Antibacterial and antiviral pharmaceutical compositions and uses thereof

Abstract

An antibacterial and antiviral pharmaceutical composition containing 4 to 14 parts by weight of Shoot Root, 9 to 28 parts by weight of Atractylodes Root, 5 to 18 parts by weight of Koju, 11 to 33 parts by weight of Spirea Root, 0.1 to 3 parts by weight of Clove, and 2 to 10 parts by weight of Mentha Root, as well as natural borneol and pharmaceutical additives. The pharmaceutical composition can be formulated into a medicinal essential oil. The pharmaceutical composition and its essential oil can be used for antibacterial and antiviral purposes, for fever reduction by removing heat from the body and / or for treating respiratory diseases, and can also be used to prepare medicines exhibiting anti-SARS-CoV-2 properties.

Description

[Technical field]

[0001] The present application is in the field of medicine, specifically, antibacterial and antiviral pharmaceutical compositions and uses thereof. [Background technology]

[0002] Respiratory diseases are common clinical diseases that occur frequently due to microbial infections, including bacterial infections, viral infections, fungal infections, etc., and are characterized by high incidence, significant harm, high recurrence, and increasing serious drug resistance.

[0003] Among them, bacterial infection is a common cause of respiratory diseases, and the pathogenic bacteria that cause respiratory bacterial infections are various, and Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae and the like are common. Clinical chronic lung diseases include chronic obstructive pulmonary disease, bronchial asthma, pulmonary tuberculosis, etc., and when these bacterial infections occur concomitantly, these diseases may rapidly worsen or even lead to death. Chemical drugs represented by antibiotics currently have obvious therapeutic effects, but their side effects are severe, they are prone to superinfection, and drug resistance is becoming increasingly serious.

[0004] Respiratory tract viruses are the main pathogens that invade the respiratory tract and cause local lesions in the respiratory tract or only use the respiratory tract as an entry portal, mainly causing lesions in respiratory tissues and organs, and are the main pathogens causing acute respiratory tract infections. Statistics show that more than 90% of acute respiratory tract infections are caused by viruses, and upper respiratory tract infections in particular are clinically common and frequently occurring diseases. Currently, more than 10 viruses are known to cause acute respiratory tract infections, and these viruses are generally spread through droplets, which is an easy route of infection, and often result in high morbidity and excess mortality due to the lack of effective and sustained immunity. Especially during influenza epidemics and epidemics, they often cause complications and severe cases in children and the elderly. Vaccines can be used for prevention, but they are only effective against influenza viruses, not against other respiratory tract viruses, and the duration of immunity is relatively limited. As for treatment, so far no Western medicine has been found that has low toxicity and side effects while ensuring good therapeutic effects. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is an urgent need to provide a drug that can effectively exhibit antibacterial and antiviral properties and has good therapeutic effects on respiratory diseases. [Means for solving the problem]

[0006] Thus, the present application provides an antibacterial and antiviral pharmaceutical composition and its use in the preparation of antibacterial, antiviral, antipyretic by removing heat from the body and / or treating respiratory diseases.

[0007] To achieve the above objectives, the present application uses the following technical solutions: Great Cuckoo1. An antibacterial and antiviral pharmaceutical composition comprising the raw materials: 4-14 parts by weight of Pogostemi Herba, 9-28 parts by weight of Atractylodis Lanceae Rhizoma, 5-18 parts by weight of Moslae Herba, 11-33 parts by weight of Artemisiae Folium, 0.1-3 parts by weight of Caryophylii Flos, and 2-10 parts by weight of Mentha Herba.

[0008] Great Cuckoo The fragrance of the herb dissolves dampness, harmonizes the stomach and prevents vomiting, dissipates heat, and releases it to the outside; the herb Atractylodes dries out dampness, strengthens the spleen, dissipates wind, and eliminates cold; the herb Koju removes heat, expels dampness, has antibacterial properties, and relieves inflammation; and the herb Gaiyou warms the meridians, dissipates dampness, suppresses coughs, dissipates phlegm, and improves asthma. Great Cuckoo The combination of Atractylodes Root, Koju and Gaiyo is useful for reducing inflammation, suppressing coughs, improving asthma, clearing phlegm, removing fever and increasing antibacterial and antiviral effects.

[0009] Clove is used to warm the stomach, reduce qi reflux, moisten the kidneys, harmonize the properties of the medicinal substances mentioned above, and enhance the antibacterial and antiviral effects.

[0010] Mint dissipates impurities with its aroma and has antiviral, analgesic, antipruritic and bactericidal effects, which can reduce foamy phlegm in the airways, expand the effective airway lumen and enhance the expectorant effect.

[0011] Preferably, the antibacterial and antiviral pharmaceutical composition further comprises 0.01 to 0.2 parts by weight of natural borneol.

[0012] Natural borneol is capable of clearing all natural orifices, relieving pathogenic inflammation, and also relieving swelling and pain, and its addition can further improve the antibacterial and antiviral effects of the composition.

[0013] More preferably, the antibacterial and antiviral pharmaceutical composition comprises Great Cuckoo The raw materials include 8 to 10 parts by weight of Atractylodes Root, 18 to 20 parts by weight of Atractylodes Root, 10 to 12 parts by weight of Koju, 20 to 24 parts by weight of Gaiyo, 0.4 to 2.5 parts by weight of Clove, 4 to 8 parts by weight of Mint, and 0.05 to 0.15 parts by weight of natural borneol.

[0014] Preferably, the above-mentioned pharmaceutical composition further comprises a pharmaceutical excipient.

[0015] Preferably, the pharmaceutical composition further comprises 0.5 to 1.5 parts by weight of a pharmaceutical additive, which can be selected from sucralose and / or sodium benzoate.

[0016] Antibacterial and antiviral medicinal essential oils are 1) Great Cuckoo adding water to each of the Atractylodes Root, Koju, Gaiyo, Clove and Mint, distilling the mixture to obtain essential oils from each of the above-mentioned raw materials, and then mixing the essential oils from each of the above-mentioned raw materials to obtain a mixed essential oil; or Great Cuckoo , Atractylodes Root, Koju, Gaiyo, Clove, and Mint, and then adding water and distilling to obtain total essential oil; or Great Cuckoo , Atractylodes Root, Koju, Gaiyo, Clove, and Mint are mixed and placed in a supercritical carbon dioxide extraction device, liquid carbon dioxide is introduced, and essential oil is extracted by supercritical extraction to obtain total essential oil; 2) adding natural borneol to the mixed essential oil or the whole essential oil, and heating to dissolve the natural borneol to obtain a medicinal essential oil; or Add natural borneol to a solvent at 50-60°C, dissolve it by stirring, cool it to room temperature, and then add it to the mixed essential oil or the whole essential oil to obtain a medicinal essential oil. It is prepared by a method comprising:

[0017] Preferably, the heating temperature after adding natural borneol does not exceed 60°C.

[0018] Preferably, the process for preparing the essential oil of each of the raw materials comprises: Great Cuckoo Add the mixture to 5 to 8 times its own weight in water, leave it to soak overnight, and then steam distill it for 4 to 8 hours. Add the soju to 5 to 8 times its own weight in water, soak for 20 to 60 minutes, then steam distill for 4 to 6 hours. The kouju is added to 5 to 8 times its own weight in water, soaked for 1 to 3 hours, and then steam distilled for 2 to 4 hours. The mussels are added to 6 to 10 times their weight in water, soaked for 4 to 8 hours, and then steam distilled for 4 to 6 hours. The cloves are added to 5 to 8 times their weight in water, soaked overnight, and then steam distilled for 4 to 8 hours. The process involves adding mint to 5 to 8 times its own weight in water, soaking it for 1 to 3 hours, and then steam distilling it for 4 to 6 hours.

[0019] Preferably, the method for preparing the total essential oil comprises: Great Cuckoo Mix the ginger, soju, kouju, chinese ginger, clove, and peppermint, add them to 5 to 10 times their weight in water, soak for 1 to 8 hours, and then steam distill for 2 to 8 hours; or Great Cuckoo The method involves mixing Atractylodes Root, Koju, Gaiyo, Clove, and Mint and placing them into a supercritical carbon dioxide extraction device, then introducing liquid carbon dioxide to extract essential oils at an extraction pressure of 20 to 40 MPa, an extraction temperature of 40 to 70°C, a separation temperature of 30 to 60°C, and an extraction time of 0.5 to 10 hours.

[0020] 1. An antibacterial and antiviral medicinal fragrance comprising: Great Cuckoo The present invention provides an antibacterial and antiviral medicinal aromatic perfume obtained by mixing atractylodes rhizome, kouju, chinese ginger, clove, and peppermint, adding the mixture to 5 to 12 times its own weight in water, soaking overnight, and then distilling the mixture.

[0021] There is provided a use of a pharmaceutical composition or a medicinal essential oil or a medicinal fragrance in the preparation of a medicine for antibacterial, antiviral, antipyretic by removing heat from the body and / or treating respiratory diseases.

[0022] The pharmaceutical composition, medicinal essential oil, medicinal aromatic perfume or medicines prepared therefrom of the present application have good therapeutic effects on fever and respiratory diseases, such as rhinitis, pharyngitis, pneumonia, cough and asthma, effectively exhibit antibacterial and antiviral properties, and can alleviate various symptoms caused by bacterial infection and viral infection.

[0023] Furthermore, the aforementioned viruses include SARS-CoV-2.

[0024] Furthermore, rhinitis in traditional Chinese medicine is generally classified into four types, consisting of three deficiency symptoms and one substantial symptom. Among them, the three deficiency symptoms include: 1) Lung Qi deficiency: The nose opens the lungs, and lung deficiency naturally causes nasal obstruction and also causes some nasal symptoms. 2) Spleen Qi deficiency: The spleen is the mother of the lungs, and spleen deficiency also causes lung deficiency, which results in nasal symptoms including nasal itching, clear nasal discharge, persistent clear nasal discharge, sneezing, and persistent sneezing. 3) Kidney Yang deficiency and Kidney Qi deficiency: The kidneys and lungs are connected to each other, and a weak kidney also causes nasal symptoms. The above three deficiency symptoms are actually related to the lungs. In addition, substantial symptoms in the classification of syndromes are related to the fever of the lung meridian, belong to heat syndrome, and are also caused by the heat of the lungs themselves. The pharmaceutical composition of the present application can effectively treat the above four types of rhinitis.

[0025] Preferably, the above-mentioned medicaments can be prepared into a number of dosage forms, such as sprays, medicinal distillates, aerosols, dropping pills, soft capsules, nasal drops or spray inhalation preparations.

[0026] Preferably, the method for preparing the spray comprises: Dilute the medicinal essential oil with a diluent, mix evenly, and filter to obtain a spray; or Add natural borneol and pharmaceutical additives to a solvent at 50-60℃, stir to dissolve, cool to room temperature, add mixed essential oil or whole essential oil to it, dilute with diluent, stir uniformly, and filter to obtain a spray. The method for preparing a pharmaceutical distillate comprises: Add pharmaceutical additives to the medicinal aromatic fragrance and mix evenly to obtain a medicinal distillate; The method for preparing the aerosol includes: Diluting the medicinal essential oil with a diluent, stirring uniformly, filtering, filling into a pressurized container, capping, and filling with a propellant to obtain an aerosol; or Add natural borneol and pharmaceutical additives to a solvent at 50-60°C, stir to dissolve, cool to room temperature, add mixed essential oils or whole essential oils to it, dilute with a diluent, stir uniformly, filter, fill into a pressurized container, cover, and fill with a propellant to obtain an aerosol. The preparation method of the drop pills is: The medicinal essential oil is prepared into an essential oil microcapsule powder by microencapsulation technology, and then mixed with a drop pill matrix to obtain a drop pill; The method for preparing the soft capsules is as follows: encapsulating the medicinal essential oil with a soft capsule material to obtain a soft capsule; The method for preparing the nasal drops is as follows: Dissolving medicinal essential oil in anhydrous ethanol solution of gel matrix material, adding diluent, stirring uniformly, and drying under reduced pressure to remove anhydrous ethanol to obtain oil gel nasal drops; The method for preparing a nebulized formulation for inhalation comprises the steps of: The method involves diluting medicinal essential oils with diluents, stirring uniformly, filtering and filling.

[0027] More preferably, the method for preparing the dropping pills is as follows: A. Mixing emulsifier, corn protein and sunflower phospholipid in medicinal essential oil, and their mass fractions in the medicinal essential oil are 0.5-1.5%, 0.5-2% and 0.1-2%, respectively. B. Dissolve sodium starch octenylsuccinate in water to prepare an aqueous solution with a mass fraction of 15 to 50%. The two solutions prepared in CA and B are mixed in a weight ratio of 1:1-2, emulsified while stirring using an ultra-high speed stirrer at 10,000-14,000 r / min, and homogenized 2-3 times using a high-pressure homogenizer. The homogenized solution obtained in DC is spray dried to obtain the essential oil microcapsule powder. E. The essential oil microcapsule powder and the drop pill matrix are prepared into drop pills with a mass ratio of 1:2, and the drop pill matrix material is polyethylene glycol 4000:polyethylene glycol 6000=1:1.

[0028] Preferably, the solvent is any one of medium chain triglyceride, 1 to 98% ethanol, absolute ethanol, propylene glycol, glycerol, and edible oil; The diluent is any one of medium chain triglyceride, 1 to 98% ethanol, anhydrous ethanol, propylene glycol, glycerol, and edible oil; The propellant can be nitrogen gas, carbon dioxide, heptafluoropropane (HFC-227ea), tetrafluoroethane (HFC-134a), 1,3,3,3-tetrafluoropropene (HFO-1234ze), 2,3,3,3-tetrafluoropropene (HFO-1234yf), or compressed air.

[0029] Preferably, the ratio of the liquid phase to the gas phase in the aerosol can be selected from any one of 2:8, 3:7, 4:6, 5:5 and 6:4, and the pressure in the pressurized container is 0.6 to 1 MPa.

[0030] More preferably, the edible oil may be selected from hemp seed oil, sacha inchi oil, olive oil, coconut oil, pomegranate seed oil, wheat germ oil, walnut oil, avocado oil, linseed oil, grape seed oil, tea oil, perilla oil, pumpkin seed oil, sunflower seed oil, canola seed oil, cranberry seed oil, astaxanthin oil, deep sea fish oil, soybean oil, peanut oil, sesame oil, camellia oil, corn oil, Princepia utilis seed oil, borage oil, evening primrose oil, palm oil and the like.

[0031] Preferably, the gel matrix material may be selected from ethyl cellulose and the like.

[0032] Furthermore, the methods for preparing sprays, medicinal distillates, aerosols, drop pills, soft capsules, nasal drops, nebulizer formulations and the like according to the present application are not limited to the methods mentioned above.

[0033] In summary, the pharmaceutical composition of the present application can be used to prepare medicines for antibacterial, antiviral, and antipyretic and / or respiratory disease treatment by removing heat from the body, and has good therapeutic effects on fever and respiratory diseases, such as rhinitis, pharyngitis, pneumonia, cough and asthma, and has remarkable efficacy against diseases caused by bacterial infection and viral infection. The pharmaceutical composition of the present application has good applicability in treating mild COVID-19 (mild clinical symptoms, no signs of pneumonia in imaging diagnosis, and positive nucleic acid test) and ordinary COVID-19 (fever and respiratory symptoms, macroscopic signs of pneumonia in imaging diagnosis, signs of viral pneumonia, and positive nucleic acid test). [Brief description of the drawings]

[0034] [Figure 1] Figure 2 shows the toxic effect of the spray of Example 4 on Vero E6 cells, CC50>1 / 100 (v / v). [Diagram 2] Shows the toxic effect of remdesivir on Vero E6 cells, CC50>200μM. [Diagram 3] Shows the inhibitory effect of the spray of Example 4 on cell death induced by SARS-CoV-2 (co-addition of drug and virus), IC50>1 / 100 (v / v). [Figure 4] Figure 1 shows the inhibitory effect of remdesivir on cell death induced by SARS-CoV-2 (co-addition of drug and virus), IC50=1.118±0.052 μM. [Diagram 5] Shows the inhibitory effect of the spray of Example 4 on cell death induced by SARS-CoV-2 (post-infection drug addition), IC50>1 / 100 (v / v). [Figure 6] Figure 2 shows the inhibitory effect of remdesivir on cell death induced by SARS-CoV-2 (drug added post-infection), IC50=0.825±0.128 μM. [Figure 7] Shows the lethal effect of 1 / 100 (v / v) spray against SARS-CoV-2. [Figure 8] Showing the lethal effect of H2O2 (0.01%) against SARS-CoV-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The technical solutions in the embodiments of the present application are clearly and fully described below, and the general principles defined in this specification may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to these embodiments shown in this specification, but is to be accorded the widest scope consistent with the principles and novel features disclosed in this specification.

[0036] In the examples, the Asian mouse lung-adapted strain of influenza A virus A / FM / 1 / 34 (H1N1) (FM1) was provided by the Institute of Virology, Chinese Academy of Preventive Medicine and stored in liquid nitrogen in our laboratory, A / PR / 8 / 34 (H1N1) and A / Aichi / 2 / 1968 (H3N2) were purchased from the American Type Culture Collection (ATCC), and the 2009 H1N1 novel influenza A virus strain (A / Guangzhou / GIRD07 / 09, H1N1, GenBank No. HM014332.1) and influenza B virus (B / Guangzhou / GIRD08 / 09, FluB) were provided by the State Key Laboratory of Respiratory Diseases, Guangzhou Medical University. The clinical isolates from the Department of Virology, University of South China, H9N2 influenza A virus (A / Ahicken / Guangdong / 1996, H9N2), H6N2 influenza A virus (A / Duck / Guangdong / 2009, H6N2), and H7N3 influenza A virus (A / Duck / Guangdong / 1994, H7N3) were provided by the College of Veterinary Medicine, South China Agricultural University. In vitro anti-SARS-CoV-2 activity tests were commissioned to the Kunming Institute of Zoology, Chinese Academy of Sciences. EXAMPLES

[0037] Example 1 In the antibacterial and antiviral pharmaceutical composition, the raw material is 885 g Great Cuckoo, 1,869g of Atractylodes orbiculatus, 1,064g of Koju, 2,174g of Gaiyo, 46g of Clove, 532g of Mint, and 10g of natural borneol.

[0038] The above-mentioned pharmaceutical composition was used to prepare the medicinal essential oil, and the preparation method was as follows.

[0039] In the form of herbal decoction pieces (drinking pieces) Great Cuckoo was crushed, sieved through a 20 mesh sieve, immersed in 6 times its weight of water overnight, and then steam distilled for 6 hours to obtain the obtained Great Cuckoo The essential oil was recovered and the extraction yield was 2.26%.

[0040] Crude powder of Atractylodes monadelpha was collected and soaked in 6 times its weight in water for 0.5 hours, then steam distilled for 5 hours to recover the Atractylodes monadelpha essential oil, with an extraction rate of 1.07%.

[0041] Koju was harvested and soaked in 6 times its weight in water for 2 hours, then steam distilled for 3 hours, and the resulting Koju essential oil was recovered, with an extraction rate of 0.94%.

[0042] The mussels were harvested, roughly crushed, and soaked in 8 times its weight in water for 6 hours, and then steam distilled for 5 hours to recover the mussel essential oil, with an extraction rate of 0.46%.

[0043] Clove in the form of herbal decoction pieces was soaked in 6 times its weight of water overnight, and then steam distilled for 6 hours to recover the resulting clove essential oil, with an extraction rate of 10.88%.

[0044] The mint in the form of herbal decoction pieces was immersed in 6 times its weight of water for 2 hours, and then steam distilled for 5 hours to recover the resulting mint essential oil, with an extraction rate of 0.84%.

[0045] Extracted Great CuckooThe essential oils, Soujutsu essential oil, Koju essential oil, Gaiyo essential oil, Clove essential oil, and Mentha essential oil were mixed, natural borneol was added, and heated appropriately (not exceeding 60°C) to completely dissolve the natural borneol, and the medicinal essential oil was obtained.

[0046] Example 2 In the antibacterial and antiviral pharmaceutical composition, the raw material is 663.5 g Great Cuckoo , 1,402g of Atractylodes orbiculatus, 798g of Koju, 1,630.5g of Gaiyo, 32g of Clove, 106.5g of Mint, and 2.5g of Natural Borneol.

[0047] The preparation method for preparing the above-mentioned pharmaceutical composition into medicinal essential oil was as follows:

[0048] Each raw material was ground into coarse powder, mixed together and placed into HA220-40-48 supercritical carbon dioxide extraction equipment, then liquid carbon dioxide was introduced and the essential oil was extracted at an extraction pressure of 28-30Mpa, an extraction temperature of 55℃, a separation temperature of 45℃ and an extraction time of 3 hours, obtaining 106.5g of extract (total essential oil); then, natural borneol was added and heated appropriately (not exceeding 60℃) to completely dissolve the natural borneol and obtain the medicinal essential oil.

[0049] Example 3 An efficacy investigation test was conducted on the medicinal essential oil of Example 1.

[0050] Doses for mice in efficacy studies were 65 mg / kg, 130 mg / kg and 260 mg / kg, respectively.

[0051] The doses for guinea pigs in the efficacy study were 32 mg / kg, 64 mg / kg, and 128 mg / kg, respectively.

[0052] The doses for rats in the efficacy study were 37 mg / kg, 74 mg / kg, and 148 mg / kg, respectively.

[0053] 1. Cough induction test in mice using concentrated ammonia water (cough suppressant effect) 1.1 Experimental Method ICR mice, half male and half female, were used. They were randomly divided into six groups according to body weight level: normal control group, model control group, pentoxyverine citrate 20mg / kg group, and medicinal essential oil low dose group (65mg / kg), medium dose group (130mg / kg), and high dose group (260mg / kg), with 14 mice in each group. Mice were intragastrically administered 20mL / kg (mixed with soybean oil to the corresponding concentration) once a day for 7 days. Mice in normal control and model control groups were intragastrically administered the same amount of soybean oil.

[0054] 1.2 Inspection of indicators One hour after the final administration, the mice were placed in a 500mL transparent sealed container, and concentrated ammonia water was sprayed uniformly into the container for 20 seconds using an ultrasonic nebulizer. The abdominal contraction and mouth opening phenomenon in the mice were regarded as coughing, and the coughing latency and the number of coughs in the mice within 2 minutes after being placed in the container were recorded.

[0055] 1.3 Experimental results Compared with the model control group, the cough latency period of mice was significantly longer and the cough frequency of mice was significantly reduced in each dose group of medicinal essential oils (P<0.01). The results are shown in Table 1.

[0056] [Table 1]

[0057] Under the experimental conditions and planned dosages of this study, the medicinal essential oils had obvious cough suppressant effects.

[0058] 2. Effect on asthma-induced guinea pigs by the combination of acetylcholine and histamine phosphate (improvement of asthma) 2.1 Experimental Method A set of half male and half female conventionally raised guinea pigs was used. Each guinea pig was placed in a glass cover (volume 4 L), and an equal volume of 0.1% histamine phosphate and 2% acetylcholine chloride was sprayed into the glass cover using an ultrasonic nebulizer for 15 seconds, and the pre-administration asthma latency (time from spraying to convulsion) of each guinea pig was recorded. Guinea pigs with asthma latency of 120 seconds or less were considered eligible, whereas those exceeding the asthma latency were considered insensitive and were excluded.

[0059] The selected eligible guinea pigs were randomly divided into five groups according to pre-treatment asthma latency: model control group, aminophylline 60 mg / kg group, and medicinal essential oil low dose group (32 mg / kg), medium dose group (64 mg / kg), and high dose group (128 mg / kg), with 12 guinea pigs in each group. The guinea pigs were intragastrically administered 4 mL / kg (mixed with soybean oil to the corresponding concentration) once a day for 7 days. The model control group guinea pigs were intragastrically administered the same amount of soybean oil.

[0060] 2.2. Inspection of indicators One hour after the final dose, the post-challenge asthma latency of the guinea pigs was recorded using the same method: if the guinea pigs did not show any asthmatic symptoms within 3 minutes, it was calculated as 180 seconds.

[0061] 2.3 Experimental results Compared with the model control group, the post-administration asthma latency period and the extension value of the post-administration asthma latency period (post-administration latency period - pre-administration latency period) of guinea pigs in each dose group of medicinal essential oils significantly increased (P<0.01). The results are shown in Table 2.

[0062] [Table 2]

[0063] Under the experimental conditions and planned dosages of this study, medicinal essential oils had obvious asthma improving effects.

[0064] 3. Effect on airway secretion induced by intraperitoneal administration of phenol red in mice (phlegm reduction effect) 3.1 Experimental Method ICR mice, half male and half female, were randomly divided into five groups according to body weight level: model control group, ambroxol hydrochloride 30mg / kg group, and medicinal essential oil low dose group (65mg / kg), medium dose group (130mg / kg), and high dose group (260mg / kg), with 14 mice in each group. Mice were intragastrically administered 20mL / kg (mixed with soybean oil to the corresponding concentration) once a day for 7 days. Mice in the model control group were intragastrically administered the same amount of soybean oil.

[0065] 3.2 Inspection of indicators Thirty minutes after the final administration, the mice were intraperitoneally administered 20 mL / kg of 5% phenol red saline, and 30 minutes later, the mice were sacrificed, the trachea was exposed, the tissue around the trachea was peeled off, and a portion of the trachea from the thyroid cartilage to the tracheal bifurcation was cut off and immersed in a test tube filled with 2 mL of 5% NaHCO3 solution and shaken to thoroughly wash out the secreted phenol red. The mouse was left overnight to obtain a clear supernatant, which was used to measure the absorbance value of the tracheal eluate at 546 nm.

[0066] 3.3 Experimental results Compared with the model control group, the airway secretion of phenol red in mice in each dose group of medicinal essential oils was significantly increased (P<0.01). The results are shown in Table 3.

[0067] [Table 3]

[0068] Under the experimental conditions and planned dosages of this study, medicinal essential oils had obvious phlegm-reducing effects.

[0069] 4. Effect on the amount of sputum discharged from the capillary of rats (sputum reduction effect) 4.1 Experimental Method Half male and half female SD rats were randomly divided into five groups according to body weight level: model control group, 200 mg / kg carbocisteine ​​group, and low (37 mg / kg), medium (74 mg / kg), and high (148 mg / kg) doses of medicinal essential oils, with 12 rats in each group. The rats were intragastrically administered 10 mL / kg (mixed with soybean oil to the corresponding concentration) once a day for 7 days. The rats in the model control group were intragastrically administered the same amount of soybean oil.

[0070] 4.2 Inspection of indicators Forty minutes after the final administration, the rats were anesthetized and fixed in a supine position. The skin in the center of the rat's neck was cut and the trachea was isolated. A glass capillary was inserted between the two cricoid cartilages in the center of the lower edge of the thyroid cartilage through a small hole made with a sharp injection needle, and the capillary was brought into contact with the bottom of the trachea. The sputum in the trachea was absorbed into the capillary. As soon as the capillary was filled with sputum, it was replaced with another capillary of the same diameter. After continuous collection for 2 hours, the length of the liquid column in the capillary was measured using a graduated scale to determine the amount of sputum excreted.

[0071] 4.3 Experimental results Compared with the model control group, the expectoration volume of rats in each dose group of medicinal essential oils was significantly increased (P<0.05 or P<0.01). The results are shown in Table 4.

[0072] [Table 4]

[0073] Under the experimental conditions and planned dosages of this study, medicinal essential oils had obvious phlegm-reducing effects.

[0074] 5. Effect on lipopolysaccharide (LPS)-induced fever model in rats (antipyretic effect) 5.1 Experimental Method Half of the rats were male and half female. The rats were subjected to rectal temperature measurement twice a day for 2 days, and the average value was used as the basal body temperature. Rats with a single body temperature of more than 38°C or a temperature difference between two measurements of more than 0.5°C were excluded. Eligible rats were randomly divided into 6 groups according to body temperature level: normal control group, model control group, aspirin 200mg / kg group, and medicinal essential oil low dose group (37mg / kg), medium dose group (74mg / kg), and high dose group (148mg / kg), with 12 rats in each group. The rats were intragastrically administered 10mL / kg (mixed with soybean oil to the corresponding concentration) once a day for 7 days. The normal control and model control rats were intragastrically administered the same amount of soybean oil.

[0075] 5.2 Inspection of indicators The rats were fasted for 12 hours before the experiment, but were given water. On the day of the experiment, the rats' basal body temperature was measured. One hour after the final administration, the rats in the normal control group were intraperitoneally administered sterile saline, and the rats in the other groups were intraperitoneally administered LPS (20 μg / kg) to model the rats. After modeling, the rats' condition and body temperature changes were recorded for 7 hours, and the body temperature was measured once every hour. The difference between the hourly body temperature and the basal body temperature (ΔT) was used as an index.

[0076] 5.3 Experimental results The normal control rats had no obvious change in body temperature at 7 hours, whereas the model control rats had a gradual increase in body temperature after LPS injection, which was significantly different from the model control group (P<0.01) and reached a peak value at 6 hours. Compared with the model control group, the body temperature in each medicinal essential oil dosage group was significantly decreased from 2 hours to 7 hours (P<0.05 or P<0.01). The results are shown in Table 5.

[0077] [Table 5]

[0078] Under the experimental conditions and planned dosages of this study, medicinal essential oils had obvious antipyretic effects.

[0079] 6. Effect on acute pharyngitis model in rats 6.1 Experimental Method Half male and half female Wistar rats were used, the rats were held by hand and fixed, the lower jaws of the rats were opened and fixed using tweezers, the spray head of the spray was extended from the mouth to the throat of the rats, and 25% ammonia water was sprayed once (about 0.2 mL) into the throat of the rats, and also sprayed twice a day for three consecutive days to induce acute inflammation formation of the rat pharyngeal mucosa by irritation. After three consecutive days of modeling, the rats were randomly divided into a model control group, a dexamethasone 5 mg / kg group, and a medicinal essential oil low dose group (37 mg / kg), a medium dose group (74 mg / kg), and a high dose group (148 mg / kg), with 12 rats in each group. Another 12 rats from the same batch were used as normal control groups. The rats were intragastrically administered 10 mL / kg (mixed with soybean oil to the corresponding concentration) once a day for three days. Rats in the normal control and model control groups were intragastrically administered the same amount of soybean oil.

[0080] 6.2 Inspection of indicators One hour after the final administration, the rats were anesthetized, and the rats were subjected to abdominal aortic blood sampling, then sacrificed, and the serum was separated, and the serum IL-6 and TNF-α concentrations were measured by ELISA.

[0081] 6.3. Experimental Results Compared with the normal control group, the serum IL-6 and TNF-α contents of the model control group rats were significantly increased (P<0.01). Compared with the model control group, each dose group of medicinal essential oils could significantly reduce the serum IL-6 and TNF-α contents of rats (P<0.05 or P<0.01). The results are shown in Table 6.

[0082] [Table 6]

[0083] Under the experimental conditions and planned doses of this study, medicinal essential oils could significantly inhibit the inflammatory response in acute pharyngitis model rats.

[0084] 7. Protective effect against virus-induced death in mice 7.1 Experimental Method Before use, the allantoic cavity of a 9-day-old chicken embryo was inoculated with an Asian mouse lung-adapted strain of influenza A virus A / FM / 1 / 34 (H1N1) (FM1), which was then cultured in the usual manner. After 48 hours, the allantoic fluid was collected aseptically and subcultured twice to obtain the median lethal dose (LD) for ICR mice. 50 ) was measured. Half of the ICR mice, male and female, were randomly divided into six groups according to body weight level: normal control group, model control group, ribavirin 100 mg / kg group, and medicinal essential oil low dose group (65 mg / kg), medium dose group (130 mg / kg), and high dose group (260 mg / kg), with 16 mice in each group. The mice were lightly anesthetized with diethyl ether, and 25 μL of virus solution was inoculated into the nasal cavity of each mouse, and the infectious dose was LD 50 The dose of each medicinal essential oil was 25 times that of the control group, whereas the same amount of sterile saline was dripped into the nasal cavity of the normal control group. Mice in each medicinal essential oil group were intragastrically administered 20mL / kg (mixed with soybean oil to the corresponding concentration), and ribavirin group mice were intraperitoneally administered once a day for 7 days. Mice in the normal control and model control groups were intragastrically administered the same amount of soybean oil.

[0085] 7.2 Inspection of indicators Mice were observed daily for 14 days for disease symptoms, and time to death, number of deaths, and body weight were recorded. Mice alive on the 14th day were counted as 14 days, and mice that died within 24 hours of infection were considered not to have died from infection.

[0086] 7.3 Experimental results The mice in the normal control group were vigorous and agile, had smooth and shiny fur, normal respiratory rate, normal food and water intake, and daily weight gain. Mice infected with influenza virus showed head shaking (head spasms), rapid respiratory rate mainly via abdominal breathing, slowed behavior, curving, hair standing up (ruffled hair), and a significant decrease in food and water intake 24 hours after infection. Mice began to die from the fourth day, reaching a peak on the seventh day, and the condition of the mice in each treatment group was obviously better than that of the model group.

[0087] No mice died in the normal control group, while the survival rate of mice in the model control group was only 6.2%. Compared with the model control group, the survival rates of mice in the low-dose, medium-dose and high-dose medicinal essential oil groups were 25.0%, 37.5% and 56.2%, respectively, which were significantly increased (P<0.05 or P<0.01). The results are shown in Table 7.

[0088] [Table 7]

[0089] Under the experimental conditions and planned dosages of this study, medicinal essential oils could obviously improve the survival rate of mice infected with influenza virus FM1, and had obvious antiviral effects.

[0090] 8. Effect on viral pneumonia in influenza virus-infected mice 8.1 Experimental Method Before use, the allantoic cavity of a 9-day-old chicken embryo was inoculated with an Asian mouse lung-adapted strain of influenza A virus A / FM / 1 / 34 (H1N1) (FM1), which was then cultured in the usual manner. After 48 hours, the allantoic fluid was collected aseptically and subcultured twice to obtain the median lethal dose (LD) for ICR mice. 50) was measured. Half of the ICR mice, male and female, were randomly divided into six groups according to body weight level: normal control group, model control group, oseltamivir 27.5 mg / kg group, and medicinal essential oil low dose group (65 mg / kg), medium dose group (130 mg / kg), and high dose group (260 mg / kg), with 14 mice in each group. The mice were lightly anesthetized with diethyl ether, and each mouse was inoculated with 20 μL of virus solution into the nasal cavity, with the infectious dose being LD 50 The doses of essential oils were 25 times higher than those of the control group, whereas the same amount of sterile saline was dripped into the nasal cavity of the normal control group. Mice in each dose group of medicinal essential oils were intragastrically administered 20mL / kg (mixed with soybean oil to the corresponding concentration) once a day for 7 days. Mice in the normal control and model control groups were intragastrically administered the same amount of soybean oil.

[0091] 8.2 Inspection of indicators One hour after the final administration, the mice were weighed, sacrificed, dissected, and the whole lungs were removed, and the lung index value and the lung index inhibition rate were calculated. The lung tissue was prepared into a 10% lung tissue homogenate with saline, and the prepared lung tissue homogenate was subjected to low-temperature centrifugation, and the contents of IL-8 and TNF-α in the lung tissue were measured by ELISA.

[0092] Lung index = [lung weight (g) / body weight (g)] × 100; The lung index inhibition rate=(mean lung index of the model control group-mean lung index of the test group) / mean lung index of the model control group.

[0093] 8.3 Experimental results Compared with the normal control group, the lung index of the mice in the model control group was significantly increased (P<0.01), and compared with the model control group, the lung index of each medicinal essential oil dosage group was significantly decreased (P<0.05 or P<0.01). The results are shown in Table 8.

[0094] [Table 8]

[0095] Compared with the normal control group, the IL-8 and TNF-α contents in the mouse lung tissue homogenate of the model control group were significantly increased (P<0.01), and compared with the model control group, the IL-8 and TNF-α contents in the mouse lung tissue homogenate of each medicinal essential oil dosage group were significantly decreased (P<0.05 or P<0.01). The results are shown in Table 9.

[0096] [Table 9]

[0097] Under the experimental conditions and planned dosages of this study, medicinal essential oils could obviously ameliorate viral pneumonia induced by H1N1 influenza.

[0098] 9. In vitro antiviral effect study 9.1 Experimental Method The cell line selected was canine kidney cells (MDCK) and the seven virus strains were A / PR / 8 / 34 (H1N1), A / Aichi / 2 / 1968 (H3N2), 2009 H1N1 novel influenza A virus strain (A / Guangzhou / GIRD07 / 09, H1N1), influenza B virus (B / Guangzhou / GIRD08 / 09, FluB), H9N2 influenza A virus (A / Ahicken / Guangdong / 1996, H9N2), H6N2 influenza A virus (A / Duck / Guangdong / 2009, H6N2), and H7N3 influenza A virus (A / Duck / Guangdong / 1994, H7N3).

[0099] The seven influenza viruses mentioned above were propagated in 9- to 11-day-old chicken embryos, and allantoic fluid was harvested, hemagglutination titers were measured, and stored at -80°C.

[0100] The inhibition of influenza virus strains, including H1N1, H3N2, 2009 H1N1 new type A, FluB, H9N2, H6N2 and H7N3, by medicinal essential oils was investigated by cytopathic effect inhibition assay. MDCK cells were routinely cultured in MEM medium containing two antibiotics (dual antibiotics) and 10% fetal bovine serum. Viruses were adsorbed on 96-well plates coated with monolayers of MDCK cells for 2 hours, and the test groups were added with different concentrations of medicinal essential oils (equal volume), whereas the positive control group (ribavirin) and the untreated control group were set at 100 μL / well and incubated at 37°C, 5% CO2 for 2 days.

[0101] 9.2 Inspection of indicators The degree of cytopathic effect was recorded on a 6-point scale: "-", normal cell growth without cytopathic effect; "±", cytopathic effect in less than 10% of the total cell monolayer; "+", cytopathic effect in less than 25% of the total cell monolayer; "++", cytopathic effect in less than 50% of the total cell monolayer; "+++", cytopathic effect in less than 75% of the total cell monolayer; and "++++", cytopathic effect in more than 75% of the total cell monolayer.

[0102] The half inhibitory concentration (IC 50 ) is calculated, and the selection index SI is (TC 50 / I C 50 ) where SI<1 indicates ineffectiveness, SI:1-2 indicates high toxicity and low efficacy, and SI>2 indicates low toxicity and high efficacy.

[0103] 9.3 Experimental results The non-toxic concentration of medicinal essential oils for MDCK cells is 50 mg / mL, and the median toxic concentration (TC 50 The IC values ​​of medicinal essential oils against H1N1, H3N2, 2009 H1N1 novel A, FluB, H9N2, H6N2 and H7N3 were greater than 50mg / mL. 50The values ​​were 18.4 mg / mL, 36.2 mg / mL, 24.2 mg / mL, 28.5 mg / mL, 48.3 mg / mL, 45.5 mg / mL and 30.2 mg / mL, respectively. The results are shown in Table 10.

[0104] [Table 10]

[0105] Under the experimental conditions and planned dosages of this study, medicinal essential oils have obvious inhibitory effects on all seven influenza virus strains, including H1N1, H3N2, 2009 H1N1 novel A, FluB, H9N2, H6N2 and H7N3, among which they show stronger inhibitory effects on H1N1, 2009 H1N1 novel A and FluB strains.

[0106] 10. Protective effect against pneumococcus infection in mice 10.1 Experimental Method Half male and half female ICR mice were randomly divided into 6 groups according to body weight level: normal control group, model control group, Coptis Rhizome 20g / kg group, and medicinal essential oil low dose group (65mg / kg), medium dose group (130mg / kg), and high dose group (260mg / kg), with 20 mice in each group. Mice were intragastrically administered 20mL / kg (mixed with soybean oil to the corresponding concentration) once a day for 7 days. Mice in the model control group were intragastrically administered the same amount of soybean oil.

[0107] 10.2 Inspection of indicators One hour after administration on the fourth day, mice other than the normal control group were intraperitoneally administered 0.5 mL of pneumococcal solution per mouse for three days, and the deaths of mice in each group were observed within seven days.

[0108] 10.3 Experimental Results Compared with the normal control group, the death rate of the pneumococcus-infected mice was significantly increased during 7 days, with a survival rate of 20% (P<0.01); and compared with the model control group, the death rate of the mice in the medium and high dose groups of medicinal essential oils was significantly decreased during 7 days (P<0.05 or P<0.01). The results are shown in Table 11.

[0109] [Table 11]

[0110] Under the experimental conditions and planned dosages of this study, medicinal essential oils could obviously improve the survival rate of mice infected with pneumococcus, and had obvious antibacterial effects.

[0111] 11. Effects on ovalbumin (OVA)-induced allergic rhinitis in rats 11.1 Experimental Method All male SD rats were randomly divided into 6 groups according to body weight level: normal control group, model control group, rhinitis control 0.4g / kg group, and medicinal essential oil low dose group (37mg / kg), medium dose group (74mg / kg) and high dose group (148mg / kg), with 15 mice in each group. Rats other than the normal control group were immunized intraperitoneally with a mixture of 0.3mg ovalbumin (OVA), 30mg Al(OH)3 and 1mL saline, and boosted on the 15th day with 200μg (50μL) of 4% OVA administered to each nostril of the rats by nasal drop. Each group was immunized once a day for 14 days from the 15th day after sensitization. Rats were intragastrically administered 10 mL / kg (mixed with soybean oil to the corresponding concentration), and normal and model control rats were intragastrically administered the same amount of soybean oil.

[0112] 11.2 Inspection of indicators One hour after the final administration, the rats in each group were anesthetized by intraperitoneal administration of 0.35 g / kg of 10% chloral hydrate, and blood was collected from the abdominal aorta of the rats and centrifuged at 4°C and 3000 r / min for 15 minutes to separate serum, which was then stored at -70°C. Histamine (HIS), interleukin-4 (IL-4) and tumor necrosis factor (TNF-α) were measured according to the kit instructions.

[0113] 11.3 Experimental Results Compared with the normal control group, the serum HIS, IL-4 and TNF-α concentrations of the model control rats were significantly increased (P<0.01); compared with the model control group, the serum HIS and IL-4 concentrations of the medium and high dose groups of medicinal essential oils were significantly decreased (P<0.05 and P<0.01); and the serum TNF-α concentrations of rats in each dose group of medicinal essential oils were significantly decreased (P<0.05 or P<0.01). The results are shown in Table 12.

[0114] [Table 12]

[0115] Under the experimental conditions and planned dosages of this study, medicinal essential oils could obviously improve OVA-induced allergic rhinitis in rats and reduce the inflammatory response.

[0116] Example 4 The proportions of the raw materials of the pharmaceutical composition are the same as those in Example 1, and the pharmaceutical composition is prepared according to the method of Example 1. Great Cuckoo Essential oils, soujutsu essential oil, kouju essential oil, gaio essential oil, clove essential oil, and mentha essential oil were prepared and mixed to obtain a mixed essential oil.

[0117] 200mL of medium-chain triglyceride was taken and heated to 50℃-60℃, and natural borneol was added. When natural borneol was completely dissolved, the mixture was cooled to room temperature, and then added to the mixed essential oil to obtain medicinal essential oil. The medicinal essential oil was diluted with medium-chain triglyceride to adjust the total volume to 1000mL, stirred uniformly, and filtered to obtain a pale yellow to yellow transparent oily solution with fragrant odor and slight bitterness, and a relative density (Chinese Pharmacopoeia General Rule 0601) of 0.933-0.961, and then filled into each bottle with 20mL each to obtain the spray. If the spray was stored in an airtight place away from light, its effective duration could be 24 months.

[0118] Further, the spray was identified as follows:

[0119] (1) 15 mL of the product was collected, 50 mL of petroleum ether (60°C-90°C) was added, mixed uniformly, and extracted with 10 mL of 2 mol / L sodium hydroxide solution. The aqueous layer was collected, adjusted to pH 2.0 with 2 mol / L hydrochloric acid solution, and extracted by shaking with 10 mL of petroleum ether (60°C-90°C). The petroleum ether layer was separated and concentrated to 0.5 mL to prepare the test solution. Standard substances of patchouli alcohol (patchoolol) and pogostone were collected, and ethyl acetate was added to each to prepare a solution containing 4 mg of standard substance per mL as the standard substance solution. According to thin-layer chromatography (Chinese Pharmacopoeia General Rules 0502), 2μL of test solution and 1μL of standard solution were aspirated and spotted on the same silica gel G thin-layer plate, and petroleum ether (60℃~90℃)-ethyl acetate-formic acid (10:2:1) was used as the developing solvent to develop the silica gel G thin-layer plate, and the silica gel G thin-layer plate was taken out, dried, and developed by immersing in 5% iron(III) chloride ethanol solution and heating until the spots were clearly visible. In the chromatogram of the test sample, the same purple-blue spots appeared at the positions corresponding to the patchouli alcohol standard, and the same color spots appeared at the positions corresponding to the pogostone standard.

[0120] (2) Content determination: Measured according to gas chromatography (Chinese Pharmacopoeia General Rules 0521).

[0121] Preparation of standard solutions: Appropriate amounts of eugenol and patchouli alcohol standards were precisely weighed and n-hexane was added to prepare solutions containing 0.3 mg eugenol per mL and 0.3 mg patchouli alcohol per mL.

[0122] Preparation of test solution: 1 mL of the product was precisely weighed and placed in a 10 mL measuring flask, and n-hexane was added up to the mark, followed by shaking uniformly to obtain a test solution.

[0123] Chromatography conditions and system suitability test: Capillary chromatography column with 100% dimethylpolysiloxane as stationary phase (column length: 30 m, inner diameter: 0.32 mm, thickness: 0.25 μm); Column temperature is programmed temperature rise, initial temperature 70 ° C., hold for 5 min, heat to 110 ° C. at 5 ° C. / min, heat to 280 ° C. at 10 ° C. / min, then hold for 4 min, heat to 300 ° C. at 10 ° C. / min, hold for 5 min. According to the peak of borneol, the theoretical plate number should be more than 40000.

[0124] 1 μL of the standard solution and the test solution were precisely sampled and injected into the gas chromatograph for measurement.

[0125] Test chromatograms showed the presence of chromatographic peaks with retention times corresponding to the chromatographic peak retention times of standards for patchouli alcohol, carvacrol, eucalyptol, borneol, isoborneol, eugenol and menthol.

[0126] The product is eugenol (C 10 H 12 Contains 3.75 mg of clove per mL calculated as O2 and patchouli alcohol (C 15 H26 O) was calculated. Great Cuckoo Contains more than 5.2 mg per mL.

[0127] The spray showed the effects of dispelling impurities through aroma, diffusing the lungs and regulating qi, dispelling dampness and eliminating phlegm, and cleansing the throat. The spray could be used for mild, normal and convalescent COVID-19 cases, as well as nasal congestion, runny nose, cough and sore throat caused by cold or flu.

[0128] The spray can be sprayed directly into the throat or applied to the nasal cavity. For mild and common COVID-19, the spray is sprayed into the throat twice in the morning, afternoon and evening, 6 pumps each time; for COVID-19 recovery stage, the spray is sprayed into the throat once in the morning, afternoon and evening, 3 pumps each time; for cough and pharyngitis caused by cold or flu, the spray is sprayed into the throat twice a day, 3 pumps each time; for nasal obstruction, runny nose, the spray can be sprayed onto a cotton swab and applied to the nasal cavity.

[0129] In vitro anti-SARS-CoV-2 activity tests were conducted on the above-mentioned sprays.

[0130] 1. Toxicity test of test samples against VeroE6 cells 1.1 Experimental Method Vero E6 cells were cultured in DMEM high glucose complete medium (purchased from Sangon Biotech Co., Ltd., Shanghai, product number: E600005-0500, batch number FC11FC0255) containing 10% fetal bovine serum (FBS), 100 IU / mL penicillin, and 100 μg / mL streptomycin and without phenol red. The cells were subcultured once the day before the experiment, so that the cells were in logarithmic growth phase.

[0131] Vero E6 cells were cultured at 2 × 10 4The cells were inoculated into a 96-well plate at 100 μL / well and incubated at 37° C., 5% CO2 overnight. When the cell monolayer had grown to about 70%, various concentrations of test samples (diluted spray with DMEM medium containing 3% FBS, prepared at the time of use) or Remdesivir (stock solution dissolved in DMSO to a final concentration of 100 mM / mL, stored at 4° C., standard solution diluted with DMEM medium containing 3% FBS, prepared at the time of use) were added to the wells at 100 μL / well, and three replicate wells were set up, whereas drug-free control wells were set up and incubated at 37° C., 5% CO2 for 3 days.

[0132] 1.2 Inspection of indicators Cell viability was detected using a CCK8 kit (purchased from Beyotime Biotechnology Co., Ltd. (Shanghai), product number: C0039, batch number: 121819200616). OD values ​​were measured using a Synergy 2 multimode microplate reader at a measurement wavelength of 450 nm and a reference wavelength of 630 nm.

[0133] According to the experimental results, the dose-response fitting curve was plotted by Origin 7.5, and the 50% inhibitory concentration (CC) of the samples on cell proliferation was calculated by the Reed-Muench method. 50 ) was calculated. Cell proliferation viability (%) = OD value of experimental wells / OD value of control wells × 100.

[0134] 1.3 Experimental results The results are shown in Tables 13 and 14. As shown in Figures 1 and 2, the CC of the spray on Vero E6 cells 50 CC of remdesivir against Vero E6 cells was more than 1 / 100 (v / v). 50 was greater than 200 μM.

[0135] [Table 13]

[0136] [Table 14]

[0137] 2. Inhibition test of test samples against SARS-CoV-2-induced VeroE6 cell death (co-addition of drug and virus to cell groups) 2.1 Experimental Method In the P2 laboratory, Vero E6 cells were cultured at 2 × 10 4 Cells were seeded at 100 cells / well into 96-well plates and incubated overnight at 37° C., 5% CO2; when the cell monolayer had grown to approximately 70%, they were transferred to the P3 laboratory for use.

[0138] According to the toxicity test results of the test samples and remdesivir on Vero E6 cells, various concentration gradients were set up, with three replicate wells for each gradient, and in the P3 laboratory, 50μL of pre-prepared test sample / remdesivir and 50μL of virus diluted supernatant (MOI 0.1) were added simultaneously into each well of the cell culture plate, a negative control without drug and virus, and a positive control without drug only were set up, and the cells were cultured at 37℃, 5% CO2 for 3 days.

[0139] 2.2 Inspection of indicators Cell viability was detected by CCK8 kit, and OD values ​​were measured using a Bio-Tek microplate reader at a measurement wavelength of 450 nm and a reference wavelength of 630 nm.

[0140] According to the experimental results, the dose-response fitting curve was plotted by Origin 7.5, and the 50% effective concentration (IC) of the samples for virus inhibition was calculated by the Reed-Muench method. 50 ) was calculated, and the therapeutic index (TI) value of anti-SARS-CoV-2 activity was calculated as TI=CC 50 / I C 50and the inhibition rate (%) of SARS-CoV-2-induced cell death = (OD value of test well - OD value of positive control well) / (OD value of negative control well - OD value of positive control well) x 100.

[0141] 2.3 Experimental results The results are shown in Tables 15 and 16, and the IC of the spray group is shown in Figures 3 and 4. 50 The IC of the remdesivir group exceeded 1 / 100 (v / v). 50 was 1.118 ± 0.052 μM, with a TI of over 178.89.

[0142] [Table 15]

[0143] [Table 16]

[0144] 3. Inhibition test of test samples against SARS-CoV-2-induced VeroE6 cell death (addition of drugs 1 hour after virus infection of cells) 3.1 Experimental Method In the P2 laboratory, Vero E6 cells were cultured at 2 × 10 4 Cells were seeded at 100 cells / well into 96-well plates and incubated overnight at 37° C., 5% CO2; when the cell monolayer had grown to approximately 70%, they were transferred to the P3 laboratory for use.

[0145] According to the toxicity test results of the test samples and Remdesivir on Vero E6 cells, various concentration gradients were set up, with three replicate wells for each gradient, and in the P3 laboratory, 50μL of virus diluted supernatant (MOI 0.1) was added to each well of the cell culture plate, and a negative control without drug and virus, and a positive control without drug only were set up. After incubation at 37℃ in a 5% CO2 incubator for 1 hour, 50μL / well of the pre-prepared test samples or Remdesivir were added to the test wells, respectively, and the cells were cultured at 37℃, 5% CO2 for 3 days.

[0146] 3.2 Inspection of indicators Cell viability was detected by CCK8 kit, and OD values ​​were measured using a Bio-Tek microplate reader at a measurement wavelength of 450 nm and a reference wavelength of 630 nm.

[0147] According to the experimental results, the dose-response fitting curve was plotted by Origin 7.5, and the 50% effective concentration (IC) of the samples for virus inhibition was calculated by the Reed-Muench method. 50 ) was calculated, and the therapeutic index (TI) value of anti-SARS-CoV-2 activity was calculated as TI=CC 50 / I C 50 and the inhibition rate (%) of SARS-CoV-2-induced cell death = (OD value of test well - OD value of positive control well) / (OD value of negative control well - OD value of positive control well) x 100.

[0148] 3.3 Experimental results The results are shown in Tables 17 and 18, and the IC of the spray group is shown in Figures 5 and 6. 50 was more than 1 / 100 (v / v), and the IC 50 was 0.825±0.128 μM and the TI was greater than 242.42.

[0149] [Table 17]

[0150] [Table 18]

[0151] 4. Direct viral lethality test 4.1 Experimental Method In the P2 laboratory, Vero E6 cells were cultured at 2 × 10 4 Cells were seeded at 100 cells / well into 96-well plates and incubated overnight at 37° C., 5% CO2; when the cell monolayer had grown to approximately 70%, they were transferred to the P3 laboratory for use.

[0152] In a 96-well plate, 100 μL of test sample or 0.01% HO diluted 1 / 100 (v / v) was mixed with 100 μL of SARS-CoV-2 diluent (MOI 0.1), a positive control without drug, and a negative control without virus were set up, and the plate was incubated at 37°C, 5% CO2 for 0, 5, 10, 30 and 60 minutes.

[0153] The medium in the cell culture plate was removed by suction, and the above-mentioned mixed solution of drug and virus was added at 200 μL / well, and the plate was cultured at 37° C. for 3 days in a 5% CO 2 incubator.

[0154] 4.2 Inspection of indicators Cell viability was detected by CCK8 kit, and OD values ​​were measured using a Bio-Tek microplate reader at a measurement wavelength of 450 nm and a reference wavelength of 630 nm.

[0155] The inhibition rate (%) of SARS-CoV-2-induced cell death = (OD value of test well - OD value of positive control well) / (OD value of negative control well - OD value of positive control well) × 100.

[0156] 4.3 Experimental results The results are shown in Tables 19 and 20 and Figures 7 and 8.

[0157] [Table 19]

[0158] [Table 20]

[0159] In summary, the spray of the present application has an inhibitory effect on SARS-CoV-2-induced Vero E6 cell death when incubated with cells and viruses, and the inhibition rate can reach 26.67% when the concentration is 1 / 100 (v / v); when the spray is administered 1 hour after pre-culture of cells and viruses, it has an inhibitory effect on SARS-CoV-2-induced Vero E6 cell death similar to that of the simultaneous incubation group, and the inhibition rate at a concentration of 1 / 100 (v / v) can reach 23.94%; and the spray at a concentration of 1 / 100 (v / v) has a lethal effect on SARS-CoV-2, and the inhibition rate at 60 minutes is 19.53%.

[0160] Example 5 In the pharmaceutical composition, the raw material is 445 g Great Cuckoo , 934g of Atractylodes orbiculatus, 532g of Koju, 1087g of Gaiyo, 2g of Clove, 745g of Mint, and 15g of Natural Borneol.

[0161] According to the same method as in Example 1, the above-mentioned pharmaceutical composition was prepared into medicinal essential oils.

[0162] The medicinal essential oil was diluted with medium-chain triglyceride to adjust the total volume to 1000 mL, stirred uniformly, filtered, filled into a spray bottle according to the specification of 20 mL / bottle, and tightly clamped with a spray pump to obtain the spray.

[0163] Example 6 In the pharmaceutical composition, the raw material is 885 g Great Cuckoo, 1,869g of soju, 1,064g of kouju, 2,174g of gaiyo, 46g of clove, and 532g of mint.

[0164] Great Cuckoo , Atractylodes macrocarpa, Koju, Gaiyo, Clove, and Mint were collected, cut up, mixed, and added to eight times their weight in water. After soaking overnight, they were extracted by steam distillation, and approximately 14,000 mL of distillate, i.e., aromatic perfume, was recovered.

[0165] A pharmacodynamics investigation experiment was conducted on aromatic perfume.

[0166] In the pharmacodynamics study, the doses for mice were 12 mL / kg, 24 mL / kg, and 40 mL / kg, respectively.

[0167] 1. Protective effect against virus-induced death in mice Experimental Method Before use, the allantoic cavity of a 9-day-old chicken embryo was inoculated with an Asian mouse lung-adapted strain of influenza A virus A / FM / 1 / 34 (H1N1) (FM1), which was then cultured in the usual manner. After 48 hours, the allantoic fluid was collected aseptically and subcultured twice to obtain the median lethal dose (LD) for ICR mice. 50 ) was measured. Half of the ICR mice, male and female, were randomly divided into six groups according to body weight level: normal control group, model control group, ribavirin 100 mg / kg group, and low dose (12 mL / kg), medium dose (24 mL / kg), and high dose (40 mL / kg) groups of aromatic perfume, with 20 mice in each group. The mice were lightly anesthetized with diethyl ether, and 25 μL of virus solution was inoculated into the nasal cavity of each mouse, with the infectious dose being LD 50The dose of each group was 25 times that of the control group, whereas the same amount of sterile saline was dripped into the nasal cavity of the normal control group. The mice in each group were administered with 6 mL / kg, 12 mL / kg, and 20 mL / kg, half the total dose, twice a day, with a 6-hour interval between doses. The mice in the ribavirin group were administered with ribavirin intraperitoneally once a day for 7 days, and the normal control group and model control group were administered with the same amount of distilled water intragastrically.

[0168] 1.2 Inspection of indicators Mice were observed daily for 14 days for disease symptoms, and time to death, number of deaths, and body weight were recorded. Mice alive on the 14th day were counted as 14 days, and mice that died within 24 hours of infection were considered not to have died from infection.

[0169] 1.3 Experimental results No mice died in the normal control group, while the survival rate of mice in the model control group was only 10.0%. Compared with the model control group, the survival rates of the low-dose, medium-dose and high-dose groups of aromatic perfume were 30.0%, 40.0% and 55.0%, respectively, which were significantly increased (P<0.05 or P<0.01). The results are shown in Table 21.

[0170] [Table 21]

[0171] From the table above, it can be seen that aromatic water can significantly improve the survival rate of mice infected with influenza virus FM1, and has obvious antiviral effects.

[0172] 2. Effect on viral pneumonia in influenza virus-infected mice 2.1 Experimental Method Before use, the allantoic cavity of a 9-day-old chicken embryo was inoculated with an Asian mouse lung-adapted strain of influenza A virus A / FM / 1 / 34 (H1N1) (FM1), which was then cultured in the usual manner. After 48 hours, the allantoic fluid was collected aseptically and subcultured twice to obtain the median lethal dose (LD) for ICR mice. 50 ) was measured. Half of the ICR mice, male and female, were randomly divided into six groups according to body weight level: normal control group, model control group, oseltamivir 27.5 mg / kg group, and low dose (12 mL / kg), medium dose (24 mL / kg), and high dose (40 mL / kg) groups of aromatic perfume, with 14 mice in each group. The mice were lightly anesthetized with diethyl ether, and 20 μL of virus solution was inoculated into the nasal cavity of each mouse, and the infectious dose was LD 50 The mice in the normal control group received 25 times the amount of sterile saline in the nasal cavity, whereas the same amount of sterile saline was dripped into the nasal cavity of the mice in the normal control group. The mice in each dose group were administered with the aromatic fragrance twice a day for seven days, with a 6-hour interval between doses, and the respective doses were 6mL / kg, 12mL / kg, and 20mL / kg, half the total dose. The normal control group and the model control group received the same amount of distilled water intragastrically.

[0173] 2.2 Inspection of indicators One hour after the final administration, the mice were weighed, sacrificed, dissected, and the whole lungs were removed, and the lung index value and the lung index inhibition rate were calculated. The lung tissue was prepared into a 10% lung tissue homogenate with saline, and the prepared lung tissue homogenate was subjected to low-temperature centrifugation, and the contents of IL-8 and TNF-α in the lung tissue were measured by ELISA.

[0174] Lung index = [lung weight (g) / body weight (g)] × 100; The lung index inhibition rate=(mean lung index of the model control group-mean lung index of the test group) / mean lung index of the model control group.

[0175] 2.3 Experimental results Compared with the normal control group, the lung index of the mice in the model control group was significantly increased (P<0.01), and compared with the model control group, the lung index of each aromatic dosage group was significantly decreased (P<0.05 or P<0.01). The results are shown in Table 22.

[0176] [Table 22]

[0177] Compared with the normal control group, the IL-8 and TNF-α content in the mouse lung tissue homogenate of the model control group was significantly increased (P<0.01); compared with the model control group, the TNF-α content in the mouse lung tissue homogenate of each dose group of Feng Shui was significantly decreased (P<0.05 or P<0.01); and the IL-8 content in the mouse lung tissue homogenate of the medium dose group and high dose group of Feng Shui was significantly decreased (P<0.05 or P<0.01). The results are shown in Table 23.

[0178] [Table 23]

[0179] In summary, aromatic perfume could obviously ameliorate viral pneumonia induced by H1N1 influenza.

[0180] 3. Protective effect against pneumococcus infection in mice 3.1 Experimental Method Half male and half female ICR mice were randomly divided into 6 groups according to body weight level: normal control group, model control group, Shuanghuanglian 20g / kg group, and low dose (12mL / kg), medium dose (24mL / kg) and high dose (40mL / kg) groups of Fanghui, with 20 mice in each group. Mice in each dose group were administered Fanghui twice a day for 7 days, with a 6-hour interval between doses, and the respective doses were 6mL / kg, 12mL / kg and 20mL / kg, half the total dose. The normal control group and the model control group were intragastrically administered the same amount of distilled water.

[0181] 3.2 Inspection of indicators One hour after administration on the fourth day, mice other than the normal control group were intraperitoneally administered 0.5 mL of pneumococcal solution per mouse for three days, and the deaths of mice in each group were observed within seven days.

[0182] Experimental Results Compared with the normal control group, the death rate of the mice infected with pneumococcus increased significantly in 7 days, with a survival rate of 20% (P<0.01); and compared with the model control group, the death rate of the mice in the medium and high dose groups of medicinal fragrance in 7 days was significantly reduced (P<0.05 or P<0.01). The results are shown in Table 24.

[0183] [Table 24]

[0184] From the above table, it can be seen that aromatic perfume can significantly improve the survival rate of mice infected with Streptococcus pneumoniae, and has obvious antibacterial effect.

[0185] Example 7 In the pharmaceutical composition, the raw material is 885 g Great Cuckoo , 1,869g of Atractylodes Root, 1,064g of Koju, 2,174g of Gaiyo, 46g of Clove, 532g of Mint, 35g of Sucralose, and 35g of Sodium Benzoate.

[0186] Great Cuckoo , Atractylodes Root, Koju, Gaiyou, Clove, and Mint were weighed, cut, mixed, and added to 8 times its weight in water. After soaking overnight, it was distilled by steam distillation, the distillate was collected, sucralose and sodium benzoate were added, heated (not exceeding 60°C), stirred uniformly, filled according to the specification of 20mL / bottle, sealed, and after filling, sterilized by moist heat sterilization at 121°C for 30 minutes to obtain a pharmaceutical distillate.

[0187] Example 8 According to the method of Example 1, a medicinal essential oil was prepared, diluted with anhydrous ethanol to adjust the total volume to 1000 mL, stirred uniformly, filtered, and filled into, for example, a pressure-resistant aluminum bottle according to the specification of 20 mL / bottle, capped, and a certain amount of nitrogen gas was filled into the aluminum bottle at a pressure of 0.8 MPa to obtain an aerosol.

[0188] Example 9 In the antibacterial and antiviral pharmaceutical composition, the raw material is 960 g Great Cuckoo , 1,920g of Atractylodes orbiculatus, 1,200g of Koju, 2,160g of Gaiyo, 240g of Clove, 720g of Mint, and 10g of Natural Borneol.

[0189] The above-mentioned pharmaceutical composition was used to prepare the medicinal essential oil, and the preparation method was the same as that in Example 1.

[0190] The preparation method of medicinal essential oil drop pills was as follows: A. Emulsifier, corn protein and sunflower phospholipids are mixed into the prepared pharmaceutical essential oil, and their mass fractions in the pharmaceutical essential oil are 1%, 1.5% and 0.5%, respectively. B. Starch sodium octenyl succinate was dissolved in water to prepare an aqueous solution with a mass fraction of 20%. C. The two solutions prepared in A and B were mixed in a weight ratio of 1:2, emulsified by stirring using an ultra-high speed stirrer at 12000 r / min, and homogenized three times using a high-pressure homogenizer. D. The homogenized solution obtained in C was spray dried to obtain essential oil microcapsule powder. E. The essential oil microcapsule powder and the drop pill matrix were prepared into drop pills with a mass ratio of 1:2, and the drop pill matrix material was polyethylene glycol 4000:polyethylene glycol 6000=1:1.

[0191] The drop pills were 0.32g per 10 tablets, and each tablet contained 7mg of medicinal essential oil.When applied to treat mild, normal or convalescent COVID-19, or nasal congestion, runny nose, cough and sore throat caused by cold or flu, and the like, 14 drop pills were taken once in the morning / afternoon / night.

[0192] Pharmacodynamics experiment was carried out for the medicinal essential oil drop pills of this embodiment.For the experimental groups, the dose was 1.10g / kg for mice, 0.56g / kg for rats, and 0.48g / kg for guinea pigs.Control group 1, control group 2 and control group 3 were set up.

[0193] Among these, the drop pills of control group 1 were prepared according to the method of the experimental group, except that Gaiyou was replaced with Ephedrae Herba, Mint was replaced with Angelicae Dahuricae Radix, and clove and natural borneol were not added.

[0194] Control group 2 drop pills: Great Cuckoo It was prepared according to the method of the experimental group, except that was replaced by Banrankon (Isatidis Radix) and Soujutsu was replaced by Boufu (Saposhnikoviae Radix).

[0195] The drop pills of control group 3 were prepared according to the method of the experimental group, except that Koju was replaced by Sanshishi (Gardeniae Fructus) and Gaiyou was replaced by Qinpi (Citri Reticulatae Pericarpium).

[0196] 1. Cough induction test in mice using concentrated ammonia water (cough suppressant effect) 1.1 Experimental Method ICR mice, half male and half female, were used. The mice were randomly divided into 7 groups according to body weight level: normal control group, model control group, pentoxyverine citrate 20mg / kg group, medicinal essential oil drop pill group, control group 1, control group 2 and control group 3, with 14 mice in each group. The mice were intragastrically administered 20mL / kg (the drop pills were crushed into powder and suspended in 0.5% CMC-Na to the concentration corresponding to each administration group) once a day for 7 days. The mice in the normal control group and model control group were intragastrically administered the same amount of 0.5% CMC-Na solution.

[0197] 1.2 Inspection of indicators One hour after the final administration, the mice were placed in a 500mL transparent sealed container, and concentrated ammonia water was sprayed uniformly into the container for 20 seconds using an ultrasonic nebulizer. The abdominal contraction and mouth opening phenomenon in the mice were regarded as coughing, and the coughing latency and the number of coughs in the mice within 2 minutes after being placed in the container were recorded.

[0198] Experimental Results Compared with the model control group, in the medicinal essential oil drop pill group, as well as in control group 1, control group 2 and control group 3, the mouse cough latency period was significantly longer, and the mouse cough frequency was significantly reduced (P<0.01); compared with the medicinal essential oil drop pill group, in control group 1, control group 2 and control group 3, the mouse cough latency period was significantly shorter, and the mouse cough frequency was significantly increased (P<0.05). The results are shown in Table 25.

[0199] [Table 25]

[0200] Under the experimental conditions and planned dosages of this experiment, the medicinal essential oil drop pill group, as well as control group 1, control group 2 and control group 3, had obvious cough suppression effect, while the cough suppression effect of the medicinal essential oil drop pill group was significantly better than that of control group 1, control group 2 and control group 3.

[0201] 2. Effect on asthma-induced guinea pigs by the combination of acetylcholine and histamine phosphate (improvement of asthma) 2.1 Experimental Method A group of normally bred guinea pigs, half male and half female, were used. Each guinea pig was placed in a glass cover (volume 4 L), and an equal mixture of 0.1% histamine phosphate and 2% acetylcholine chloride was sprayed into the glass cover for 15 seconds using an ultrasonic sprayer, and the pre-administration asthma latency period (time from spraying to convulsion) of each guinea pig was recorded. Guinea pigs with asthma latency periods of 120 seconds or less were considered eligible, whereas those exceeding the asthma latency period were considered insensitive and were excluded.

[0202] The selected eligible guinea pigs were randomly divided into 6 groups according to pre-administration asthma incubation period: model control group, aminophylline 60mg / kg group, medicinal essential oil drop pill group, control group 1, control group 2 and control group 3, with 12 guinea pigs in each group. The guinea pigs were intragastrically administered 4mL / kg (drop pills were crushed into powder and suspended with 0.5% CMC-Na to the corresponding concentration of each administration group) once a day for 7 days. The guinea pigs in the model control group were intragastrically administered the same amount of 0.5% CMC-Na solution.

[0203] 2.2 Inspection of indicators One hour after the final dose, the post-challenge asthma latency of the guinea pigs was recorded using the same method: if the guinea pigs did not show any asthmatic symptoms within 3 minutes, it was calculated as 180 seconds.

[0204] 2.3 Experimental results Compared with the model control group, the medicinal essential oil drop pill group, as well as control group 1, control group 2 and control group 3, the post-administration asthma latency period of guinea pigs and the extension value of the post-administration asthma latency period (post-administration latency period-pre-administration latency period) significantly increased (P<0.01); compared with the medicinal essential oil drop pill group, the post-administration asthma latency period of guinea pigs and the extension value of the post-administration asthma latency period (post-administration latency period-pre-administration latency period) significantly decreased (P<0.05). The results are shown in Table 26.

[0205] [Table 26]

[0206] Under the experimental conditions and planned dosage of this experiment, the medicinal essential oil drop pill group, as well as control group 1, control group 2 and control group 3, had obvious asthma improving effects, while the asthma improving effects of the medicinal essential oil drop pill group were obviously superior to those of control group 1, control group 2 and control group 3.

[0207] 3. Effect on the amount of sputum discharged from the capillaries of rats (sputum reduction effect) 3.1 Experimental Method Half of the SD rats, male and female, were randomly divided into 6 groups according to body weight level: model control group, carbocisteine ​​0.2g / kg group, medicinal essential oil drop pill group, control group 1, control group 2, and control group 3, with 12 rats in each group. The rats were intragastrically administered 10mL / kg (the drop pills were crushed into powder and suspended in 0.5% CMC-Na to the corresponding concentration for each administration group) once a day for 7 days. The rats in the model control group were intragastrically administered the same amount of 0.5% CMC-Na solution.

[0208] 3.2 Inspection of indicators 40 minutes after the final administration, the rats were anesthetized and fixed in a supine position. The skin in the center of the rat's neck was cut and the trachea was isolated. A glass capillary was inserted between the two cricoid cartilages in the center of the lower edge of the thyroid cartilage through a small hole made with a sharp injection needle, and the capillary was allowed to come into contact with the bottom of the trachea. The sputum in the trachea was absorbed into the capillary. As soon as the capillary was filled with sputum, it was replaced with another capillary of the same diameter. After continuous collection for 2 hours, the length of the liquid column in the capillary was measured using a graduated scale to determine the amount of sputum excreted.

[0209] 3.3 Experimental results Compared with the model control group, the expectoration amount of rats in medicinal essential oil drop pill group, as well as control group 1, control group 2 and control group 3 increased significantly (P<0.05 or P<0.01); and compared with the medicinal essential oil drop pill group, the expectoration amount of rats in control group 1, control group 2 and control group 3 decreased significantly (P<0.05). The results are shown in Table 27.

[0210] [Table 27]

[0211] Under the experimental conditions and planned dosages of this experiment, the medicinal essential oil drop pill group, as well as control group 1, control group 2 and control group 3, had obvious phlegm-reducing effect, while the phlegm-reducing effect of the medicinal essential oil drop pill group was obviously better than that of control group 1, control group 2 and control group 3.

[0212] 4. Protective effect against pneumococcus infection in mice (antibacterial effect) 4.1 Experimental Method ICR mice, half male and half female, were randomly divided into 7 groups according to body weight level: normal control group, model control group, Shuanghuanglian 20g / kg group, medicinal essential oil drop pill group, control group 1, control group 2 and control group 3, with 20 mice in each group. Mice were intragastrically administered 20mL / kg (drop pills were crushed into powder and suspended in 0.5% CMC-Na to the corresponding concentration for each administration group) once a day for 7 days. Mice in the model control group were intragastrically administered the same amount of 0.5% CMC-Na solution.

[0213] 4.2 Inspection of indicators One hour after administration on the fourth day, mice other than the normal control group were intraperitoneally administered 0.5 mL of pneumococcal solution per mouse for three days, and the deaths of mice in each group were observed within seven days.

[0214] 4.3 Experimental results Compared with the normal control group, the death rate of mice infected with pneumococcus increased significantly in 7 days, with a survival rate of 20% (P<0.01); and compared with the model control group, the mortality rate of mice in the medicinal essential oil drop pill group, as well as control group 1, control group 2 and control group 3 in 7 days was significantly reduced (P<0.05). Compared with the medicinal essential oil drop pill group, the mortality rate of mice in control group 1, control group 2 and control group 3 in 7 days was increased, but there was no statistically significant difference (P>0.05). The results are shown in Table 28.

[0215] [Table 28]

[0216] Under the experimental conditions and planned dosage of this experiment, the medicinal essential oil drop pill group, as well as control group 1, control group 2 and control group 3, could obviously improve the survival rate of mice infected with Streptococcus pneumoniae, and had obvious antibacterial effects. The antibacterial effect of the medicinal essential oil drop pill group was slightly better than that of control group 1, control group 2 and control group 3.

[0217] 5. Comparative study of protective effects against virus-induced death in mice 5.1 Experimental Method Before use, the allantoic cavity of a 9-day-old chicken embryo was inoculated with an Asian mouse lung-adapted strain of influenza A virus A / FM / 1 / 34 (H1N1) (FM1), which was then cultured in the usual manner. After 48 hours, the allantoic fluid was collected aseptically and subcultured twice to obtain the median lethal dose (LD) for ICR mice. 50 ) was measured. Half of the ICR mice were randomly divided into 6 groups according to body weight level: normal control group, model control group, ribavirin 100 mg / kg group, medicinal essential oil drop pill group 1.10 g / kg group, commercial drop pill group 1.73 g / kg group, and commercial spray 20 mL / kg group (maximum dose), with 24 mice in each group. The mice were lightly anesthetized with diethyl ether, and 25 μL of virus solution was inoculated into the nasal cavity of each mouse, with the infectious dose being LD 50 The amount of saline solution was 25 times that of the nasal cavity of the normal control group, whereas the same amount of sterile saline solution was dripped into the nasal cavity of the normal control group. The mice in the medicinal essential oil dripping pill group, commercial dripping pill group, and commercial spray group were intragastrically administered 20mL / kg (the dripping pills were crushed into powder and suspended in 0.5% CMC-Na to the concentration corresponding to each administration group), and the mice in the ribavirin group were intraperitoneally administered once a day for 7 days. The mice in the normal control group and model control group were intragastrically administered the same amount of 0.5% CMC-Na solution.

[0218] Here, the commercially available drop pills contain the medicinal ingredients cholic acid, Nacre, hyodeoxycholic acid, Gardeniae Fructus, Bubali Cornu, Banlankon, Baicalin and Lonicerae Flos, and the commercially available sprays contain the medicinal ingredients Litsea lancilimba essential oil, Litsea lancilimba essential oil and menthol.

[0219] 5.2 Inspection of indicators Mice were observed daily for 14 days for disease symptoms, and time to death, number of deaths, and body weight were recorded. Mice alive on the 14th day were counted as 14 days, and mice that died within 24 hours of infection were considered not to have died from infection.

[0220] 10.3 Experimental Results The mice in the normal control group were vigorous and agile, had smooth and shiny fur, normal respiratory rate, normal food and water intake, and daily weight gain. Mice infected with influenza virus showed head-shaking behavior, rapid respiratory rate mainly via abdominal breathing, slowed behavior, curving, erect hair, and a significant decrease in food and water intake 24 hours after infection. Mice began to die from the fourth day, reaching a peak on the seventh day, and the condition of the mice in each treatment group was obviously better than that of the model group.

[0221] No mice died in normal control group, while the survival rate of mice in model control group was only 6.2%; compared with model control group, the survival rates of mice in medicinal essential oil dropping pill group, commercial dropping pill group and commercial spray group were 62.5%, 41.7% and 37.5%, respectively, which were significantly increased (P<0.05 or P<0.01); and compared with medicinal essential oil dropping pill group, the survival rates of mice in commercial dropping pill group and commercial spray group were significantly decreased (P<0.05 or P<0.01). The results are shown in Table 29.

[0222] [Table 29]

[0223] Under the experimental conditions and planned dosages of this experiment, the medicinal essential oil dropping pills group, the commercial dropping pills and the commercial spray could significantly improve the survival rate of mice infected with influenza virus FM1, and the antiviral effect of the medicinal essential oil dropping pills was significantly better than that of the commercial dropping pills and the commercial spray.

[0224] 6. Comparative study of viral pneumonia in influenza virus-infected mice 6.1 Experimental Method Before use, the allantoic cavity of a 9-day-old chicken embryo was inoculated with an Asian mouse lung-adapted strain of influenza A virus A / FM / 1 / 34 (H1N1) (FM1), which was then cultured in the usual manner. After 48 hours, the allantoic fluid was collected aseptically and subcultured twice to obtain the median lethal dose (LD) for ICR mice. 50 ) was measured. Half of the ICR mice were randomly divided into 6 groups according to body weight level: normal control group, model control group, oseltamivir 27.5 mg / kg group, medicinal essential oil drop pill group, commercial drop pill group 1.10 g / kg group, commercial spray 20 mL / kg group (maximum dose), with 14 mice in each group. The mice were lightly anesthetized with diethyl ether, and 20 μL of virus solution was inoculated into the nasal cavity of each mouse, with the infectious dose being LD 50 The amount of sterilized saline solution was 25 times that of the normal control group, whereas the same amount of sterile saline solution was dripped into the nasal cavity of the normal control group. The mice in the medicinal essential oil drop pill group, commercial drop pill group, and commercial spray group were intragastrically administered 20mL / kg, and the mice in the ribavirin group were intraperitoneally administered once a day for 7 days. The normal control group and model control group mice were intragastrically administered the same amount of 0.5% CMC-Na solution.

[0225] 6.2 Inspection of indicators One hour after the final administration, the mice were weighed, sacrificed, dissected, and the whole lungs were removed, and the lung index value and the lung index inhibition rate were calculated. The lung tissue was prepared into a 10% lung tissue homogenate with saline, and the homogenate was centrifuged at low temperature, and the contents of IL-8 and TNF-α in the lung tissue were measured by ELISA.

[0226] Lung index = [lung weight (g) / body weight (g)] × 100; The lung index inhibition rate=(mean lung index of the model control group-mean lung index of the test group) / mean lung index of the model control group.

[0227] 6.3 Experimental results Compared with the normal control group, the lung index of the mice in the model control group was significantly increased (P<0.01); compared with the model control group, the lung index of the mice in the medicinal essential oil dropping pill group, the commercial dropping pill group and the commercial spray group was significantly decreased (P<0.05 or P<0.01); and compared with the medicinal essential oil dropping pill group, the lung index of the mice in the commercial dropping pill group and the commercial spray group was significantly increased (P<0.05). The results are shown in Table 30.

[0228] [Table 30]

[0229] Compared with the normal control group, the IL-8 and TNF-α content in the mouse lung tissue homogenate of the model control group is significantly increased (P<0.01); compared with the model control group, the IL-8 and TNF-α content in the mouse lung tissue homogenate of the medicinal essential oil drop pill group, the commercial drop pill group and the commercial spray group is significantly decreased (P<0.05 or P<0.01); and compared with the medicinal essential oil drop pill group, the IL-8 and TNF-α content in the mouse lung tissue homogenate of the commercial spray group is significantly increased (P<0.05). The results are shown in Table 31.

[0230] [Table 31]

[0231] Under the experimental conditions and planned dosages of this study, the medicinal essential oil dropping pills, commercial dropping pills and commercial spray can obviously improve viral pneumonia caused by H1N1 influenza, and the antiviral effect of the medicinal essential oil dropping pills is significantly better than that of the commercial dropping pills and commercial spray.

[0232] Example 10 The method for preparing soft capsules of medicinal essential oils was as follows:

[0233] (1) Preparation of medicinal essential oils According to the method of Example 1, Great Cuckoo Essential oils, Soujutsu essential oil, Koju essential oil, Gaiyo essential oil, Clove essential oil, and Mentha essential oil were prepared respectively. Great Cuckoo essential oil, 20g of soju essential oil, 10g of kouju essential oil, 10g of gaio essential oil, 5g of clove essential oil, and 5g of mentha essential oil were weighed and mixed uniformly to obtain a mixed essential oil for use.

[0234] 10 g of natural borneol was weighed and placed in a container having an appropriate capacity, 200 mL of medium chain triglyceride was added, stirred and heated (not exceeding 60° C.) to facilitate dissolution of the natural borneol until it was completely dissolved for further use.

[0235] The mixed essential oils were added into the natural borneol medium chain triglyceride solution and stirred, then medium chain triglyceride was added up to 1000 mL and mixed evenly for further use.

[0236] (2) Preparation of capsule material Gelatin, glycerol and purified water were weighed in a mass ratio of 1:1:0.5, put into a gelatin melting barrel, melted and mixed at 70°C, and vacuum degassed for further use.

[0237] (3) Preparation of soft capsules Using the YWJ100-IP soft capsule production line, soft capsules were prepared, each containing 0.3g of content by adjusting the parameters in the soft capsule machine, and thus soft capsules of medicinal essential oils were obtained.

[0238] Example 11 The method for preparing medicinal essential oil nasal drops was as follows:

[0239] The proportions of the raw materials of the pharmaceutical composition are the same as those in Example 1, and the pharmaceutical composition is prepared according to the method of Example 1. Great Cuckoo Essential oils, Soujutsu essential oil, Koju essential oil, Gaiyo essential oil, Clove essential oil, and Mint essential oil were prepared and then mixed to obtain a mixed essential oil. 1g of natural borneol was added to 7g of the mixed essential oil, stirred, and heated appropriately (not exceeding 60°C) to completely dissolve the natural borneol, and a medicinal essential oil was obtained as Liquid A.

[0240] As liquid B, 100 mL of a 10% ethyl cellulose absolute ethanol solution was prepared.

[0241] Solution A and solution B were mixed, 100 mL of medium chain triglyceride was added, stirred and mixed for 30 minutes, and dried under reduced pressure to remove absolute ethanol, resulting in a pale yellow to yellow oily gel.

[0242] Example 12 The method for preparing the medicinal essential oil formulation for aerosol inhalation was as follows.

[0243] The proportions of the raw materials of the pharmaceutical composition are the same as those in Example 1, and the pharmaceutical composition is prepared according to the method of Example 1. Great Cuckoo The essential oils, souju essential oil, kouju essential oil, gaio essential oil, clove essential oil, and mentha essential oil were prepared and then mixed to obtain a mixed essential oil.

[0244] 70g of the mixed essential oil was weighed, 10g of natural borneol was added, heated appropriately (not exceeding 60°C) and stirred until the natural borneol was completely dissolved to obtain the medicinal essential oil.

[0245] The medicinal essential oil was diluted with medium-chain triglyceride to adjust the total volume to 1000 mL, stirred uniformly, and filtered to obtain a pale yellow to yellow transparent oily solution with an aromatic odor, slight bitterness, and a relative density (Chinese Pharmacopoeia General Rules 0601) of 0.933 to 0.961, and then 20 mL was filled into each bottle. When used, the medicinal solution was injected into a nebulizer for nebulization inhalation.

[0246] The above examples are only some of the embodiments of the present application, but not all of the embodiments. Based on the examples of the present application, all other examples obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

Claims

1. 1. An antibacterial and antiviral medicinal essential oil comprising essential oil derived from raw materials consisting of 4 to 14 parts by weight of Pogostemi Herba, 18 to 28 parts by weight of Atractylodes Lanceae Rhizoma, 5 to 18 parts by weight of Moslae Herba, 11 to 33 parts by weight of Artemisiae Folium, 0.1 to 3 parts by weight of Caryophilii Flos, and 2 to 10 parts by weight of Mentae Herba.

2. 1. An antibacterial and antiviral medicinal fragrance comprising a fragrance derived from raw materials consisting of 4 to 14 parts by weight of Pogostemi Herba, 18 to 28 parts by weight of Atractylodes Lanceae Rhizoma, 5 to 18 parts by weight of Moslae Herba, 11 to 33 parts by weight of Artemisiae Folium, 0.1 to 3 parts by weight of Caryophilii Flos, and 2 to 10 parts by weight of Mentae Herba.

3. An antibacterial and antiviral pharmaceutical composition comprising the antibacterial and antiviral medicinal essential oil of claim 1 or the antibacterial and antiviral medicinal fragrance of claim 2.

4. The antibacterial and antiviral pharmaceutical composition according to claim 3, wherein the pharmaceutical composition further comprises 0.01 to 0.2 parts by weight of natural borneol.

5. 5. The antibacterial and antiviral pharmaceutical composition according to claim 3 or 4, wherein the pharmaceutical composition further comprises a pharmaceutical excipient.

6. The antibacterial and antiviral pharmaceutical composition according to any one of claims 3 to 5, wherein the pharmaceutical composition is formulated into a dosage form selected from the group consisting of sprays, pharmaceutical distillates, aerosols, drop pills, soft capsules, nasal drops or nebulizer formulations.

7. 2. A method for preparing the antibacterial and antiviral medicinal essential oil of claim 1, comprising the steps of: 1) adding water to each of Kokushitsu, Soujutsu, Koju, Gaiyou, Clove, and Mint, distilling the mixture to obtain essential oils from each of the raw materials, and then mixing the essential oils from each of the raw materials to obtain a mixed essential oil; or Mixing Kokushi, Soujutsu, Koju, Gaiyou, Clove and Mint, then adding water and distilling to obtain total essential oil; or A step of mixing Kokushitsu, Soujutsu, Koju, Gaiyou, Clove and Mint, and putting them into a supercritical carbon dioxide extraction device, introducing liquid carbon dioxide, and performing supercritical extraction to extract essential oils, thereby obtaining total essential oils; 2) adding natural borneol to the mixed essential oil or the total essential oil, and heating to dissolve the natural borneol to obtain a medicinal essential oil; or adding natural borneol to a solvent at 50-60°C, dissolving it by stirring, cooling it to room temperature, and then adding it to the mixed essential oil or the total essential oil to obtain a medicinal essential oil.

8. The method for preparing the essential oils from each of the above raw materials is as follows: The above-mentioned Chinese yew is added to water of 5 to 8 times its own weight, and then soaked overnight, and then distilled by steam distillation for 4 to 8 hours; The soju is added to 5 to 8 times its own weight in water, soaked for 20 to 60 minutes, and then distilled by steam distillation for 4 to 6 hours. The kouju is added to 5 to 8 times its own weight in water, soaked for 1 to 3 hours, and then distilled by steam distillation for 2 to 4 hours. The above-mentioned shellfish is added to 6 to 10 times its own weight of water, and is soaked for 4 to 8 hours, and then is distilled by steam distillation for 4 to 6 hours. The cloves are added to 5 to 8 times their weight in water, soaked overnight, and then steam distilled for 4 to 8 hours. Add the mint to 5 to 8 times its own weight in water, soak for 1 to 3 hours, and then steam distill it for 4 to 6 hours; or The method for preparing the total essential oil comprises: Mix the Kokkuri Root, Soujutsu, Koju, Gaiyou, Clove, and Mint, add to water 5 to 10 times its own weight, soak for 1 to 8 hours, and then distill by steam distillation for 2 to 8 hours; or the Kokushitsu, Soujutsu, Koju, Gaiyou, Clove and Mint are mixed and placed in the supercritical carbon dioxide extraction device, and then liquid carbon dioxide is introduced to extract essential oil at an extraction pressure of 20-40 MPa, an extraction temperature of 40-70°C, a separation temperature of 30-60°C and an extraction time of 0.5-10 hours; The method according to claim 7.

9. A method for preparing the antibacterial and antiviral medicinal fragrance of claim 2, comprising the steps of: The method includes the steps of mixing Kokkuri Root, Soujutsu, Koju, Gaiyou, Clove and Mint, adding them to water 5 to 12 times their weight, soaking them overnight, and then distilling them to obtain a medicinal aromatic fragrance.

10. A pharmaceutical composition according to any one of claims 3 to 6, or a medicinal essential oil according to claim 1, or a medicinal aromatic fragrance according to claim 2, for use in antibacterial, antiviral, antipyretic and / or respiratory disease treatment by removing heat from the body.

11. The pharmaceutical composition, or the medicinal essential oil, or the medicinal aromatic for use according to claim 10, wherein the pharmaceutical composition, or the medicinal essential oil, or the medicinal aromatic is formulated as a dosage form selected from a spray, a medicinal distillate, an aerosol, a drop pill, a soft capsule, a nasal drop or a spray inhalation preparation.

12. Use of the pharmaceutical composition according to any one of claims 3 to 6, the medicinal essential oil according to claim 1 or the medicinal aromatic perfume according to claim 2 in the preparation of a medicine for antibacterial, antiviral, antipyretic and / or respiratory disease treatment, said medicine being formulated in a dosage form selected from spray, medicinal distillate, aerosol, drop pill, soft capsule, nasal drops or nebulized inhalation preparation; The method for preparing the spray comprises: Dilute the medicinal essential oil with a diluent, mix evenly, and filter to obtain a spray; or Add natural borneol and pharmaceutical additives to a solvent at 50-60°C, dissolve them by stirring, cool to room temperature, add the mixed essential oil or the whole essential oil, dilute with a diluent, stir uniformly, and filter to obtain a spray; The method for preparing the pharmaceutical distillate comprises the steps of: Adding pharmaceutical additives to the medicinal aromatic and perfume, and stirring uniformly to obtain a medicinal distillate; The method for preparing the aerosol comprises the steps of: Diluting the medicinal essential oil with a diluent, stirring uniformly, filtering, filling into a pressurized container, capping, and filling with a propellant to obtain an aerosol; or Add natural borneol and pharmaceutical additives to a solvent at 50-60°C, dissolve them by stirring, cool to room temperature, add the mixed essential oil or the whole essential oil, dilute with a diluent, stir uniformly, filter, fill into a pressurized container, close the lid, and fill with a propellant to obtain an aerosol; The method for preparing the drop pills includes: The medicinal essential oil is prepared into an essential oil microcapsule powder by microencapsulation technology, and then mixed with a drop pill matrix to obtain a drop pill; The method for preparing the soft capsule comprises: encapsulating the medicinal essential oil with a soft capsule material to obtain a soft capsule; The method for preparing the nasal drops comprises the steps of: Dissolving said medicinal essential oil in anhydrous ethanol solution of gel matrix material, adding diluent, stirring uniformly, and drying under reduced pressure to remove anhydrous ethanol to obtain an oil gel nasal drop; The method for preparing the formulation for nebulization comprises the steps of: The use thereof is to dilute the medicinal essential oil with a diluent, stir uniformly, filter and fill.

13. The solvent is any one of medium chain triglyceride, 1-98% ethanol, absolute ethanol, propylene glycol, glycerol, and edible oil; the diluent is any one of medium chain triglycerides, 1-98% ethanol, absolute ethanol, propylene glycol, glycerol, or edible oil; or The propellant is nitrogen gas, carbon dioxide, heptafluoropropane, tetrafluoroethane, 1,3,3,3-tetrafluoropropene, 2,3,3,3-tetrafluoropropene, or compressed air; 13. The use according to claim 12.

14. A pharmaceutical composition according to any one of claims 3 to 6, or a medicinal essential oil according to claim 1, or a medicinal fragrance according to claim 2, for use in the treatment of COVID-19.

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