorbed and stored at -80°C, avoiding repeated freezing and thawing. When measuring CRP, the blood was diluted 3000 times with saline.

[0175] (2) After equilibrating to room temperature, the micropore plate was removed from the sealed bag. Different concentrations of standard and sample solutions were placed in the corresponding wells, 100 μL per well, and incubated at room temperature for 2 hours. The plate was washed five times with washing solution. 100 μL of biotinylated antibody was added and incubated at room temperature for 60 minutes. The plate was washed five times, and 100 μL of horseradish peroxidase was added and incubated for 20 minutes in the dark. After washing the plate five times, a color developer was added and incubated for 20 minutes. Finally, 50 μL of stop solution was added, and the absorbance at 450 nm was measured within 15 minutes using an enzyme meter. The results were calculated.

[0176] Table 20. Therapeutic effect of genipin-1-β-D gentiobioside in MP-infected mouse pneumonia model JPEG2026502403000021.jpg79128Note: Comparison with normal control group ## p<0.01, compared with the model control group ** p<0.01.

[0177] According to the results in Table 20, the serum CRP content of mice in the MP model control group increased significantly, with a significant difference compared with the normal control group (p<0.01). After 4 days of aerosol administration of high and low doses of genipin-1-β-D gentiobioside, the inflammatory factor content CRP level of pneumonia mice decreased significantly, with a significant difference compared with the model control group (p<0.01).

[0178] 2.4 Mouse lung histopathological changes (HE staining) Normal control group: The alveolar surface was smooth, without obvious deformation or rupture, the arrangement of thin bronchial epithelial cells was orderly, and no edema, degeneration, or necrosis was observed. The pulmonary interstitium showed no fibrous tissue proliferation or inflammatory cell infiltration, and the tissue structure was normal. Model control group: endoscopic observation showed extensive alveolar congestion, edema, and exudation, some alveolar walls ruptured, alveolar fusion changes formed, the alveolar spaces were slightly or moderately thickened, accompanied by a large amount of inflammatory cell infiltration, mainly composed of neutral granular cells, with a small amount of lymphocytes, giant cells, and plasma cells, and slight or mild edema and degeneration of bronchial mucosal epithelial cells, with a small amount of necrotic exfoliated epithelium and inflammatory cells in the lumen, which were significantly different from the normal control group (P<0.01). BD-77 high-dose group: The mice's alveoli showed slight or mild focal congestion and edema, the alveolar walls showed slight or mild thickening, accompanied by a small amount of inflammatory cell infiltration, mainly consisting of neutral granular cells. Some animals' bronchial epithelium showed slight or mild edema, and the arrangement of bronchial epithelial cells was relatively regular, which was significantly reduced compared with the model group (P<0.1). BD-77 low-dose group: Under microscopic observation, the alveolar walls of the mice were slightly or mildly thickened, accompanied by a small amount of inflammatory cell infiltration, which was significantly reduced compared to the model group (P<0.1). Alveolar multifocality was slightly or mildly congested and edematous, which was reduced compared to 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.

[0179] Table 21. Therapeutic effect on BD-77 MP-infected mouse pneumonia model JPEG2026502403000022.jpg70136Pharmacological Test Example 7: Therapeutic effect of intravenous injection of genipin-1-β-D gentiobioside in an LPS-induced mouse pneumonia model

[0180] 1. Test materials 1.1 Drug: Genipin-1-β-D gentiobioside preparation, Specification: 5ml, 375mg, Lot number: 230608, Properties: Light yellow liquid, Storage: Store at room temperature. 1.2 Animals: ICR mice, SPF grade, body weight 18 - 20 g, 60 mice, half male and half female, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., animal production license number: SCXK(Beijing)2021 - 0006, housed in an ABSL - 2 biosafety laboratory. 1.3 Reagents and equipment: Escherichia coli endotoxin (LPS) is produced by Sigma, lot number: lot#057M4013V, isofluroroalkane, Jiangsu Hengfengqiang Biotechnology Co., Ltd., lot number: 20211202. Production date: 20211224, expiration date: 20231223. MC 1.8 type biosafety cabinet, Thermo company. BSA3202S - CW, BSA323S - CW electronic balances, Sartorius Instrument Co., Ltd. AL - 204 type METTLER TOLEDO electronic balance, Mettler Toledo Instruments (Shanghai) Co., Ltd.

[0181] 2 Test methods and results: 2.1 Effects on mouse lung index and lung index inhibition rate Sixty healthy mice, 18 - 20 g, half male and half female, were selected, stratified by body weight, and then randomly grouped into a normal control group, a model control group, three dose groups of genipin - 1 - β - D - genthiobioside at 150 mg / kg / d, 75 mg / kg / d, 37.5 mg / kg / d, and a positive control drug group of sivelestat sodium at 50 mg / kg / d, with 10 mice in each group, half male and half female. After grouping, each administration group was administered by intravenous injection at 20 ml / kg, once a day for 2 consecutive days. The normal control group and the model control group were injected with physiological saline. One hour after the administration on the second day, except for the normal control group, the remaining groups were lightly anesthetized with isofluroroalkane, and the mice were given intranasally with an LPS physiological saline solution at a concentration of 20 mg / ml, 0.05 ml per mouse, resulting in a mouse pneumonia model. Six hours after infection, the animals were sacrificed and dissected, the whole lungs were weighed, and the lung index (lung index = lung weight / body weight × 100%) was calculated.

[0182] Table 22 Effects of genipin - 1 - β - D - genthiobioside on LPS - induced mouse pneumonia model JPEG2026502403000023.jpg79135 Note: Compared with the normal control group ## p < 0.01, compared with the model control group** p<0.01.

[0183] According to the results in Table 22, 6 hours after the mice inhaled LPS, the lung index of the model control group mice increased significantly, with a significant difference compared with the normal control group (p<0.01), while the lung index of the high and medium dose groups of genipin-1-β-D gentiobioside decreased significantly, with a significant difference compared with the model control group (p<0.01, p<0.05). This indicates that the 150mg / kg and 75mg / kg dose groups of genipin-1-β-D gentiobioside have a protective effect on LPS-induced pneumonia in mice.

[0184] 2.2 Measurement of inflammatory factors in mouse lung tissue (ELISA method) (1) Sample collection and storage: Tissue homogenized slurry sample: After weighing the lung tissues of mice 1, 2, 3, 6, 7, and 8, the lung tissues were collected and stored at -4°C. The tissues were homogenized using an ultrasonic cell disrupter and centrifuged at -4°C and 1000 rpm in a low-temperature high-speed centrifuge for 10 minutes. The supernatant was aspirated, aliquoted, and stored in a refrigerator at -80°C as a spare. Repeated freezing and thawing was avoided.

[0185] (2) Remove the micropore plate from the sealed bag after equilibrating to room temperature. Add different concentrations of standard samples, experimental samples, or quality control products to the corresponding wells, 100 μL per well. The reaction wells were sealed with a sealing sheet and incubated at room temperature for 2 hours. The plate was washed with washing solution, and this was repeated four times. After the final wash, the plate was turned upside down and patted dry with absorbent paper to remove any remaining liquid. 100 μL of enzyme target antibody was added to each well. The reaction wells were sealed with a sealing sheet and incubated at room temperature for 2 hours. Repeat the fourth step of the washing plate procedure, adding 100 μL of chromogenic substrate to each well and incubating at room temperature for 30 minutes, while keeping the plate protected from light. Within 30 minutes of adding 100 μL of the terminal solution to each well, the absorbance at 450 nm was measured using an enzyme meter. The results were calculated.

[0186] Table 23. Effect of genipin-1-β-D-gentiobioside on LPS-induced pneumonia in mice JPEG2026502403000024.jpg102153Note: Comparison with normal control group ## p<0.01, compared with the model control group * p<0.05, ** p<0.01.

[0187] According to the results in Table 23, 6 hours after the mice inhaled LPS, the contents of IL-6, IL-10, and TNF-α in the lung tissue of the model control group mice were significantly elevated, with significant differences compared with the normal control group (p<0.01, p<0.05). The genipin-1-β-D gentiobioside-treated groups significantly reduced the contents of IL-6, IL-10, and TNF-α in all groups, with significant differences compared with the model control group (p<0.01, p<0.05). Genipin-1-β-D gentiobioside at doses of 150 mg / kg and 75 mg / kg demonstrated protective effects against LPS-induced pneumonia in mice.

Claims

1. Use of genipin-1-β-D gentiobioside in the preparation of a medicament for treating airway damage caused by coronavirus, respiratory syncytial virus, or Mycoplasma pneumoniae infection.

2. The use of genipin-1-β-D gentiobioside in preparing a medicament according to claim 1, characterized in that genipin-1-β-D gentiobioside is used to prepare a medicament for treating pneumonia inflammatory damage caused by coronavirus, respiratory syncytial virus, or Mycoplasma pneumoniae infection.

3. The use of genipin-1-β-D gentiobioside in a drug preparation according to claim 1, characterized in that genipin-1-β-D gentiobioside is used to prepare a drug for treating pneumonia caused by coronavirus infection and having a death-protecting effect.

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

5. The administration route of the genipin-1-β-D gentiobioside includes any one of atomized inhalation, oral administration, injection, sublingual, spray, and anal administration; The use of genipin-1-β-D gentiobioside in preparing a drug according to claim 1, characterized in that the drug dosage form includes any one of an inhalant, an oral administration, an injection, a spray, a film, and a suppository.

6. A nebulized inhalant containing genipin-1-β-D gentiobioside for realizing the use according to any one of claims 1 to 5, The atomized inhalant containing genipin-1-β-D gentiobioside is characterized by comprising the following raw materials: genipin-1-β-D gentiobioside, a pH adjuster for adjusting the pH to 4.5 to 7.0, an osmotic pressure adjuster that accounts for 0 to 0.9% by weight of genipin-1-β-D gentiobioside, and a solvent.

7. The method for preparing the atomized inhalant comprises: Measure out 40% to 90% (v / v) of the total volume of the solution prepared as needed with water for injection to obtain a first solution; adding an osmolality adjuster to the first solution under the condition that the temperature of the first solution is 20 to 80°C, and stirring the mixture uniformly to obtain a second solution; adding a pH adjuster 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; 7. The atomized inhalant containing genipin-1-β-D gentiobioside according to claim 6, further comprising: replenishing the third solution with a solvent to a constant volume equal to the total volume of the solutions prepared as needed; and uniformly stirring the mixture to obtain an atomized inhalant of genipin-1-β-D gentiobioside.

8. An injection containing genipin-1-β-D gentiobioside for realizing the use according to any one of claims 1 to 5, The injection containing genipin-1-β-D gentiobioside is characterized in that it contains the following raw materials: genipin-1-β-D gentiobioside, a pH adjuster that adjusts the pH value to 4.5-7.0, an osmotic adjuster that adjusts the osmotic pressure to isotonicity, and an injection solvent.

9. The method for preparing the injection comprises: Measure out 40% to 90% (v / v) of the total volume of the solution prepared as needed with water for injection to obtain a first solution; adding an osmolality adjuster to the first solution under a condition where the temperature of the first solution is 20 to 80°C, and stirring the mixture uniformly to obtain a second solution; adding a pH adjuster 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; 9. The injection containing genipin-1-β-D gentiobioside according to claim 8, further comprising: replenishing the third solution with a solvent to a constant volume equal to the total volume of the solutions prepared as needed; and uniformly stirring the mixture to obtain an injection of genipin-1-β-D gentiobioside.

10. A method for preparing genipin-1-β-D gentiobioside, which extracts genipin-1-β-D gentiobioside for use according to any one of claims 1 to 5, comprising: a. Collecting gardenia herbs, extracting them with water, concentrating the extract under reduced pressure, and adjusting the content of the herbs in the extract to 0.03-0.2 g / ml; b. The extract is passed through a large-pore resin column, first eluted with 1-5 column volumes of deionized water, then eluted with 1-5 column volumes of 10-20% ethanol, the ethanol eluate is collected, the ethanol is recovered under reduced pressure, concentrated to 0.1 times the volume of the crude drug, and ethanol is added to an alcohol concentration of 90%, allowed to stand, precipitated, filtered, the alcohol precipitate supernatant is passed through a neutral alumina column, and then eluted with 1-8 column volumes of 50-90% ethanol, the ethanol eluate with a volume concentration of 50-60% is collected, the ethanol is recovered under reduced pressure, and dried to obtain a crude product. c) Purifying the crude product two or three times by hot dissolving in ethanol and recrystallizing, drying the purified product, and then removing the ethanol to obtain highly purified genipin-1-β-D gentiobioside.

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

5.

12. 11. The method for preparing genipin-1-β-D gentiobioside according to claim 10, characterized in that in step b, the gardenia extract is loaded onto an NKA-9 macroporous resin column, and first eluted with 2 column volumes of deionized water. The sample solution and the effluent are collected, and then the sample X-5 macroporous resin is loaded onto the column, and first eluted with 1 column volume of deionized water, and then eluted with 5 column volumes of 10% ethanol by volume. The ethanol eluate is collected, and the ethanol is recovered under reduced pressure. The resulting mixture is concentrated to a relative density of 1.08-1.15 at 60°C, and then ethanol is added to a volume concentration of 90% ethanol. The mixture is allowed to stand, precipitated, and filtered. The supernatant of the alcohol precipitate is passed through a neutral alumina column, and then eluted with 6 column volumes of 90% ethanol by volume and 4 column volumes of 60% ethanol by volume. The 60% ethanol eluate is collected, and the ethanol is recovered under reduced pressure. The mixture is 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 crude product is dissolved in ethanol and recrystallized by adding the crude product to 0.5 to 1 times absolute ethanol, heated to reflux to dissolve, and then filtered by hot filtration, allowed to stand to precipitate, and then extracted to obtain a purified product.

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