WO2021218945A1 - 一种抗菌抗病毒药用组合物及其应用 - Google Patents

一种抗菌抗病毒药用组合物及其应用 Download PDF

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WO2021218945A1
WO2021218945A1 PCT/CN2021/090137 CN2021090137W WO2021218945A1 WO 2021218945 A1 WO2021218945 A1 WO 2021218945A1 CN 2021090137 W CN2021090137 W CN 2021090137W WO 2021218945 A1 WO2021218945 A1 WO 2021218945A1
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volatile oil
medicinal
control group
group
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PCT/CN2021/090137
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English (en)
French (fr)
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林瑞超
董汛
崔涛
王京昆
孙敏
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云南白药集团股份有限公司
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Priority to KR1020227034107A priority Critical patent/KR20230002351A/ko
Priority to JP2022566110A priority patent/JP2023532621A/ja
Priority to EP21796550.8A priority patent/EP4144358A4/en
Priority to US17/905,390 priority patent/US20230126992A1/en
Publication of WO2021218945A1 publication Critical patent/WO2021218945A1/zh

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Definitions

  • the invention belongs to the technical field of medicine, and more specifically relates to an antibacterial and antiviral medicinal composition and its application.
  • Respiratory system diseases are common and frequently-occurring clinical diseases, and are common in microbial infections, including bacteria, viruses, fungi, etc.; they have the characteristics of high morbidity, great harm, easy relapse, and increasingly serious drug resistance.
  • bacterial infection is a common cause of respiratory diseases, and there are many pathogenic bacteria that cause respiratory bacterial infections, among which Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae are more common.
  • chronic lung diseases include chronic obstructive pulmonary disease, bronchial asthma, tuberculosis, etc. Once combined with these bacterial infections, the disease will deteriorate rapidly and even die.
  • the current chemical drugs represented by antibiotics have significant curative effects, their side effects are large, they are prone to double infections, and their drug resistance is becoming more and more serious.
  • Respiratory tract viruses are a large group of viruses that can invade the respiratory tract and cause local respiratory tract disease or only use the respiratory tract as an intrusion portal, and mainly cause respiratory tract tissue and organ disease. They are the main pathogens that cause acute respiratory infections. According to statistics, more than 90% of acute respiratory tract infections are caused by viruses, especially the above respiratory tract infections are common and frequently-occurring clinical diseases. At present, there are more than 10 kinds of viruses known to cause acute respiratory infections. These viruses are generally transmitted by air droplets. The infection is easy to achieve, and due to the lack of effective and long-lasting immunity, they often cause high morbidity and excess mortality; Especially in the high-incidence season and outbreak period of influenza, it often causes complications and severe cases in children and the elderly.
  • vaccines can now be used for prevention, they are only effective against influenza viruses, and are not effective against other respiratory viruses, and the immunity period is relatively limited. In terms of treatment, so far, no western medicine has been found to have low toxic and side effects while ensuring good curative effects.
  • the present invention provides an antibacterial and antiviral medicinal composition and its application in the preparation of antibacterial, antiviral, heat-clearing and fever-reducing drugs and/or treatment of respiratory diseases.
  • An antibacterial and antiviral medicinal composition in parts by weight, includes the following raw materials:
  • Dingxiang warms the stomach, reduces adverse effects, and damps the kidneys. It harmonizes various medicines and enhances antibacterial and antiviral effects.
  • Peppermint is aromatic, has antiviral, analgesic, antipruritic, and sterilizing effects. It can reduce the foamy phlegm in the respiratory tract, enlarge the effective ventilation cavity, and enhance the expectorant effect.
  • the aforementioned antibacterial and antiviral pharmaceutical composition further comprises 0.01-0.2 parts of natural borneol in parts by weight.
  • Natural borneol can pass through various orifices, dispel stagnant fire, reduce swelling and relieve pain, and its addition can further improve the antibacterial and antiviral effects of the composition.
  • an antibacterial and antiviral pharmaceutical composition in parts by weight, includes the following raw materials:
  • the above-mentioned pharmaceutical composition further includes pharmaceutical excipients.
  • the above-mentioned pharmaceutical composition further includes 0.5-1.5 parts by weight of pharmaceutical excipients, and the pharmaceutical excipients can be sucralose and/or sodium benzoate.
  • An antibacterial and antiviral medicinal volatile oil which preparation includes the following steps:
  • mix patchouli, atractylodes, elm, wormwood, clove, and mint and place it in a supercritical carbon dioxide extraction device, pass liquid carbon dioxide to extract the volatile oil, and perform supercritical extraction to obtain the total volatile oil;
  • the heating temperature after adding natural borneol does not exceed 60°C.
  • the preparation method of the volatile oil of each raw material is as follows:
  • the preparation method of total volatile oil is as follows:
  • Patchouli, Atractylodes, Elsholtzia, Artemisia argyi, Clove, Mint add 5-10 times the weight of water, soak for 1-8h, steam distillation to extract 2-8h; or, Patchouli, Atractylodes, Elsholtzia, Artemisia argyi, The cloves and peppermint are mixed and placed in a supercritical carbon dioxide extraction device. Liquid carbon dioxide is introduced to extract the volatile oil.
  • the extraction pressure is 20-40Mpa
  • the extraction temperature is 40-70°C
  • the separation temperature is 30-60°C
  • the extraction time is 0.5-10h.
  • the medicinal composition, medicinal volatile oil, medicinal aromatic water or the medicament prepared by the present invention has a good therapeutic effect on fever and respiratory diseases such as rhinitis, pharyngitis, pneumonia, cough and asthma, and can effectively antibacterial, antiviral, and relieve Various symptoms caused by bacterial and viral infections.
  • viruses include SARS-CoV-2.
  • the TCM classification is generally divided into four types, with three deficiency symptoms and one true symptom.
  • the three deficiency syndromes include: 1) Weakness of lung qi: the lung opens up in the nose, which naturally causes the nasal orifice to be blocked, causing some nasal symptoms; 2) Insufficiency of the spleen: the spleen is the mother of the lungs, and the deficiency of the spleen causes lung deficiency.
  • Symptoms that cause the nose including itchy nose, clear nasal discharge, continuous clean water, sneezing, and constant sneezing; 3) Insufficiency of kidney yang, deficiency of kidney qi: the kidneys are connected to the lungs, and the weakness of the kidneys can also cause nasal symptoms; The three deficiency symptoms are actually related to the lungs.
  • the actual symptom in the classification is pulmonary meridian fever, which is a fever, which is also caused by its own lung heat.
  • the pharmaceutical composition of the present invention can effectively treat the above four types of rhinitis.
  • the above-mentioned drugs can be made into a variety of dosage forms such as sprays, lotions, aerosols, drop pills, soft capsules, nose drops or atomizers.
  • the preparation method of the spray is as follows:
  • the preparation method of lotion is as follows:
  • the preparation method of the aerosol is as follows:
  • the medicinal volatile oil is diluted with a diluent, stir it evenly, filter, fill it into a pressure-resistant container, press the cap, and fill it with a propellant to obtain an aerosol;
  • the preparation method of dripping pills is as follows:
  • microencapsulation technology to make the medicinal volatile oil into volatile oil microcapsule powder, and mix it with the dripping pill matrix to make the dripping pill;
  • the preparation method of soft capsules is as follows:
  • the preparation method of nasal drops is as follows:
  • the preparation method of the atomizer is as follows:
  • the preparation method of the dropping pills is as follows:
  • the solvent is any one of medium-chain triglycerides, 1-98% alcohol, absolute ethanol, propylene glycol, glycerin or edible oil;
  • the diluent is any one of medium-chain triglycerides, 1-98% alcohol, absolute ethanol, propylene glycol, glycerin or edible oil;
  • the propellant is nitrogen, carbon dioxide, heptafluoropropane (HFC-227ea), tetrafluoroethane (HFC-134a), 1,3,3,3-tetrafluoropropene (HFO-1234ze), 2,3,3,3-tetra Fluoropropene (HFO-1234yf) or compressed air.
  • HFC-227ea heptafluoropropane
  • HFC-134a tetrafluoroethane
  • HFO-1234ze 1,3,3,3-tetrafluoropropene
  • HFO-1234yf 2,3,3,3-tetra Fluoropropene
  • the ratio of the liquid phase to the gas phase in the aerosol can be any of 2:8, 3:7, 4:6, 5:5, 6:4, and the pressure in the pressure vessel is 0.6-1 MPa.
  • the edible oil can be hemp seed oil, vine fruit oil, olive oil, coconut oil, pomegranate seed oil, wheat germ oil, walnut oil, avocado oil, linseed oil, grape seed oil, tea seed oil, Perilla seed 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, thorn oil, glaze Lettuce oil, evening primrose oil, palm oil, etc.
  • the gel matrix material can be selected from materials such as ethyl cellulose.
  • preparation methods of sprays, lotions, aerosols, drop pills, soft capsules, nasal drops or atomizers in the present invention are not limited to the above methods.
  • the pharmaceutical composition of the present invention can be used to prepare antibacterial, antiviral, heat-clearing and fever-reducing drugs and/or drugs for the treatment of respiratory diseases. It is good for fever and respiratory diseases such as rhinitis, pharyngitis, pneumonia, cough and asthma.
  • fever and respiratory diseases such as rhinitis, pharyngitis, pneumonia, cough and asthma.
  • the therapeutic effect is significant for diseases caused by bacterial and viral infections.
  • the mild type of new coronary pneumonia the clinical symptoms are mild, there is no manifestation of pneumonia in imaging, the nucleic acid test is positive
  • the normal type of new coronary pneumonia with fever and respiratory symptoms, the imaging can see the manifestations of pneumonia, manifested as viral pneumonia, nucleic acid The test is positive
  • Figure 1 shows the toxic effect of the spray in Example 4 on Vero E6 cells, with CC 50 >1/100(v/v);
  • Figure 2 shows the toxic effect of Remdesivir on Vero E6 cells, with CC 50 >200 ⁇ M
  • Figure 3 shows the inhibitory effect of the spray of Example 4 on SARS-COV-2 induced cell death (drug and virus are added at the same time), IC 50 >1/100 (v/v);
  • Figure 5 shows the inhibitory effect of the spray of Example 4 on SARS-COV-2 induced cell death (addition of drugs after infection), IC 50 >1/100 (v/v);
  • Figure 7 shows the killing effect of 1/100 (v/v) spray on SARS-CoV-2
  • Figure 8 shows the killing effect of H 2 O 2 (0.01%) on SARS-CoV-2.
  • influenza virus Asian type A mouse lung-adapted strain 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), A/Aichi/2/1968 (H3N2) were purchased from the American Classical Culture Collection (ATCC); 2009 new influenza A H1N1 strain (A/Guangzhou/GIRD07/09, H1N1, Genebank No.
  • influenza B virus (B/Guangzhou/GIRD08/09, FluB) is a clinical isolate from the Virus Laboratory of the State Key Laboratory of Respiratory Diseases, Guangzhou Medical University; influenza A H9N2 virus (A/Ahicken/Guangdong/1996, H9N2), A H6N2 influenza virus (A/Duck/Guangdong/2009, H6N2) and A H7N3 influenza virus (A/Duck/Guangdong/1994, H7N3) are gifted by the School of Veterinary Medicine, South China Agricultural University. The in vitro anti-SARS-CoV-2 activity experiment was commissioned by the Kunming Institute of Zoology, Chinese Academy of Sciences.
  • the raw materials include 885g patchouli, 1869g atractylodes, 1064g of Elsholtzia, 2174g of mugwort, 46g of clove, 532g of mint, and 10g of natural borneol.
  • the preparation method is as follows:
  • Patchouli medicinal materials were crushed through a 20-mesh sieve, soaked overnight with 6 times the weight of water, and extracted by steam distillation for 6 hours. The patchouli volatile oil was collected and the extraction rate reached 2.26%.
  • the crude powder of Atractylodes japonicus is taken, soaked in 6 times the weight of water for 0.5h, and extracted by steam distillation for 5h, and the volatile oil of Atractylodes Rhizome is collected, with an extraction rate of 1.07%.
  • the wormwood leaves were taken, coarsely crushed, soaked in 8 times the weight of water for 6 hours, and extracted by steam distillation for 5 hours, and the volatile oil of the wormwood leaves was collected, and the extraction rate reached 0.46%.
  • Clove medicinal pieces were taken, soaked in 6 times the weight of water overnight, and extracted by steam distillation for 6 hours. The volatile oil of clove was collected, and the extraction rate reached 10.88%.
  • the patchouli volatile oil, atractylodes volatile oil, elmwood volatile oil, mugwort volatile oil, clove volatile oil, and peppermint volatile oil obtained by the above extraction are mixed, natural borneol is added, and the natural borneol is properly heated (not higher than 60°C) to completely dissolve the natural borneol to obtain the medicinal volatile oil.
  • the raw materials include patchouli 663.5g, atractylodes 1402g, elmwood 798g, mugwort leaf 1630.5g, clove 32g, mint 106.5g, and natural borneol 2.5g.
  • the raw materials are pulverized into coarse powder, and after mixing, they are placed in the HA220-40-48 supercritical carbon dioxide extraction device, and the volatile oil is extracted with liquid carbon dioxide.
  • the extraction pressure is 28-30Mpa
  • the extraction temperature is 55°C
  • the separation temperature is 45°C
  • the extraction time is 3h.
  • To obtain 106.5 g of the extract (total volatile oil) add natural borneol, and appropriately heat (not higher than 60°C) to completely dissolve the natural borneol to obtain volatile oil for medicine.
  • mice doses in the efficacy study were 65 mg/kg, 130 mg/kg and 260 mg/kg, respectively.
  • the doses of guinea pigs in the efficacy study were 32 mg/kg, 64 mg/kg and 128 mg/kg, respectively.
  • the rat doses in the efficacy study were 37mg/kg, 74mg/kg and 148mg/kg
  • ICR mice are divided into males and females. According to body weight, they were randomly divided into 6 groups, each with 14 animals, namely: normal control group, model control group, pentovirine citrate 20mg/kg group, medicinal volatile oil low-dose (65mg/kg) group, In the medium-dose (130mg/kg) group of volatile oil for medicinal use and the high-dose (260mg/kg) group of volatile oil for medicinal use, mice were given 20mL/kg intragastrically (using soybean oil to make the corresponding concentration), once a day, continuous 7d. The normal control group and the model control group were given the same amount of soybean oil by gavage.
  • mice were put into a 500mL transparent airtight container, and an ultrasonic atomizer was used to evenly spray the atomized concentrated ammonia into the container, and spray continuously for 20s; the mouse contracted abdomen and opened the mouth as the cough standard, record the small The incubation period of the mouse cough and the number of coughs within 2 minutes after being put into the container.
  • the medicinal volatile oil has an obvious antitussive effect.
  • the qualified guinea pigs were randomly divided into 5 groups according to the pre-medicine asthma incubation period, each with 12 animals: model control group, aminophylline 60mg/kg group, medicinal volatile oil low-dose (32mg/kg) group, For the medium-dose (64mg/kg) group of medicinal volatile oil and the high-dose (128mg/kg) group of medicinal volatile oil, guinea pigs were given 4mL/kg by gavage (prepared with soybean oil to the corresponding concentration), once a day for 7 days . The model control group was given the same amount of soybean oil by gavage.
  • the guinea pig’s post-medicinal asthma incubation period was recorded in the same way; if the guinea pig did not develop asthma symptoms within 3 minutes, it was calculated as 180 seconds.
  • the medicinal volatile oil has an obvious anti-asthmatic effect.
  • mice half male and half male, were randomly divided into 5 groups according to their body weight, each with 14 mice, namely the model control group, the ambroxol hydrochloride 30mg/kg group, the medicinal volatile oil low-dose (65mg/kg) group, In the medium-dose (130mg/kg) group of volatile oil for medicinal use and the high-dose (260mg/kg) group of volatile oil for medicinal use, mice were given 20mL/kg intragastrically (using soybean oil to make the corresponding concentration), once a day, continuous 7d. The model control group was given the same amount of soybean oil by gavage.
  • the medicinal volatile oil has obvious phlegm-resolving effect.
  • SD rats, half male and half male were randomly divided into 5 groups according to body weight, each with 12 rats, namely the model control group, the carbocysteine 200mg/kg group, the medicinal volatile oil low-dose (37mg/kg) group, In the middle-dose (74mg/kg) group of volatile oil for medicinal use and the high-dose (148mg/kg) group of volatile oil for medicinal use, rats were given 10mL/kg intragastrically (using soybean oil to make the corresponding concentration), once a day, continuous 7d.
  • the model control group was given the same amount of soybean oil by gavage.
  • the medicinal volatile oil has obvious phlegm-resolving effect.
  • LPS lipopolysaccharide
  • SD rats are divided into males and females.
  • the adaptive rectal temperature of SD rats is measured 2 days in advance, twice a day, and the average value is taken as the basal body temperature.
  • the rats with a single body temperature exceeding 38°C or two temperature differences exceeding 0.5°C were removed, and the qualified rats were divided into 6 groups randomly according to their body temperature, each with 12 rats, which were the normal control group, the model control group, and aspirin.
  • rats were given 10mL/kg by gavage Medicine (prepared with soybean oil to the corresponding concentration), once a day for 7 days.
  • gavage Medicine prepared with soybean oil to the corresponding concentration
  • the rats could not help but fasting.
  • the basal body temperature of the rats was measured on the day of the experiment.
  • rats in the normal control group were intraperitoneally injected with sterile normal saline, and the other groups were intraperitoneally injected with LPS (20 ⁇ g/kg).
  • LPS 20 ⁇ g/kg
  • the body temperature of rats in the normal control group did not change significantly within 7 hours.
  • the body temperature of the rats in the model control group gradually increased after injection of LPS, which was significantly different from the model control group (P ⁇ 0.01), and reached a peak at 6 hours.
  • the body temperature of each dose group of medicinal volatile oil was significantly lower in 2-7h (P ⁇ 0.05 or P ⁇ 0.01). The results are shown in Table 5.
  • Wistar rats male and female, hold the rats in their hands, use tweezers to break and fix the rat’s jaw, extend the spray nozzle of the spray device into the rat’s oral cavity to cause the pharynx, spray the rat’s pharynx with 25% ammonia water once (approximately 0.2mL), sprayed continuously for 3 days, twice a day, to cause acute inflammation of rat pharynx mucosa due to irritation.
  • the rats were randomly divided into model control group, dexamethasone 5mg/kg group, medicinal volatile oil low-dose (37mg/kg) group, medicinal volatile oil middle-dose (74mg/kg) group and medicinal Volatile oil high-dose (148mg/kg) group, 12 rats in each group, and 12 rats of the same batch as the normal control group.
  • the rats were given 10mL/kg intragastrically (using soybean oil to make the corresponding concentration). Once a day for 3 days.
  • the normal control group and the model control group were given the same amount of soybean oil.
  • the rats were anesthetized, blood was taken from the abdominal aorta, and the animals were sacrificed.
  • the serum was separated and the levels of IL-6 and TNF- ⁇ in the serum were determined by ELISA.
  • the volatile oil for medicine can significantly inhibit the inflammatory response of acute pharyngitis model rats.
  • ICR mice Influenza virus Asian type A mouse lung-adapted strain A/FM/1/34(H1N1)(FM1), inoculated into the allantoic cavity of 9-day-old chicken embryos before use, cultured routinely, fresh and aseptically collected allantoic fluid after 48h, and passaged Two times, the median lethal dose (LD 50 ) for ICR mice was determined.
  • mice After the mice were lightly anesthetized with ether, each mouse was intranasally to 25 ⁇ L virus, infection is an amount 25 times LD 50, the control groups were given sterile saline intranasally.
  • mice in each dose group of medicinal volatile oil were administered intragastrically at 20mL/kg (prepared with soybean oil to the corresponding concentration), ribavirin group was administered intraperitoneally once a day for 7 consecutive days, normal control group and model control The group gavage the same amount of soybean oil.
  • mice Observe the onset symptoms of mice daily, record the time of death, number of deaths and body weight, and observe continuously for 14 days. Those who do not die on the 14th day are counted as 14 days, and those who die within 24 hours of infection are regarded as non-infected deaths.
  • mice in the normal control group have good spirits, agility, smooth and shiny fur, normal breathing rate, normal eating and drinking, and daily weight gain. Influenza virus infects mice. After 24 hours of infection, they have head shaking, rapid breathing rate, abdominal respiration, slow movement, curling, and bulging hair, and the amount of food and water consumption are significantly reduced. From the 4th day, the mice began to die, and reached the peak on the 7th day. The state of the mice in each treatment group was significantly better than that of the model group.
  • the survival rates of the mice in the low, medium, and high dose groups of medicinal volatile oil were 25.0%, 37.5%, and 56.2, respectively. %, the survival rate was significantly increased (P ⁇ 0.05 or P ⁇ 0.01). The results are shown in Table 7.
  • the medicinal volatile oil can significantly improve the survival rate of mice infected with influenza virus FM1, and has a significant antiviral effect.
  • ICR mice Influenza virus Asian type A mouse lung-adapted strain A/FM/1/34(H1N1)(FM1), inoculated into the allantoic cavity of 9-day-old chicken embryos before use, cultured routinely, fresh and aseptically collected allantoic fluid after 48h, and passaged Two times, the median lethal dose (LD 50 ) for ICR mice was determined. ICR mice, half male and half male, were randomly divided into 6 groups according to their body weight, each with 14 mice, namely the normal control group, the model control group, the Tamiflu 27.5 mg/kg group, and the low-dose medicinal volatile oil (65 mg/kg). ) Group, medicinal volatile oil medium dose (130mg/kg) group and medicinal volatile oil high dose (260mg/kg) group.
  • mice After the mice were lightly anesthetized with ether, each mouse was intranasally in 20 ⁇ l virus, infection is an amount 25 times LD 50, the control groups were given sterile saline intranasally.
  • Mice in each dose group of medicinal volatile oil were administered intragastrically at 20mL/kg (using soybean oil to make the corresponding concentration), once a day, for 7 days, the normal control group and the model control group were intragastrically administered the same amount of soybean oil.
  • mice were weighed and sacrificed.
  • the whole lungs were dissected and weighed, and the lung index value and lung index inhibition rate were calculated.
  • the lung tissue was prepared into 10% lung tissue homogenate with normal saline, and the prepared homogenate was centrifuged at low temperature, and the content of IL-8 and TNF- ⁇ in the lung tissue was determined by ELISA.
  • Lung index [lung weight (g)/body weight (g)] ⁇ 100
  • Lung index inhibition rate (average lung index of model control group-average lung index of test group)/average lung index of model control group.
  • the medicinal volatile oil can significantly improve the viral pneumonia caused by H1N1 influenza.
  • the selected cell line was dog kidney cell (MDCK), and the 7 virus strains were A/PR/8/34 (H1N1), A/Aichi/2/1968 (H3N2), 2009 new influenza A H1N1 strain (A /Guangzhou/GIRD07/09, H1N1), influenza B virus (B/Guangzhou/GIRD08/09, FluB), influenza A H9N2 virus (A/Ahicken/Guangdong/1996, H9N2), influenza A H6N2 virus (A /Duck/Guangdong/2009, H6N2) and influenza A H7N3 (A/Duck/Guangdong/1994, H7N3).
  • influenza viruses were amplified from 9 to 11-day-old chicken embryos, allantoic fluid was collected, and the corresponding hemagglutination titer was determined, and stored at -80°C.
  • the cytopathic inhibition method was used to study the inhibitory effect of medicinal volatile oil on H1N1, H3N2, 2009 new influenza virus strains such as H1N1, FluB, H9N2, H6N2 and H7N3.
  • MDCK cells were routinely cultured.
  • the degree of cell lesions is recorded according to the following 6-level standards: "-" means that the cells grow normally and no lesions appear; “ ⁇ ” means that the cytopathic changes are less than 10% of the entire monolayer; “+” means that the cytopathic lesions are less than the entire monolayer. 25% of the cells; “++” means that the cytopathic effect is less than 50% of the entire monolayer of cells; “+++” means that the cytopathic effect is less than 75% of the entire monolayer of cells; “++++” means that the cytopathic effect is less More than 75% of the entire monolayer of cells.
  • SI ⁇ 1 means invalid
  • SI: 1 ⁇ 2 means high toxicity and low efficiency
  • SI> 2 means low toxicity and high efficiency.
  • the non-toxic concentration of medicinal volatile oil on MDCK cells is 50 mg/mL, and its half toxic concentration TC 50 is > 50 mg/mL.
  • the IC 50 of medicinal volatile oil against H1N1, H3N2, 2009 new Aflow H1N1, FluB, H9N2, H6N2 and H7N3 were 18.4mg/mL, 36.2mg/mL, 24.2mg/mL, 28.5mg/mL, 48.3mg/ mL, 45.5mg/mL and 30.2mg/mL. The results are shown in Table 10.
  • the volatile oil for medicine has obvious inhibitory effect on H1N1, H3N2, 2009 new influenza H1N1, FluB, H9N2, H6N2 and H7N3 seven influenza viruses, of which H1N1, 2009 The H1N1 and FluB strains of New Aflow have stronger effects.
  • ICR mice male and female, are divided into 6 groups randomly according to body weight, each with 20 mice, which are normal control group, model control group, Shuanghuanglian 20g/kg group, and low-dose medicinal volatile oil (65mg/kg) Group, medicinal volatile oil medium dose (130mg/kg) group and medicinal volatile oil high dose (260mg/kg) group, mice were given 20mL/kg intragastrically (using soybean oil to make the corresponding concentration), once a day , 7d consecutively.
  • the model control group was given the same amount of soybean oil by gavage.
  • mice in each group were intraperitoneally injected with 0.5 mL of pneumococcal bacteria solution, and the administration was continued for 3 days. The death of mice in each group was observed within 7 days.
  • mice infected with pneumococcus increased significantly within 7 days, and the survival rate was 20% (P ⁇ 0.01); compared with the model control group, the mice in the middle and high dose groups of medicinal volatile oil died within 7 days The number was significantly reduced (P ⁇ 0.05 or P ⁇ 0.01). The results are shown in Table 11.
  • the volatile oil for medicine can significantly improve the survival rate of pneumococcal infected mice, and has obvious antibacterial effect.
  • OVA ovalbumin
  • mice all males, were stratified and randomly divided into 6 groups according to their body weight, each with 15 rats, namely the normal control group, the model control group, the Biyankang 0.4g/kg group, and the low dose of medicinal volatile oil (37mg/kg)
  • rats in each group were treated with ovalbumin (OAV) 0.3 mg, Al(OH) 3 30mg and 1mL of normal saline were mixed and injected intraperitoneally for the first immunization.
  • OAV ovalbumin
  • Al(OH) 3 30mg and 1mL of normal saline were mixed and injected intraperitoneally for the first immunization.
  • OVA 200 ⁇ g 50 ⁇ L
  • Rats in each group were given the drug on the 15th day of sensitization, once a day for 14 consecutive days.
  • Rats were given 10 mL/kg intragastrically (using soybean oil to make the corresponding concentration), and the normal control group and the model control group were given the same amount of soybean oil intragastrically.
  • mice in each group were anesthetized by intraperitoneal injection of 10% chloral hydrate 0.35g/kg, blood was collected from the abdominal aorta, centrifuged at 4°C3000r/min for 15min, serum was separated, stored at -70°C, according to the kit instructions Determination of histamine (HIS), interleukin-4 (IL-4) and tumor necrosis factor (TNF- ⁇ ).
  • HIS histamine
  • IL-4 interleukin-4
  • TNF- ⁇ tumor necrosis factor
  • the volatile oil for medicine can significantly improve allergic rhinitis caused by OVA in rats and reduce the inflammatory response.
  • the raw material ratio of the medicinal composition is the same as in Example 1.
  • patchouli volatile oil, atractylodes volatile oil, elmwood volatile oil, mugwort volatile oil, clove volatile oil, and peppermint volatile oil are prepared according to the method of Example 1, and mixed volatile oil is obtained after mixing them.
  • Preparation of reference substance solution Take appropriate amounts of eugenol reference substance and stoichiometric alcohol reference substance, accurately weigh them, and add n-hexane to make a solution containing 0.3 mg of eugenol per 1 mL and 0.3 mg per 1 mL of eugenol reference substance. The solution is ready.
  • test solution accurately measure 1mL of this product, place it in a 10mL measuring flask, add n-hexane to the mark, shake well, and get it.
  • Chromatographic conditions and system suitability test a capillary column with 100% dimethyl polysiloxane as the stationary phase (column length is 30m, inner diameter is 0.32mm, thickness is 0.25 ⁇ m); column temperature is programmed temperature, initial temperature 70°C, hold for 5 minutes, 5°C/min to 110°C, 10°C/min to 280°C, hold for 4 minutes, 10°C/min to 300°C, hold for 5 minutes.
  • the number of theoretical boards should not be less than 40,000 according to the calculation of borneol peak.
  • the chromatograms of the test substance show the retention of the chromatographic peaks of the baiqiol reference substance, caravanol reference substance, eucalyptol reference substance, borneol reference substance, isoborneol reference substance, eugenol reference substance and menthol reference substance
  • the chromatographic peak corresponding to the time.
  • This product contains cloves per 1mL based on eugenol (C 10 H 12 O 2 ), not less than 3.75mg; containing patchouli based on fenol (C 15 H 26 O), not less than 5.2mg.
  • the spray is fragrant to dispel dirt, promotes lungs and regulates qi, dispels dampness and phlegm, and clears throat and throat. It can be used for nasal congestion, runny nose, cough, swelling and sore throat caused by new coronary pneumonia in mild, common, recovery period and cold or flu.
  • Vero E6 cells were purchased from Shenggong Bioengineering (Shanghai ) Co., Ltd.) was cultured, and the cells were subcultured once a day before the experiment, so that the cells used were in the logarithmic growth phase.
  • test samples use containing 3 %FBS DMEM medium dilution spray, used now
  • Redcivir stock solution dissolved in DMSO, final concentration of 100mM/mL, stored at 4°C, working solution diluted with 3% FBS-containing DMEM medium , Used now
  • 100 ⁇ L/well set up 3 repetitive wells; set up a control well without drugs at the same time; culture at 37°C and 5% CO 2 for 3 days.
  • the CCK8 kit (article number: C0039, batch number: 121819200616, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) was used to detect the cell survival rate.
  • the Synergy 2 multi-function microplate detector measures the OD value with a wavelength of 450nm and a reference wavelength of 630nm.
  • Origin7.5 was used to draw a dose-response fitting curve, and the 50% inhibitory cell growth concentration (CC 50 ) of the sample was calculated according to the Reed & Muench method.
  • Cell growth survival rate (%) OD value of experimental wells/OD value of control wells ⁇ 100.
  • the results are shown in Tables 13, 14 and Figures 1 and 2.
  • the spray has a CC 50 >1/100 (v/v) for Vero E6 cells, and remdesivir has a CC 50 >200 ⁇ M for Vero E6 cells.
  • the CCK8 kit was used to detect the cell viability, and the Bio-Tek microplate detector was used to determine the OD value.
  • the measurement wavelength was 450nm and the reference wavelength was 630nm.
  • Origin7.5 is used to draw a dose-response fitting curve, and the 50% effective concentration (IC 50 ) of the sample to inhibit the virus and the therapeutic index TI value (Therapeutic index) of anti-SARS-CoV-2 activity (Therapeutic index) is calculated according to the Reed&Muench method.
  • TI CC 50 /IC 50
  • Inhibition rate of cell death caused by SARS-CoV-2 (%) (OD value of experimental well-OD value of positive control well)/(OD value of negative control well-OD value of positive control well) ⁇ 100.
  • the CCK8 kit was used to detect the cell viability, and the Bio-Tek microplate detector was used to determine the OD value.
  • the measurement wavelength was 450nm and the reference wavelength was 630nm.
  • Origin7.5 is used to draw a dose-response fitting curve, and the 50% effective concentration (IC 50 ) of the sample to inhibit the virus and the therapeutic index TI value (Therapeutic index) of anti-SARS-CoV-2 activity (Therapeutic index) is calculated according to the Reed&Muench method.
  • TI CC 50 /IC 50
  • Inhibition rate of cell death caused by SARS-CoV-2 (%) (OD value of experimental well-OD value of positive control well)/(OD value of negative control well-OD value of positive control well) ⁇ 100.
  • a 96-well plate take 100 ⁇ L of the sample to be tested diluted to 1/100 (v/v) or 0.01% H 2 O 2 and mix it with 100 ⁇ L of SARS-CoV-2 diluent (MOI 0.1), and set the drug-free Positive control and negative control without virus; incubate at 37°C and 5% CO 2 for 0, 5 min, 10 min, 30 min and 60 min.
  • MOI 0.1 SARS-CoV-2 diluent
  • the CCK8 kit was used to detect the cell viability, and the Bio-Tek microplate detector was used to determine the OD value.
  • the measurement wavelength was 450nm and the reference wavelength was 630nm.
  • the spray of the present invention is incubated with cells and viruses at the same time, and has a certain inhibitory effect on the death of Vero E6 cells caused by SARS-CoV-2, and the inhibition rate can reach 26.67% at a concentration of 1/100 (v/v); After cells and virus were incubated for 1 hour, spray was added, and the inhibitory effect on SARS-CoV-2 induced Vero E6 cell death was equivalent to that of the simultaneous incubation group. At a concentration of 1/100 (v/v), the inhibition rate reached 23.94%; the concentration was 1 /100(v/v) spray has a certain killing effect on SARS-CoV-2, the inhibition rate of 60 minutes is 19.53%.
  • a medicinal composition raw materials including patchouli 445g, atractylodes 934g, elm 532g, mugwort 1087g, clove 2g, mint 745g, natural borneol 15g.
  • a medicinal composition with raw materials including 885g patchouli, 1869g atractylodes, 1064g of Elsholtzia, 2174g of mugwort, 46g of clove, and 532g of mint.
  • mice doses in the efficacy study were 12mL/kg, 24mL/kg and 40mL/kg, respectively.
  • ICR mice Influenza virus Asian type A mouse lung-adapted strain A/FM/1/34(H1N1)(FM1), inoculated into the allantoic cavity of 9-day-old chicken embryos before use, cultured routinely, fresh and aseptically collected allantoic fluid after 48h, and passaged Two times, the median lethal dose (LD50) for ICR mice was determined. ICR mice, half male and half male, were divided into 6 groups randomly according to their body weight, each with 20 mice, namely the normal control group, the model control group, the ribavirin 100mg/kg group, and the low-dose aromatic water (12mL/kg). ) Group, aromatic water dose (24mL/kg) group and aromatic water high dose (40mL/kg) group.
  • LD50 median lethal dose
  • each mouse was instilled with 25 ⁇ L of virus liquid, and the infection amount was 25 times the LD50.
  • the normal control group was instilled with the same amount of sterile normal saline.
  • Mice in each dose group of aromatic water were administered twice a day with an interval of 6 hours between each administration.
  • the dose of each administration was half of the total dose, which were 6 mL/kg, 12 mL/kg and 20 mL/kg, respectively.
  • Ribavirin The group was given intraperitoneal injection once a day for 7 consecutive days.
  • the normal control group and the model control group were given the same amount of distilled water.
  • mice Observe the onset symptoms of mice daily, record the time of death, number of deaths and body weight, and observe continuously for 14 days. Those who do not die on the 14th day are counted as 14 days, and those who die within 24 hours of infection are regarded as non-infected deaths.
  • ICR mice Influenza virus Asian type A mouse lung-adapted strain A/FM/1/34(H1N1)(FM1), inoculated into the allantoic cavity of 9-day-old chicken embryos before use, cultured routinely, fresh and aseptically collected allantoic fluid after 48h, and passaged Two times, the median lethal dose (LD50) for ICR mice was determined.
  • mice After the mice were lightly anesthetized with ether, each mouse was instilled with 20 ⁇ l of virus liquid, and the infection amount was 25 times the LD50.
  • the normal control group was instilled with the same amount of sterile normal saline. Mice in each dose group of aromatic water were administered twice a day, with an interval of 6 hours between each administration, and the dose of each administration was half of the total dose, respectively 6mL/kg, 12mL/kg and 20mL/kg, for 7 consecutive days, normal
  • the control group and the model control group were given the same amount of distilled water.
  • mice were weighed and sacrificed.
  • the whole lungs were dissected and weighed, and the lung index value and lung index inhibition rate were calculated.
  • the lung tissue was prepared into 10% lung tissue homogenate with physiological saline, and the prepared homogenate was centrifuged at low temperature and speed, and the content of IL-8 and TNF- ⁇ in the lung tissue was determined by ELISA.
  • Lung index [lung weight (g)/body weight (g)] ⁇ 100
  • Lung index inhibition rate (average lung index of model control group-average lung index of test group)/average lung index of model control group.
  • the levels of IL-8 and TNF- ⁇ in the lung tissue homogenate of the model control group were significantly increased (P ⁇ 0.01); compared with the model control group, the lung tissues of each dose group of aromatic water The content of TNF- ⁇ in the homogenate was significantly reduced (P ⁇ 0.05 or P ⁇ 0.01), and the content of IL-8 in the lung tissue homogenate of mice in the aromatic water dose group and the high-dose group was significantly reduced (P ⁇ 0.05 or P ⁇ 0.01), the results are shown in Table 23.
  • aromatic water can significantly improve viral pneumonia caused by H1N1 influenza.
  • ICR mice male and female, are divided into 6 groups randomly according to their body weight, each with 20 mice, namely normal control group, model control group, Shuanghuanglian 20g/kg group, and aromatic water low-dose (12mL/kg) group , Aromatic water dose (24mL/kg) group and aromatic water high dose (40mL/kg) group.
  • Mice in each dose group of aromatic water were administered twice a day, with an interval of 6 hours between each administration, and the dose of each administration was half of the total dose, respectively 6mL/kg, 12mL/kg and 20mL/kg, for 7 consecutive days, normal
  • the control group and the model control group were given the same amount of distilled water.
  • mice in each group were intraperitoneally injected with 0.5 mL of pneumococcal bacteria solution, and the administration was continued for 3 days. The death of mice in each group was observed within 7 days.
  • mice infected with pneumococcus increased significantly within 7 days, and the survival rate was 20% (P ⁇ 0.01); compared with the model control group, the number of deaths within 7 days of the aromatic water dose and high dose groups was obvious Decrease (P ⁇ 0.05 or P ⁇ 0.01).
  • the results are shown in Table 24.
  • a medicinal composition with raw materials including 885g patchouli, 1869g atractylodes, 1064g of Elsholtzia, 2174g of mugwort, 46g of cloves, 532g of peppermint, 35g of sucralose, and 35g of sodium benzoate.
  • Example 1 Prepare medicinal volatile oil according to the method in Example 1. Use absolute ethanol to dilute the medicinal volatile oil to adjust the total amount to 1000mL, stir well, filter, fill according to the specifications of 20mL/bottle such as a pressure-resistant aluminum can, press the cap, and fill A certain amount of nitrogen is used to make the pressure in the aluminum can 0.8Mpa to obtain an aerosol.
  • absolute ethanol to dilute the medicinal volatile oil to adjust the total amount to 1000mL, stir well, filter, fill according to the specifications of 20mL/bottle such as a pressure-resistant aluminum can, press the cap, and fill A certain amount of nitrogen is used to make the pressure in the aluminum can 0.8Mpa to obtain an aerosol.
  • the raw materials include patchouli 960g, atractylodes 1920g, Elsholtzia 1200g, mugwort 2160g, clove 240g, mint 720g, and natural borneol 10g.
  • the above-mentioned medicinal composition was used to prepare medicinal volatile oil, and the preparation method was the same as that in Example 1.
  • the preparation method of medicinal volatile oil dripping pills is as follows:
  • Each 10 pill weighs 0.32g, and each pill contains about 7mg of volatile oil for medicine. It is used for the treatment of nasal congestion, runny nose, cough and swelling and sore throat caused by the mild, common, recovery period and cold or flu of new coronary pneumonia, 14 pills each time, once in the morning, once in the evening and once in the evening.
  • the medicinal volatile oil dripping pill of this example was tested for efficacy.
  • the dose for mice was 1.10 g/kg
  • the dose for rats was 0.56 g/kg
  • the dose for guinea pigs was 0.48 g/kg.
  • control group 1 prepared dripping pills according to the method of the experimental group, and replaced the mugwort leaves with ephedra, and mint with dahurian, without adding cloves and natural borneol.
  • control group 2 prepared dripping pills according to the method of the experimental group, and replaced patchouli with Isatis indigotica, and replaced Atractylodes japonica with Fangfeng.
  • control group 3 prepared dripping pills according to the method of the experimental group, and replaced the Elsholtzia jasminoides with gardenia, and the wormwood with dried tangerine peel.
  • ICR mice are divided into males and females. According to body weight, they were randomly divided into 7 groups, each with 14 animals: normal control group, model control group, pentovirine citrate 20mg/kg group, medicinal volatile oil dripping pill group, control group 1, control group In group 2 and control group 3, mice were given 20mL/kg intragastrically. Each administration group grinds the dripping pills into powder and suspends them to the corresponding concentration with 0.5% CMC-Na, once a day for 7 days. The normal control group and the model control group were intragastrically given the same amount of 0.5% CMC-Na solution.
  • mice were put into a 500mL transparent airtight container, and an ultrasonic atomizer was used to evenly spray the atomized concentrated ammonia into the container, and spray continuously for 20s; the mouse contracted abdomen and opened the mouth as the cough standard, record the small The incubation period of the mouse cough and the number of coughs within 2 minutes after being put into the container.
  • mice in the medicinal volatile oil dripping pill group Compared with the model control group, the mice in the medicinal volatile oil dripping pill group, the control group 1, the control group 2 and the control group 3 all significantly prolonged the incubation period of coughing, and significantly reduced the number of coughs in the mice (P ⁇ 0.01), and Comparing the medicinal volatile oil dripping pill group, the mice in the control group 1, the control group 2 and the control group 3 all significantly reduced the incubation period of cough and increased the number of coughs (P ⁇ 0.05). The results are shown in Table 25.
  • the medicinal volatile oil dripping pill group, the control group 1, the control group 2 and the control group 3 have obvious cough relieving effects, but the medicinal volatile oil dripping pill group has obvious cough relieving effects.
  • the control group 1 the control group 2 and the control group 3 groups.
  • the qualified guinea pigs were stratified and randomly divided into 6 groups according to the pre-medicine asthma incubation period, each with 12 animals: model control group, aminophylline 60mg/kg group, medicinal volatile oil dripping pill group, control group 1, control group In group 2 and control group 3, guinea pigs were given 4mL/kg intragastrically. Each administration group grinds the dripping pills into powder and suspends them to the corresponding concentration with 0.5% CMC-Na, once a day for 7 days. The model control group was given the same amount of 0.5% CMC-Na solution.
  • the guinea pig’s post-medicinal asthma incubation period was recorded in the same way; if the guinea pig did not develop asthma symptoms within 3 minutes, it was calculated as 180 seconds.
  • control group 1 Compared with the model control group, the post-medicine asthma incubation period and the prolonged value of post-medicine asthma incubation period (post-medicine incubation period-pre-medicine incubation period) in the medicinal volatile oil dripping pill group, control group 1, control group 2, and control group 3 were significantly increased (P ⁇ 0.01). Compared with the medicinal volatile oil dripping pill group, the post-medicine asthma latency and the prolonged value of post-medicinal asthma latency (post-medicine latency-pre-medicine latency) of control 1, control 2 and control 3 are all significant Decrease (P ⁇ 0.05). The results are shown in Table 26.
  • the medicinal volatile oil dripping pill group, the control group 1, the control group 2 and the control group 3 have obvious anti-asthmatic effects, but the medicinal volatile oil dripping pill group has an anti-asthmatic effect It is significantly better than the control group 1 group, the control group 2 group and the control group 3 group.
  • SD rats male and female, were divided into 6 groups randomly according to their body weight, each with 12 rats, namely the model control group, the carbocysteine 0.2g/kg group, the medicinal volatile oil dripping pill group, the control group 1.
  • rats were given 10 mL/kg intragastrically.
  • Each administration group grinds the dripping pills into powder and suspends them to the corresponding concentration with 0.5% CMC-Na, once a day for 7 days.
  • the model control group was given the same amount of 0.5% CMC-Na solution.
  • the medicinal volatile oil dripping pill group, the control group 1, the control group 2 and the control group 3 have obvious phlegm resolving effects, but the medicinal volatile oil dripping pill group has the phlegm-resolving effect It is significantly better than the control group 1 group, the control group 2 group and the control group 3 group.
  • mice half male and half male, were randomly divided into 7 groups according to their body weight, each with 20 mice, namely the normal control group, the model control group, the Shuanghuanglian 20g/kg group, the medicinal volatile oil dripping pill group, and the control group 1.
  • mice were given 20mL/kg intragastrically, each administration group grinds the dripping pills into powder, and uses 0.5% CMC-Na to suspend to the corresponding concentration, once a day for 7 days.
  • the model control group was given the same amount of 0.5% CMC-Na solution.
  • mice in each group were intraperitoneally injected with 0.5 mL of pneumococcal bacteria solution, and the administration was continued for 3 days. The death of mice in each group was observed within 7 days.
  • mice infected with pneumococcus increased significantly within 7 days, and the survival rate was 20% (P ⁇ 0.01); compared with the model control group, the medicinal volatile oil dripping pill group, the control group 1, the control group 2 The number of deaths of mice in the 3 groups of the control group and the control group was significantly reduced within 7 days (P ⁇ 0.05). Compared with the medicinal volatile oil dripping pill group, the number of deaths of mice in control group 1, control group 2 and control group 3 increased in 7 days, but there was no statistical difference (P>0.05). The results are shown in Table 28.
  • the medicinal volatile oil dripping pill group, control group 1, control group 2 and control group 3 can significantly improve the survival rate of pneumococcal infection mice, and have obvious antibacterial effect .
  • the antibacterial effect of the medicinal volatile oil dripping pill group was slightly better than that of the control group 1, the control group 2 and the control group 3.
  • ICR mice Influenza virus Asian type A mouse lung-adapted strain A/FM/1/34(H1N1)(FM1), inoculated into the allantoic cavity of 9-day-old chicken embryos before use, cultured routinely, fresh and aseptically collected allantoic fluid after 48h, and passaged Two times, the median lethal dose (LD 50 ) for ICR mice was determined. ICR mice, half male and half male, were randomly divided into 6 groups according to body weight, each with 24 mice, which were normal control group, model control group, ribavirin 100mg/kg group, and medicinal volatile oil dripping pills 1.10g/ The kg group, the commercially available drip pill 1.73g/kg group and the commercially available spray 20mL/kg group (maximum dose).
  • LD 50 median lethal dose
  • mice After the mice were lightly anesthetized with ether, each mouse was intranasally to 25 ⁇ L virus, infection is an amount 25 times LD 50, the control groups were given sterile saline intranasally.
  • the mice in the medicinal volatile oil dripping pill, the commercially available dripping pill, and the commercially available spray group were administered intragastrically at a rate of 20 mL/kg.
  • the bavirin group was given intraperitoneal injection once a day for 7 consecutive days.
  • the normal control group and the model control group were given an equal amount of 0.5% CMC-Na solution.
  • the medicinal ingredients of the commercially available dripping pills are: cholic acid, mother-of-pearl, hyodeoxycholic acid, gardenia, buffalo horn, isatis root, baicalin, honeysuckle; the medicinal ingredients of the commercially available spray are Aina sesame oil and ginger Oil, menthol.
  • mice Observe the onset symptoms of mice daily, record the time of death, number of deaths and body weight, and observe continuously for 14 days. Those who do not die on the 14th day are counted as 14 days, and those who die within 24 hours of infection are regarded as non-infected deaths.
  • mice in the normal control group have good spirits, agility, smooth and shiny fur, normal breathing rate, normal eating and drinking, and daily weight gain. Influenza virus infects mice. After 24 hours of infection, they have head shaking, rapid breathing rate, abdominal respiration, slow movement, curling, and bulging hair, and the amount of food and water consumption are significantly reduced. From the 4th day, the mice began to die, and reached the peak on the 7th day. The state of the mice in each treatment group was significantly better than that of the model group.
  • the survival rates of the mice in the medicinal volatile oil dripping pills, the commercially available dripping pills, and the commercially available spray group were 62.5%, respectively. 41.7% and 37.5%, the survival rate was significantly increased (P ⁇ 0.05 or P ⁇ 0.01); compared with the medicinal volatile oil dripping pill group, the survival rate of mice in the commercially available dripping pill group and the commercially available spray group was significantly reduced (P ⁇ 0.05 or P ⁇ 0.01).
  • the results are shown in Table 29.
  • the medicinal volatile oil dripping pills, commercially available dripping pills and commercial sprays can significantly improve the survival rate of influenza virus FM1 infected mice, and the antiviral effect of the medicinal volatile oil dripping pills is significantly better In the commercially available drop pills and commercially available sprays.
  • ICR mice Influenza virus Asian type A mouse lung-adapted strain A/FM/1/34(H1N1)(FM1), inoculated into the allantoic cavity of 9-day-old chicken embryos before use, cultured routinely, fresh and aseptically collected allantoic fluid after 48h, and passaged Two times, the median lethal dose (LD 50 ) for ICR mice was determined. ICR mice, half male and half male, were divided into 6 groups randomly according to their body weight, each with 14 mice, namely the normal control group, the model control group, the Tamiflu 27.5 mg/kg group, and the medicinal volatile oil dripping pill 1.10g/kg Group, marketed dripping pills 1.73g/kg group and marketed spray 20mL/kg group (maximum dose).
  • LD 50 median lethal dose
  • mice After the mice were lightly anesthetized with ether, each mouse was intranasally in 20 ⁇ l virus, infection is an amount 25 times LD 50, the control groups were given sterile saline intranasally. Medicinal volatile oil dripping pills, commercially available dripping pills, and commercially available spray group mice were given 20mL/kg intragastrically, ribavirin group was given intraperitoneal injection once a day for 7 consecutive days, normal control group and model control group Gavage the same amount of 0.5% CMC-Na solution.
  • mice were weighed and sacrificed.
  • the whole lungs were dissected and weighed, and the lung index value and lung index inhibition rate were calculated.
  • the lung tissue was prepared into 10% lung tissue homogenate with normal saline, and the prepared homogenate was centrifuged at low temperature, and the content of IL-8 and TNF- ⁇ in the lung tissue was determined by ELISA.
  • Lung index [lung weight (g)/body weight (g)] ⁇ 100
  • Lung index inhibition rate (average lung index of model control group-average lung index of test group)/average lung index of model control group.
  • the lung index of mice in the model control group was significantly increased (P ⁇ 0.01); compared with the model control group, the lungs of the mice in the medicinal volatile oil dripping pill group, the commercially available dripping pill group, and the commercially available spray group
  • the indexes were significantly decreased (P ⁇ 0.05 or P ⁇ 0.01); compared with the medicinal volatile oil dripping pill group, the lung index of the mice in the commercially available dripping pill group and the commercially available spray group increased significantly (P ⁇ 0.05).
  • the results are shown in Table 30.
  • the medicinal volatile oil dripping pills, commercially available dripping pills and commercial sprays can significantly improve the viral pneumonia caused by H1N1 influenza, and the antiviral effect of the medicinal volatile oil dripping pills is significantly better than that Commercial drop pills and commercial sprays.
  • the preparation method of medicinal volatile oil soft capsule is as follows:
  • the volatile oil of patchouli, the volatile oil of atractylodes, the volatile oil of Elsholtzia, the volatile oil of mugwort, the volatile oil of clove and the volatile oil of peppermint were respectively prepared.
  • gelatin:glycerin:purified water at a mass ratio of 1:1:0.5, place it in a gelatinization bucket, melt and mix at 70°C, and degas in a vacuum for use.
  • the preparation method of medicinal volatile oil nose drops is as follows:
  • the raw material ratio of the medicinal composition is the same as in Example 1.
  • patchouli volatile oil, atractylodes volatile oil, elmwood volatile oil, mugwort volatile oil, clove volatile oil, and peppermint volatile oil are prepared according to the method of Example 1, and mixed volatile oil is obtained after mixing them.
  • Take 7g of the above-mentioned mixed volatile oil add 1g of natural borneol, stir and appropriately heat (not higher than 60°C) to completely dissolve the natural borneol, and obtain medicinal volatile oil as solution A.
  • the preparation method of medicinal volatile oil atomizer is as follows:
  • the raw material ratio of the medicinal composition is the same as in Example 1.
  • patchouli volatile oil, atractylodes volatile oil, elmwood volatile oil, mugwort volatile oil, clove volatile oil, and peppermint volatile oil are prepared according to the method of Example 1, and mixed volatile oil is obtained after mixing them.

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Abstract

一种抗菌抗病毒药用组合物，以重量份数计，包括如下原料：广藿香4-14份、苍术9-28份、香薷5-18份、艾叶11-33份、丁香0.1-3份和薄荷2-10份，还可以包括天然冰片和药用辅料。该药用组合物可制成药用挥发油。该药用组合物及其挥发油能抗菌、抗病毒、清热退烧和/或治疗呼吸系统疾病，可在制备抗SARS-CoV-2的药物中应用。

Description

一种抗菌抗病毒药用组合物及其应用 技术领域
本发明属于医药技术领域，更具体的说是涉及一种抗菌抗病毒药用组合物及其应用。
背景技术
呼吸系统疾病是临床常见病、多发病，常见于微生物感染，包括细菌、病毒、真菌等；具有发病率高、危害性大、容易复发、耐药性日益严重的特点。
其中，细菌感染是引起呼吸道疾病的常见原因，引起呼吸道细菌性感染的致病菌有很多，其中铜绿假单胞菌、金黄色葡萄球菌、肺炎克雷伯菌等较为常见。临床上慢性肺部疾病有慢性阻塞性肺疾病、支气管哮喘、肺结核等，一旦合并这些细菌感染将导致病情的快速恶化甚至死亡。虽然目前以抗生素为代表的化学药具有较显著疗效，但其副作用大、易致二重感染且耐药性日趋严重。
呼吸道病毒是一大类能侵犯呼吸道引起呼吸道局部病变或仅以呼吸道为侵入门户、主要引起呼吸道组织器官病变的病毒，是引起急性呼吸道传染的主要病原体。据统计，90％以上急性呼吸道感染由病毒引起，尤以上呼吸道感染为临床上的常病和多发病。目前，已知能引起急性呼吸道感染的病毒有10多种，这些病毒一般通过空气飞沫传播，感染途径容易实现，并且因缺乏有效和持久的免疫，经常引起很高的发病率和超额死亡率；特别是在流感高发季节和暴发流行期，常引起小儿及老年人的并发症和严重病例。虽然现在可以使用疫苗预防，但只对流行性感冒病毒有效，对其他呼吸道病毒的效果不理想，而且免疫效期也比较有限。在治疗方面，迄今为止，没有发现任何一种西药在保证良好疗效的同时具有较低的毒副作用。
因此，亟需提供一种能够有效抗菌、抗病毒，对呼吸系统疾病具有良好治疗效果的药物。
发明内容
有鉴于此，本发明提供了一种抗菌抗病毒药用组合物及其在制备抗菌、抗病毒、清热退烧和/或治疗呼吸系统疾病的药物中的应用。
为了实现上述目的，本发明采用如下技术方案：
一种抗菌抗病毒药用组合物，以重量份数计，包括如下原料：
广藿香4-14份、苍术9-28份、香薷5-18份、艾叶11-33份、丁香0.1-3份和薄荷2-10份。
广藿香芳香化湿，和胃止呕，祛暑解表；苍术燥湿健脾，祛风散寒；香薷清热利湿、抗菌消炎；艾叶温经祛湿、镇咳祛痰平喘；本发明将广藿香、苍术、香薷、艾叶共用，利于消炎、止咳、平喘、化痰、解热，增强抗菌、抗病毒功效。
丁香暖胃、降逆、湿肾，调和诸药同时增强抗菌、抗病毒功效。
薄荷芳香辟秽，具有抗病毒、镇痛，止痒、杀菌的作用，能减少呼吸道的泡沫痰，使有效通气腔道增大，进而增强祛痰功效。
优选地，上述抗菌抗病毒药用组合物，以重量份数计，还包括天然冰片0.01-0.2份。
天然冰片可通诸窍，散郁火，消肿止痛，其添加可进一步提高组合物的抗菌、抗病毒效果。
进一步优选地，一种抗菌抗病毒药用组合物，以重量份数计，包括如下原料：
广藿香8-10份、苍术18-20份、香薷10-12份、艾叶20-24份、丁香0.4-2.5份、薄荷4-8份，天然冰片0.05-0.15份。
优选地，上述药用组合物，还包括药用辅料。
优选地，上述药用组合物，以重量份数计还包括0.5-1.5份药用辅料，药用辅料可选用三氯蔗糖和/或苯甲酸钠。
一种抗菌抗病毒药用挥发油，其制备包括如下步骤：
1)广藿香、苍术、香薷、艾叶、丁香和薄荷分别加水蒸馏提取挥发油，得到各原料挥发油，将各原料挥发油混合得到混合挥发油；
或，将广藿香、苍术、香薷、艾叶、丁香、薄荷混合，加水蒸馏提取得到总挥发油；
或，将广藿香、苍术、香薷、艾叶、丁香、薄荷混合，置于超临界二氧化碳萃取装置中，通入液态二氧化碳萃取挥发油，进行超临界萃取，得到总挥发油；
2)向混合挥发油或总挥发油中加入天然冰片，加热使天然冰片溶解，得到药用挥发油；
或，将天然冰片加入到50-60℃溶剂中搅拌溶解，冷却至室温，再加入混合挥发油或总挥发油中，得到药用挥发油。
优选地，加入天然冰片后加热温度不超过60℃。
优选地，各原料挥发油制备方法如下：
广藿香加5-8倍重量的水，浸泡过夜后水蒸气蒸馏法提取4-8h；
苍术加5-8倍重量的水，浸泡20-60min后水蒸气蒸馏法提取4-6h；
香薷加5-8倍重量的水，浸泡1-3h后水蒸气蒸馏法提取2-4h；
艾叶加6-10倍重量的水，浸泡4-8h后水蒸气蒸馏法提取4-6h；
丁香加5-8倍重量的水，浸泡过夜后水蒸气蒸馏法提取4-8h；
薄荷加5-8倍重量的水，浸泡1-3h后水蒸气蒸馏法提取4-6h；
总挥发油制备方法如下：
广藿香、苍术、香薷、艾叶、丁香、薄荷混合后加入5-10倍重量的水，浸泡1-8h后水蒸气蒸馏法提取2-8h；或，广藿香、苍术、香薷、艾叶、丁香、薄荷混合，置于超临界二氧化碳萃取装置中，通入液态二氧化碳萃取挥发油，萃取压力20-40Mpa、萃取温度40-70℃、分离温度30-60℃，萃取时间0.5-10h。
一种抗菌抗病毒药用芳香水，将广藿香、苍术、香薷、艾叶、丁香和薄荷混合，加入5-12倍重量的水，浸泡过夜后蒸馏，得到药用芳香水。
上述药用组合物或药用挥发油或药用芳香水在制备抗菌、抗病毒、清热退烧和/或治疗呼吸系统疾病的药物中的应用。
本发明药用组合物、药用挥发油、药用芳香水或其制备的药剂对发热以及鼻炎、咽炎、肺炎、咳喘等呼吸系统疾病均有良好的治疗效果，能够有效抗菌、抗病毒，缓解细菌、病毒感染引起的各种症状。
进一步地，上述病毒包括SARS-CoV-2。
进一步地，对于鼻炎，中医分型一般分为四型，三个虚症一个实症。其中，三个虚症包括：1)肺气虚弱：肺开窍于鼻，肺虚自然造成鼻窍不通，引起一些鼻部的症状；2)脾气不足：脾为肺之母，脾虚造成肺虚也会引起鼻部的症状，包括鼻子痒、流清鼻涕、清水连连、打喷嚏、喷嚏不断；3)肾阳不足、肾气虚：肾和肺相连，肾脏出现虚弱，也会引起鼻部的症状；上述三个虚症实际均与肺相关联。另外，分型之中的实症为肺经伏热，为热症，也是由本身肺热所引起。本发明药用组合物可有效治疗上述四种分型的鼻炎。
优选地，上述药物可制成喷雾剂、露剂、气雾剂、滴丸、软胶囊、滴鼻剂或雾化剂等多种剂型。
优选地，喷雾剂的制备方法如下：
药用挥发油用稀释剂稀释后，搅拌均匀，过滤，得到喷雾剂；
或，将天然冰片与药用辅料加入到50-60℃溶剂中搅拌溶解，冷却至室温，再加入混合挥发油或总挥发油，用稀释剂稀释后，搅拌均匀，过滤，得到喷雾剂；
露剂的制备方法如下：
将药用辅料加入到药用芳香水中搅拌均匀，得到露剂；
气雾剂的制备方法如下：
药用挥发油用稀释剂稀释后，搅拌均匀，过滤，灌装到耐压容器中，压盖，充入抛射剂，得到气雾剂；
或，将天然冰片与药用辅料加入到50-60℃溶剂中搅拌溶解，冷却至室温，再加入混合挥发油或总挥发油，用稀释剂稀释后，搅拌均匀，过滤，灌装到耐压容器中，压盖，充入抛射剂，得到气雾剂；
滴丸的制备方法如下：
使用微囊包裹技术将药用挥发油制成挥发油微囊粉，与滴丸基质混合，制得滴丸；
软胶囊的制备方法如下：
使用软胶囊囊材包裹药用挥发油，制得软胶囊；
滴鼻剂的制备方法如下：
将药用挥发油溶于凝胶基质材料的无水乙醇溶液中，加入到稀释剂，搅拌均匀，减压干燥去除无水乙醇，制得油凝胶滴鼻剂；
雾化剂的制备方法如下：
将药用挥发油用稀释剂稀释后，搅拌均匀，过滤，灌装。
进一步优选地，滴丸的制备方法如下：
A、将乳化剂、玉米蛋白、葵花磷脂混合于药用挥发油中，使其在药用挥发油中的质量分数分别为0.5-1.5％，0.5-2％，0.1-2％；
B、将辛烯基琥珀酸淀粉钠溶解到水中，配成质量分数15-50％的水溶液；
C、将A和B配置好的两种溶液以1:1-2重量比混合，使用10000-14000r/min的超高速搅拌机搅拌乳化，并使用高压均质机均质2-3次；
D、对C中获得的均质溶液进行喷雾干燥，得到挥发油微囊粉；
E、按照挥发油微囊粉与滴丸基质质量比1:2的比例制备成滴丸，滴丸基质材料为聚乙二醇4000：聚乙二醇6000＝1:1。
优选地，溶剂为中链甘油三酯、1-98％酒精、无水乙醇、丙二醇、甘油或食用油中的任意一种；
稀释剂为中链甘油三酯、1-98％酒精、无水乙醇、丙二醇、甘油或食用油中的任意一种；
抛射剂为氮气、二氧化碳、七氟丙烷(HFC-227ea)、四氟乙烷(HFC-134a)、1,3,3,3-四氟丙烯(HFO-1234ze)、2,3,3,3-四氟丙烯(HFO-1234yf)或压缩空气。
优选地，气雾剂中液相与气相的比例可选用2:8、3:7、4:6、5:5、6:4任一种，耐压容器内压力为0.6-1MPa。
进一步优选地，食用油可选用火麻仁油、美藤果油、橄榄油、椰子油、石榴籽油、小麦胚芽油、核桃油、牛油果油、亚麻籽油、葡萄籽油、茶籽油、紫苏子油、南瓜籽油、葵花籽油、芥花籽油、蔓越莓籽油、虾青素油、深海鱼油、大豆油、花生油、芝麻油、山茶油、玉 米油、青刺果油、琉璃苣油、月见草油、棕榈油等等。
优选地，凝胶基质材料可选用乙基纤维素等材料。
进一步地，本发明中喷雾剂、露剂、气雾剂、滴丸、软胶囊、滴鼻剂或雾化剂等剂型的制备方法不仅限于上述方法。
综上所述，本发明药用组合物可用于制备抗菌、抗病毒、清热退烧和/或治疗呼吸系统疾病的药物，对于发热以及鼻炎、咽炎、肺炎、咳喘等呼吸系统疾病均有良好的治疗效果，对于细菌、病毒感染引起的疾病疗效显著。对于新冠肺炎轻型(临床症状轻微，影像学未见肺炎的表现，核酸检测为阳性)、新冠肺炎普通型(有发热和呼吸道症状，影像学可以看到肺炎的表现，表现为病毒性肺炎，核酸检测为阳性)的治疗有良好的应用潜力。
附图说明
图1所示为实施例4喷雾剂对Vero E6细胞的毒性作用，CC 50>1/100(v/v)；
图2所示为瑞德西韦对Vero E6细胞的毒性作用，CC 50>200μM；
图3所示为实施例4喷雾剂对SARS-COV-2致细胞死亡的抑制作用(药物、病毒同时加)，IC 50>1/100(v/v)；
图4所示为瑞德西韦对SARS-COV-2致细胞死亡的抑制作用(药物、病毒同时加)，IC 50＝1.118±0.052μM；
图5所示为实施例4喷雾剂对SARS-COV-2致细胞死亡的抑制作用(感染后加药物)，IC 50>1/100(v/v)；
图6所示为瑞德西韦对SARS-COV-2致细胞死亡的抑制作用(感染后加药物)，IC 50＝0.825±0.128μM；
图7所示为1/100(v/v)喷雾剂对SARS-CoV-2杀灭作用；
图8所示为H 2O 2(0.01％)对SARS-CoV-2的杀灭作用。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
实施例中流感病毒亚洲甲型鼠肺适应株A/FM/1/34(H1N1)(FM1)为中国预防医学科学院病毒研究所提供，本实验室液氮保存；A/PR/8/34(H1N1)、A/Aichi/2/1968(H3N2)购 于美国经典培养物收藏中心(ATCC)；2009新甲型H1N1流感病毒株(A/Guangzhou/GIRD07/09,H1N1，Genebank No.HM014332.1)、乙型流感病毒(B/Guangzhou/GIRD08/09,FluB)为广州医科大学呼吸疾病国家重点实验室病毒室临床分离株；甲型H9N2流感病毒(A/Ahicken/Guangdong/1996,H9N2)、甲型H6N2流感病毒(A/Duck/Guangdong/2009,H6N2)、甲型H7N3流感病毒(A/Duck/Guangdong/1994,H7N3)为华南农业大学兽医学院惠赠。体外抗SARS-CoV-2活性实验委托中国科学院昆明动物研究所进行。
实施例1
一种抗菌抗病毒药用组合物，原料包括广藿香885g、苍术1869g、香薷1064g、艾叶2174g、丁香46g、薄荷532g、天然冰片10g。
使用上述药用组合物制备药用挥发油，制备方法如下：
广藿香药材饮片，粉碎过20目筛，用6倍重量的水浸泡过夜，水蒸气蒸馏法提取6h，收集得到广藿香挥发油，提取率达2.26％。
取生苍术药材粗粉，用6倍重量的水浸泡0.5h，水蒸气蒸馏法提取5h，收集得到苍术挥发油，提取率达1.07％。
取香薷药材，用6倍重量的水浸泡2h，水蒸气蒸馏法提取3h，收集得到香薷挥发油，提取率达0.94％。
取艾叶，粗碎，用8倍重量的水浸泡6h，水蒸气蒸馏法提取5h，收集得到艾叶挥发油，提取率达0.46％。
取丁香药材饮片，用6倍重量的水浸泡过夜，水蒸气蒸馏法提取6h，收集得到丁香挥发油，提取率达10.88％。
取薄荷饮片，用6倍重量的水浸泡2h，水蒸气蒸馏法提取5h，收集得到薄荷挥发油，提取率达0.84％。
将上述提取得到的广藿香挥发油、苍术挥发油、香薷挥发油、艾叶挥发油、丁香挥发油、薄荷挥发油混合，加入天然冰片，适当加热(不高于60℃)使天然冰片完全溶解，得到药用挥发油。
实施例2
一种抗菌抗病毒药用组合物，原料包括广藿香663.5g、苍术1402g、香薷798g、艾叶1630.5g、丁香32g、薄荷106.5g、天然冰片2.5g。
使用上述药用组合物制备药用挥发油的方法，制备方法如下：
各原料先粉碎成粗粉，混合后，置HA220-40-48超临界二氧化碳萃取装置中，通入液态 二氧化碳萃取挥发油，萃取压力28-30Mpa、萃取温度55℃、分离温度45℃，萃取时间3h，得萃取物(总挥发油)106.5g，加入天然冰片，适当加热(不高于60℃)使天然冰片完全溶解，得到药用挥发油。
实施例3
对实施例1药用挥发油进行药效研究试验：
药效研究中的小鼠剂量分别为65mg/kg、130mg/kg和260mg/kg。
药效研究中的豚鼠剂量分别为32mg/kg、64mg/kg和128mg/kg。
药效研究中的大鼠剂量分别为37mg/kg、74mg/kg和148mg/kg
1小鼠浓氨水引咳实验(止咳作用)
1.1实验方法
ICR小鼠，雌雄各半。按照体重分层随机分为6组，每组14只，分别为：正常对照组、模型对照组、枸橼酸喷托维林20mg/kg组、药用挥发油低剂量(65mg/kg)组、药用挥发油中剂量(130mg/kg)组和药用挥发油高剂量(260mg/kg)组，小鼠按20mL/kg灌胃给药(使用大豆油配制成相应的浓度)，每天1次，连续7d。正常对照组和模型对照组灌胃等量大豆油。
1.2指标测定
末次给药后1h，将小鼠放入500mL透明密闭容器中，用超声雾化器向容器内均匀喷入雾化浓氨水，连续喷雾20s；以小鼠收缩腹部并张口作为咳嗽标准，记录小鼠咳嗽潜伏期和放入容器后2min内的咳嗽次数。
1.3实验结果
与模型对照组比较，药用挥发油各剂量组的小鼠咳嗽潜伏期均显著延长、小鼠咳嗽次数均显著减少(P<0.01)。结果见表1。
表1药用挥发油对浓氨水致小鼠咳嗽的影响(
Figure PCTCN2021090137-appb-000001
n＝14)
Figure PCTCN2021090137-appb-000002
注：与模型对照组比较， **P<0.01。
在本实验条件及所设计的剂量下，药用挥发油具有明显的止咳作用。
2对乙酰胆碱合用磷酸组胺致哮喘豚鼠的影响(平喘作用)
2.1实验方法
普通级豚鼠，雌雄各半，将豚鼠逐只放入玻璃罩(容量4L)中，用超声雾化器喷入0.1％磷酸组胺和2％氯化乙酰胆碱等容积混合液15s；逐个记录豚鼠药前哮喘潜伏期(从喷雾开始至抽搐的时间)；哮喘潜伏期未超过120s者为合格豚鼠，超过者认为不敏感，弃除。
将筛选合格的豚鼠按照药前哮喘潜伏期分层随机分为5组，每组12只，分别为：模型对照组、氨茶碱60mg/kg组、药用挥发油低剂量(32mg/kg)组、药用挥发油中剂量(64mg/kg)组和药用挥发油高剂量(128mg/kg)组，豚鼠按4mL/kg灌胃给药(使用大豆油配制成相应的浓度)，每天1次，连续7d。模型对照组灌胃等量的大豆油。
2.2指标检测
末次给药后1h，同法记录豚鼠的药后哮喘潜伏期；若豚鼠在3min内仍未出现哮喘症状，则以180s计算。
2.3实验结果
与模型对照组比较，药用挥发油各剂量组的药后哮喘潜伏期及药后哮喘潜伏期的延长值(药后潜伏期-药前潜伏期)均显著升高(P<0.01)。结果见表2。
表2药用挥发油对乙酰胆碱和磷酸组胺致豚鼠哮喘潜伏期的影响(
Figure PCTCN2021090137-appb-000003
n＝12)
Figure PCTCN2021090137-appb-000004
注：与模型对照组比较， **P<0.01。
在本实验条件及所设计的剂量下，药用挥发油具有明显的平喘作用。
3对小鼠腹腔注射酚红致气管排泌的影响(化痰作用)
3.1实验方法
ICR小鼠，雌雄各半，按照体重分层随机分为5组，每组14只，分别为模型对照组、盐酸氨溴索30mg/kg组、药用挥发油低剂量(65mg/kg)组、药用挥发油中剂量(130mg/kg)组和药用挥发油高剂量(260mg/kg)组，小鼠按20mL/kg灌胃给药(使用大豆油配制成相应的浓度)，每天1次，连续7d。模型对照组灌胃等量的大豆油。
3.2指标检测
末次给药后30min，腹腔注射5％酚红生理盐水溶液20mL/kg，30min后处死小鼠，暴露气管，剥去气管周围组织，剪下自甲状软骨下至支气管分支处的一段气管，浸入盛有2mL5％NaHCO 3溶液的试管中，震荡将其中排出的酚红充分洗出，放置过夜，得到透明上清液，于546nm处测定气管洗出液的吸收度值。
3.3实验结果
与模型对照组比较，药用挥发油各剂量组的小鼠气道酚红排泌量均显著升高(P<0.01)。结果见表3。
表3药用挥发油对小鼠气道酚红排泌的影响(
Figure PCTCN2021090137-appb-000005
n＝14)
Figure PCTCN2021090137-appb-000006
注：与模型对照组比较， **P<0.01。
在本实验条件及所设计的剂量下，药用挥发油具有明显的化痰作用。
4对大鼠毛细管排痰量的影响(化痰作用)
4.1实验方法
SD大鼠，雌雄各半，按照体重分层随机分为5组，每组12只，分别为模型对照组、羧甲司坦200mg/kg组、药用挥发油低剂量(37mg/kg)组、药用挥发油中剂量(74mg/kg)组和药用挥发油高剂量(148mg/kg)组，大鼠按10mL/kg灌胃给药(使用大豆油配制成相应的浓度)，每天1次，连续7d。模型对照组灌胃等量的大豆油。
4.2指标检测
末次给药后40min，麻醉，仰位固定，剪开颈中部皮肤，分离出气管，在甲状腺软骨下缘正中两软骨环之间用尖锐的注射针头扎一小孔，插入一根玻璃毛细管，使毛细管刚好接触气管底部表面，气管中痰液即被吸入毛细管，当毛细管被痰液充满后，立即换一根同直径的毛细管，连续收集2h，刻度尺测量毛细管内液柱长度，评价排痰量。
4.3实验结果
与模型对照组比较，药用挥发油各剂量组的大鼠排痰量均显著增加(P<0.05或P<0.01)。结果见表4。
表4药用挥发油对大鼠毛细管排痰量的影响(
Figure PCTCN2021090137-appb-000007
n＝12)
Figure PCTCN2021090137-appb-000008
注：与模型对照组比较， *P<0.05， **P<0.01。
在本实验条件及所设计的剂量下，药用挥发油具有明显的化痰作用。
5对脂多糖(LPS)诱导大鼠发热模型的影响(解热作用)
5.1实验方法
SD大鼠，雌雄各半，SD大鼠提前2日进行适应性肛温测量，每日2次，取平均值为基础体温。将单次体温超过38℃或者两次温差超过0.5℃的大鼠去除，将合格的大鼠按体温分层随机分为6组，每组12只，分别为正常对照组、模型对照组、阿司匹林200mg/kg组、药用挥发油低剂量(37mg/kg)组、药用挥发油中剂量(74mg/kg)组和药用挥发油高剂量(148mg/kg)组，大鼠按10mL/kg灌胃给药(使用大豆油配制成相应的浓度)，每天1次，连续7d。正常对照组和模型对照组灌胃等量的大豆油。
5.2指标检测
实验前12h，大鼠禁食不禁水，实验当天测量大鼠基础体温，末次给药1h后，正常对照组大鼠腹腔注射无菌生理盐水，其他各组分别腹腔注射LPS(20μg/kg)造模，造模后开始记录大鼠的状态及体温变化，每1h测量体温1次，连续记录7h，以每1h的体温与基础体温的差值(ΔT)作为指标。
5.3实验结果
正常对照组大鼠在7h内体温无明显变化，模型对照组大鼠在注射LPS后体温逐渐升高，与模型对照组比较有显著性差异(P<0.01)，在6h时达到峰值。与模型对照组比较，药用挥发油各剂量组在2～7h体温明显降低(P<0.05或P<0.01)。结果见表5。
表5药用挥发油对LPS致热大鼠的影响(
Figure PCTCN2021090137-appb-000009
n＝12)
Figure PCTCN2021090137-appb-000010
Figure PCTCN2021090137-appb-000011
注：与正常对照组比较， ##P<0.01；与模型对照组比较， *P<0.05， **P<0.01。
在本实验条件及所设计的剂量下，药用挥发油具有明显的解热作用。
6对急性咽炎模型大鼠的影响
6.1实验方法
Wistar大鼠，雌雄各半，手抓固定大鼠，以镊子掰开并固定大鼠下颚，将喷雾装置喷头伸入大鼠口腔致咽部，用25％氨水喷大鼠咽部1次(约0.2mL)，连续喷3d，每天2次，使大鼠咽部粘膜因刺激形成急性炎症。连续造模3d后，将大鼠随机分为模型对照组，地塞米松5mg/kg组，药用挥发油低剂量(37mg/kg)组、药用挥发油中剂量(74mg/kg)组和药用挥发油高剂量(148mg/kg)组，每组12只，另取同批次大鼠12只为正常对照组，大鼠按10mL/kg灌胃给药(使用大豆油配制成相应的浓度)，每天1次，连续3d。正常对照组和模型对照组灌胃等量的大豆油。
6.2指标检测
末次给药后1h，大鼠麻醉，腹主动脉取血处死动物，分离血清，采用ELISA法测定血清中IL-6和TNF-α水平。
6.3实验结果
与正常对照组比较，模型对照组大鼠血清中的IL-6和TNF-α的含量显著升高(P<0.01)。与模型对照组比较，药用挥发油各剂量组均可显著降低大鼠血清中的IL-6和TNF-α含量(P<0.05或P<0.01)。结果见表6。
表6药用挥发油对急性咽炎大鼠的影响(
Figure PCTCN2021090137-appb-000012
n＝12)
Figure PCTCN2021090137-appb-000013
注：与正常对照组比较， ##P<0.01；与模型对照组比较， *P<0.05， **P<0.01。
在本实验条件及所设计的剂量下，药用挥发油可以显著抑制急性咽炎模型大鼠的炎症反应。
7对流感病毒致小鼠死亡的保护作用
7.1实验方法
流感病毒亚洲甲型鼠肺适应株A/FM/1/34(H1N1)(FM1)，使用前接种于9日龄鸡胚尿囊腔，常规培养，48h后新鲜无菌收集尿囊液，传代2次，测定对ICR小鼠的半数致死量(LD 50)。ICR小鼠，雌雄各半，按体重分层随机分为6组，每组16只，分别为正常对照组、模型对照组、利巴韦林100mg/kg组、药用挥发油低剂量(65mg/kg)组、药用挥发油中剂量(130mg/kg)组和药用挥发油高剂量(260mg/kg)组。小鼠以乙醚轻度麻醉后，每只小鼠以25μL病毒液滴鼻，感染量为25倍LD 50，正常对照组用等量无菌生理盐水滴鼻。药用挥发油各剂量组小鼠按20mL/kg灌胃给药(使用大豆油配制成相应的浓度)，利巴韦林组腹腔注射给药，每天1次，连续7d，正常对照组和模型对照组灌胃等量的大豆油。
7.2指标检测
逐日观察小鼠的发病症状，记录死亡时间、死亡数以及体重，连续观察14d。第14d不死亡的，按14d计算，感染24h内死亡的视为非感染死亡。
7.3实验结果
正常对照组小鼠精神良好，行动敏捷，皮毛顺滑光亮，呼吸频率正常，进食及饮水量均正常，体重逐日增加。流感病毒感染小鼠，在感染24h后，出现甩头、呼吸频率加快急促，腹式呼吸为主，行动迟缓、卷缩、耸毛，饮食量和饮水量均明显减少。从第4d开始，小鼠开始出现死亡，第7d达到高峰，各给药治疗组小鼠状态均明显好于模型组。
正常对照组小鼠无死亡，模型对照组小鼠生存率仅为6.2％；与模型对照组比较，药用挥发油低、中、高剂量组的小鼠生存率分别为25.0％、37.5％、56.2％，生存率明显升高(P<0.05或P<0.01)。结果见表7。
表7药用挥发油对流感病毒FM1感染小鼠生存率的影响(n＝16)
Figure PCTCN2021090137-appb-000014
注：与正常对照组比较， ##P<0.01；与模型对照组比较， *P<0.05， **P<0.01。
在本实验条件及所设计的剂量下，药用挥发油可以明显提高流感病毒FM1感染小鼠的存活率，具有明显地抗病毒作用。
8对流感病毒感染小鼠病毒性肺炎的影响
8.1实验方法
流感病毒亚洲甲型鼠肺适应株A/FM/1/34(H1N1)(FM1)，使用前接种于9日龄鸡胚尿囊腔，常规培养，48h后新鲜无菌收集尿囊液，传代2次，测定对ICR小鼠的半数致死量(LD 50)。ICR小鼠，雌雄各半，按体重分层随机分为6组，每组14只，分别为正常对照组、模型对照组、达菲27.5mg/kg组、药用挥发油低剂量(65mg/kg)组、药用挥发油中剂量(130mg/kg)组和药用挥发油高剂量(260mg/kg)组。小鼠以乙醚轻度麻醉后，每只小鼠以20μl病毒液滴鼻，感染量为25倍LD 50，正常对照组用等量无菌生理盐水滴鼻。药用挥发油各剂量组小鼠按20mL/kg灌胃给药(使用大豆油配制成相应的浓度)，每天1次，连续7d，正常对照组和模型对照组灌胃等量的大豆油。
8.2指标检测
末次给药后1h，称取小鼠体重后处死，解剖摘取全肺称重，计算肺指数值和肺指数抑制率。将肺组织用生理盐水制备成10％肺组织匀浆，将制备好的匀浆液低温离心，采用ELISA法测定肺组织中的IL-8和TNF-α的含量。
肺指数＝[肺重量(g)/体重(g)]×100；
肺指数抑制率＝(模型对照组肺指数均值-试验组肺指数均值)/模型对照组肺指数均值。
8.3实验结果
与正常对照组比较，模型对照组小鼠的肺指数显著升高(P<0.01)；与模型对照组比较，药用挥发油各剂量组小鼠的肺指数显著降低(P<0.05或P<0.01)。结果见表8。
表8药用挥发油对流感病毒FM1感染小鼠肺炎肺指数的影响(
Figure PCTCN2021090137-appb-000015
n＝14)
Figure PCTCN2021090137-appb-000016
Figure PCTCN2021090137-appb-000017
注：与正常对照组比较， ##P<0.01；与模型对照组比较， *P<0.05， **P<0.01。
与正常对照组比较，模型对照组小鼠肺组织匀浆中的IL-8和TNF-α的含量显著升高(P<0.01)；与模型对照组比较，药用挥发油各剂量组小鼠肺组织匀浆中的IL-8和TNF-α的含量显著降低(P<0.05或P<0.01)。结果见表9。
表9药用挥发油对流感病毒FM1感染小鼠肺炎IL-8和TNF-α的影响(
Figure PCTCN2021090137-appb-000018
n＝14)
Figure PCTCN2021090137-appb-000019
注：与正常对照组比较， ##P<0.01；与模型对照组比较， *P<0.05， **P<0.01。
在本实验条件及所设计的剂量下，药用挥发油可以明显改善H1N1流感所致的病毒性肺炎。
9体外抗病毒作用研究
9.1实验方法
选用的细胞株为狗肾细胞(MDCK)，7株病毒株分别为A/PR/8/34(H1N1)、A/Aichi/2/1968(H3N2)、2009新甲型H1N1流感病毒株(A/Guangzhou/GIRD07/09,H1N1)、乙型流感病毒(B/Guangzhou/GIRD08/09,FluB)、甲型H9N2流感病毒(A/Ahicken/Guangdong/1996,H9N2)、甲型H6N2流感病毒(A/Duck/Guangdong/2009,H6N2)和甲型H7N3流感病毒(A/Duck/Guangdong/1994,H7N3)。
以上7种流感病毒以9～11日龄鸡胚扩增，收集尿囊液，测定其相应的血凝效价，-80℃保存。
采用细胞病变抑制法研究药用挥发油对H1N1、H3N2、09年新甲流H1N1、FluB、H9N2、H6N2和H7N3等流感病毒毒株的抑制作用。采用含有双抗及10％胎牛血清的MEM培养基，常规培养MDCK细胞。长满单层MDCK细胞的96孔板，吸附病毒2h，试验组加入含有不同浓度的药用挥发油(等体积)，同时设定阳性对照组(利巴韦林)和空白对照组，100μL/孔，于37℃、5％CO 2环境下，培养2天。
9.2指标检测
细胞的病变程度按以下6级标准记录：“-”为细胞生长正常，无病变出现；“±”为细胞病变少于整个单层细胞的10％；“+”为细胞病变少于整个单层细胞的25％；“++”为细胞病变少于整个单层细胞的50％；“+++”为细胞病变少于整个单层细胞的75％；“++++”为细胞病变少于整个单层细胞的75％以上。
用Reed-Muench法计算半数抑制浓度(IC 50)，并以选择指数SI表示(TC 50/IC 50)，判断标准：SI＜1表示无效；SI：1～2表示高毒低效；SI＞2表示低毒高效。
9.3实验结果
药用挥发油对MDCK细胞的无毒浓度为50mg/mL，其半数有毒浓度TC 50＞50mg/mL。药用挥发油对H1N1、H3N2、09年新甲流H1N1、FluB、H9N2、H6N2和H7N3的IC 50分别为18.4mg/mL、36.2mg/mL、24.2mg/mL、28.5mg/mL、48.3mg/mL、45.5mg/mL和30.2mg/mL。结果见表10。
表10药用挥发油体外抗流感病毒作用
Figure PCTCN2021090137-appb-000020
在本实验条件及所设计的剂量下，药用挥发油对H1N1、H3N2、09年新甲流H1N1、FluB、H9N2、H6N2和H7N3七种流感病毒均有明显的抑制作用，其中对H1N1，09年新甲流H1N1和FluB毒株作用较强。
10对肺炎双球菌感染小鼠的保护作用
10.1实验方法
ICR小鼠，雌雄各半，按照体重分层随机分为6组，每组20只，分别为正常对照组、模型对照组、双黄连20g/kg组、药用挥发油低剂量(65mg/kg)组、药用挥发油中剂量(130mg/kg)组和药用挥发油高剂量(260mg/kg)组，小鼠按20mL/kg灌胃给药(使用大豆油配制成相应的浓度)，每天1次，连续7d。模型对照组灌胃等量的大豆油。
10.2指标检测
第4天给药后1h，除正常对照组外，其余各组腹腔注射肺炎双球菌菌液0.5mL/只，继续给药3d，观察各组小鼠7日内死亡情况。
10.3实验结果
与正常对照组比较，肺炎双球菌感染的小鼠7日内死亡明显增加，存活率为20％(P<0.01)；与模型对照组比较，药用挥发油中剂量和高剂量组小鼠7日内死亡数明显降低(P<0.05或P<0.01)。结果见表11。
表11药用挥发油对肺炎双球菌感染小鼠生存率的影响(n＝20)
Figure PCTCN2021090137-appb-000021
注：与正常对照组比较， ##P<0.01；与模型对照组比较， *P<0.05， **P<0.01。
在本实验条件及所设计的剂量下，药用挥发油可以明显提高肺炎双球菌感染小鼠的存活率，具有明显地抑菌作用。
11对卵白蛋白(OVA)致大鼠过敏性鼻炎的影响
11.1实验方法
SD大鼠，全雄，按照体重分层随机分为6组，每组15只，分别为正常对照组、模型对照组、鼻炎康0.4g/kg组、药用挥发油低剂量(37mg/kg)组、药用挥发油中剂量(74mg/kg)组和药用挥发油高剂量(148mg/kg)组，除正常对照组外，其余各组大鼠以卵白蛋白(OAV)0.3mg、Al(OH) 330mg以及生理盐水1mL混合后腹腔注射初次免疫，第15天每侧鼻孔给予4％OVA200μg(50μL)滴鼻加强免疫。各组大鼠于致敏第15d开始给药，每天1次，连续14d。大鼠按10mL/kg灌胃给药(使用大豆油配制成相应的浓度)，正常对照组和模型对照组灌胃等量的大豆油。
11.2指标检测
末次给药后1h，各组大鼠10％水合氯醛0.35g/kg腹腔注射麻醉，腹主动脉采血，于4℃3000r/min离心15min，分离血清，-70℃保存，根据试剂盒说明书进行组胺(HIS)，白介素-4(IL-4)和肿瘤坏死因子(TNF-α)的测定。
11.3实验结果
与正常对照组比较，模型对照组大鼠血清中的HIS、IL-4和TNF-α水平均显著升高 (P<0.01)；与模型对照组比较，药用挥发油中剂量组和高剂量组大鼠血清中的HIS和IL-4水平均显著降低(P<0.05或P<0.01)，药用挥发油各剂量组大鼠血清中的TNF-α水平均显著降低(P<0.05或P<0.01)。结果见表12。
表12药用挥发油对OVA致大鼠过敏性鼻炎的影响(
Figure PCTCN2021090137-appb-000022
n＝15)
Figure PCTCN2021090137-appb-000023
注：与正常对照组比较， ##P<0.01；与模型对照组比较， *P<0.05， **P<0.01。
在本实验条件及所设计的剂量下，药用挥发油可以明显改善OVA致大鼠过敏性鼻炎，降低炎症反应。
实施例4
药用组合物原料配比同实施例1，按照实施例1方法制备广藿香挥发油、苍术挥发油、香薷挥发油、艾叶挥发油、丁香挥发油、薄荷挥发油，将其混合后得到混合挥发油。
取200mL中链甘油三酯，加热至50-60℃，添加天然冰片，使天然冰片完全溶解，冷却至室温，再加入混合挥发油中，得到药用挥发油；使用中链甘油三酯稀释药用挥发油调整总量至1000mL，搅匀，滤过，获得浅黄色至黄色澄明油状溶液，气芳香，微苦，相对密度(药典通则0601)0.933～0.961；灌装，每瓶20mL，即得喷雾剂。避光、密闭贮藏，有效期可达24个月。
进一步地，对喷雾剂进行鉴别：
(1)取本品15mL，加石油醚(60～90℃)50mL，混合均匀，用2mol/L氢氧化钠溶液10mL提取，取水层用2mol/L盐酸溶液调节pH值至2.0，再用石油醚(60～90℃)10mL振摇提取，分取石油醚层并浓缩至0.5mL，作为供试品溶液。另取百秋李醇对照品和广藿香酮对照品，分别加乙酸乙酯制成每lmL各含4mg的溶液，作为对照品溶液。照薄层色谱法(药典通则0502)试验，吸取供试品溶液2μL、对照品溶液1μL，分别点于同一硅胶G薄层板上，以石油醚(60～90℃)-乙酸乙酯-甲酸(10:2:1)为展开剂，展开，取出，晾干，5％三氯化铁乙醇溶液浸渍显色，加热至斑点显色清晰。供试品色谱中在与百秋李醇对照品相应的位置上，显相同的紫蓝色斑点；在与广藿香酮对照品相应的位置上，显相同颜色的斑点。
(2)含量测定：照气相色谱法(药典通则0521)测定
对照品溶液的制备：取丁香酚对照品和百秋李醇对照品适量，精密称定，分别加正己烷制成每lmL含丁香酚0.3mg的溶液，每lmL含百秋李醇0.3mg的溶液，即得。
供试品溶液的制备：精密量取本品1mL，置10mL的量瓶中，加正己烷至刻度线，摇匀，即得。
色谱条件与系统适用性试验：以100％二甲基聚硅氧烷为固定相的毛细管色谱柱(柱长为30m，内径为0.32mm，厚度为0.25μm)；柱温为程序升温，初始温度70℃，保持5min，5℃/min升至110℃，10℃/min升至280℃，保持4min，10℃/min升至300℃，保持5min。理论板数按龙脑峰计算应不低于40000。
分别精密吸取对照品溶液与供试品溶液各1μL，注入气相色谱仪进行测定。
供试品色谱中呈现与百秋李醇对照品、香荆芥酚对照品、桉油精对照品、龙脑对照品、异龙脑对照品、丁香酚对照品和薄荷脑对照品色谱峰保留时间相对应的色谱峰。
本品每1mL含丁香以丁香酚(C 10H 12O 2)计，不少于3.75mg；含广藿香以百秋李醇(C 15H 26O)计，不少于5.2mg。
本喷雾剂芳香辟秽、宣肺理气、祛湿化痰、清咽利喉。可用于新冠肺炎的轻型、普通型、恢复期及感冒或流感引起的鼻塞、流涕、咳嗽和咽喉肿痛等。
使用时可直接喷喉或涂抹鼻腔。新冠肺炎轻型、普通型，喷喉，早中晚各2次，6揿/次；新冠肺炎恢复期，喷喉，早中晚各1次，3揿/次；感冒或流感引起的咳嗽和咽喉肿痛，喷喉，每天2次，3揿/次；鼻塞，流涕，可适量噴于棉签上涂抹鼻腔。
对上述喷雾剂进行体外抗SARS-CoV-2活性实验：
1待测样品对VeroE6细胞的毒性实验
1.1实验方法
Vero E6细胞以含10％胎牛血清FBS、100IU/ml青霉素、100μg/ml链霉素的无酚红DMEM高糖完全培养基(货号：E600005-0500，批号FC11FC0255，购自生工生物工程(上海)股份有限公司)进行培养，实验前1天将细胞传代一次，使所用细胞处于对数生长期。
96孔板内，2×10 4个/孔接种Vero E6细胞，37℃、5％CO 2培养过夜，待单层细胞长至70％左右时，分别加入不同浓度的待测样品(使用含3％FBS的DMEM培养基稀释喷雾剂，现用现配)或瑞德西韦(储存液溶解于DMSO，终浓度为100mM/mL，4℃保存，工作液用含3％FBS的DMEM培养基稀释，现用现配)，100μL/孔，设3个重复孔；同时设置不含药物的对照孔；37℃、5％CO 2培养3天。
1.2指标检测
采用CCK8试剂盒(货号：C0039，批号：121819200616，购自上海碧云天生物技术有限公司)检测细胞存活率。Synergy 2多功能微孔板检测仪测定OD值，测定波长为450nm，参考波长为630nm。
根据实验结果，采用Origin7.5绘制剂量反应拟合曲线，按Reed&Muench法计算出样品50％抑制细胞生长浓度(CC 50)。细胞生长存活率(％)＝实验孔OD值/对照孔OD值×100。
1.3实验结果
结果如表13、14，图1、2所示，喷雾剂对Vero E6细胞CC 50>1/100(v/v)，瑞德西韦对Vero E6细胞CC 50>200μM。
表13喷雾剂对Vero E6细胞的毒性作用
Figure PCTCN2021090137-appb-000024
表14瑞德西韦对Vero E6细胞的毒性作用
Figure PCTCN2021090137-appb-000025
Figure PCTCN2021090137-appb-000026
2待测样品对SARS-CoV-2致Vero E6细胞死亡的抑制实验(药物和病毒同步加入细胞组)
2.1实验方法
P2实验室内以2×10 4个/孔接种Vero E6细胞到96孔板，37℃、5％CO 2培养过夜，待单层细胞长至70％左右时，转移至P3实验室待用。
根据待测样品、瑞德西韦对VeroE6细胞的毒性实验结果分别设不同浓度梯度，每个梯度三个重复孔；P3实验室内，在细胞培养板内，每孔同时加入预先配制的待测样品/瑞德西韦50μL和50μL病毒稀释上清(MOI 0.1)；设置不含药物和病毒的阴性对照、不含药物的阳性对照；37℃、5％CO 2培养3天。
2.2指标检测
采用CCK8试剂盒检测细胞存活率，Bio-Tek微孔板检测仪测定OD值，测定波长为450nm，参考波长为630nm。
根据实验结果，采用Origin7.5绘制剂量反应拟合曲线，按Reed&Muench法计算出样品抑制病毒的50％有效浓度(IC 50)及抗SARS-CoV-2活性的治疗指数TI值(Therapeutic index)为：TI＝CC 50/IC 50；SARS-CoV-2致细胞死亡的抑制率(％)＝(实验孔OD值-阳性对照孔OD值)/(阴性对照孔OD值-阳性对照孔OD值)×100。
2.3实验结果
结果如表15、16，图3、4所示，喷雾剂组IC 50>1/100(v/v)，瑞德西韦组IC 50＝1.118±0.052μM，TI>178.89。
表15喷雾剂对SARS-CoV-2致Vero E6细胞死亡的抑制作用
Figure PCTCN2021090137-appb-000027
Figure PCTCN2021090137-appb-000028
表16瑞德西韦对SARS-COV-2致Vero E6细胞死亡的抑制作用
Figure PCTCN2021090137-appb-000029
3待测样品对SARS-CoV-2致Vero E6细胞死亡的抑制实验(病毒感染细胞1h后加入药物组)
3.1实验方法
P2实验室内以2×10 4个/孔接种Vero E6细胞到96孔板，37℃、5％CO 2培养过夜，待单层细胞长至70％左右时，转移至P3实验室待用。
根据待测样品、瑞德西韦对VeroE6细胞的毒性实验结果分别设不同浓度梯度，每个梯度三个重复孔；P3实验室内，在细胞培养板内，每孔加入50μL病毒稀释上清(MOI 0.1)，设置不含药物和病毒的阴性对照、不含药物的阳性对照。37℃、5％CO 2培养箱孵育1h后，实验孔分别加入预先配制的待测样品或瑞德西韦，50μL/孔；37℃、5％CO 2培养3天。
3.2指标检测
采用CCK8试剂盒检测细胞存活率，Bio-Tek微孔板检测仪测定OD值，测定波长为450nm，参考波长为630nm。
根据实验结果，采用Origin7.5绘制剂量反应拟合曲线，按Reed&Muench法计算出样品抑制病毒的50％有效浓度(IC 50)及抗SARS-CoV-2活性的治疗指数TI值(Therapeutic index)为：TI＝CC 50/IC 50；SARS-CoV-2致细胞死亡的抑制率(％)＝(实验孔OD值-阳性对照孔OD值)/(阴性对照孔OD值-阳性对照孔OD值)×100。
3.3实验结果
结果如表17、18，图5、6所示，喷雾剂组IC 50>1/100(v/v)，瑞德西韦组IC 50＝0.825±0.128μM，TI>242.42。
表17喷雾剂对SARS-CoV-2致Vero E6细胞死亡的抑制作用
Figure PCTCN2021090137-appb-000030
表18瑞德西韦对SARS-CoV-2致Vero E6细胞死亡的抑制作用
Figure PCTCN2021090137-appb-000031
Figure PCTCN2021090137-appb-000032
4直接杀病毒实验
4.1实验方法
P2实验室内以2×10 4个/孔接种Vero E6细胞到96孔板，37℃、5％CO 2培养过夜，待单层细胞长至70％左右时，转移至P3实验室待用。
96孔板内，取稀释至1/100(v/v)的待测样品或0.01％H 2O 2100μL，与100μL SARS-CoV-2稀释液(MOI 0.1)混合，同时设置不含药物的阳性对照和不含病毒的阴性对照；37℃、5％CO 2下孵育0、5min、10min、30min和60min。
吸去细胞培养板中的培养基，分别加入上述药物和病毒混合液，200μL/孔，37℃、5％CO 2培养箱中培养三天。
4.2指标检测
采用CCK8试剂盒检测细胞存活率，Bio-Tek微孔板检测仪测定OD值，测定波长为450nm，参考波长为630nm。
SARS-CoV-2致细胞死亡的抑制率(％)＝(实验孔OD值-阳性对照孔OD值)/(阴性对照孔OD值-阳性对照孔OD值)×100。
4.3实验结果
结果如表19、20，图7、8所示。
表19喷雾剂对SARS-CoV-2杀灭作用
Figure PCTCN2021090137-appb-000033
表20 H 2O 2(0.01％)对SARS-CoV-2杀灭作用
Figure PCTCN2021090137-appb-000034
综上所述，本发明喷雾剂与细胞、病毒同时孵育，对SARS-CoV-2致Vero E6细胞死亡有一定的抑制作用，浓度1/100(v/v)时抑制率可达26.67％；细胞和病毒预先孵育1h后加入喷雾剂，对SARS-CoV-2致Vero E6细胞死亡的抑制作用较同时孵育组相当，浓度1/100(v/v)时抑制率达23.94％；浓度为1/100(v/v)的喷雾剂对SARS-CoV-2具有一定的杀灭作用，60分钟的抑制率为19.53％。
实施例5
一种药用组合物，原料包括广藿香445g、苍术934g、香薷532g、艾叶1087g、丁香2g、薄荷745g、天然冰片15g。
使用上述药用组合物制备药用挥发油的方法同实施例1。
使用中链甘油三酯稀释药用挥发油调整总量至1000mL，搅匀，滤过，按20mL/瓶的规格灌装入喷雾剂瓶中，拧紧喷雾剂泵，即得喷雾剂。
实施例6
一种药用组合物，原料包括广藿香885g、苍术1869g、香薷1064g、艾叶2174g、丁香46g、薄荷532g。
称取广藿香、苍术、香薷、艾叶、丁香、薄荷，切段，混合，加入8倍质量的水浸泡过夜后，采用水蒸气蒸馏法蒸馏，收集馏出液约14000mL，即为芳香水。
对上述芳香水进行药效研究试验：
药效研究中的小鼠剂量分别为12mL/kg、24mL/kg和40mL/kg。
1对流感病毒致小鼠死亡的保护作用
1.1实验方法
流感病毒亚洲甲型鼠肺适应株A/FM/1/34(H1N1)(FM1)，使用前接种于9日龄鸡胚尿囊腔，常规培养，48h后新鲜无菌收集尿囊液，传代2次，测定对ICR小鼠的半数致死量(LD50)。ICR小鼠，雌雄各半，按体重分层随机分为6组，每组20只，分别为正常对照组、模型对照组、利巴韦林100mg/kg组、芳香水低剂量(12mL/kg)组、芳香水中剂量(24mL/kg)组和芳香水高剂量(40mL/kg)组。小鼠以乙醚轻度麻醉后，每只小鼠以25μL病毒液滴鼻，感染量为25倍LD50，正常对照组用等量无菌生理盐水滴鼻。芳香水各剂量组小鼠每日给药两次，每次给药间隔6h，每次给药剂量为总剂量的一半，分别为6mL/kg、12mL/kg和20mL/kg，利巴韦林组腹腔注射给药，每天1次，连续7d，正常对照组和模型对照组灌胃等量的蒸馏水。
1.2指标检测
逐日观察小鼠的发病症状，记录死亡时间、死亡数以及体重，连续观察14d。第14d不死亡的，按14d计算，感染24h内死亡的视为非感染死亡。
1.3实验结果
正常对照组小鼠无死亡，模型对照组小鼠生存率仅为10.0％；与模型对照组比较，芳香水低、中、高剂量组的小鼠生存率分别为30.0％、40.0％、55.0％，生存率明显升高(P<0.05或P<0.01)。结果见表21。
表21芳香水对流感病毒FM1感染小鼠生存率的影响(n＝20)
Figure PCTCN2021090137-appb-000035
注：与正常对照组比较， ##P<0.01；与模型对照组比较，*P<0.05，**P<0.01。
由上表可知，芳香水可以明显提高流感病毒FM1感染小鼠的存活率，具有明显地抗病毒作用。
2对流感病毒感染小鼠病毒性肺炎的影响
2.1实验方法
流感病毒亚洲甲型鼠肺适应株A/FM/1/34(H1N1)(FM1)，使用前接种于9日龄鸡胚尿囊腔，常规培养，48h后新鲜无菌收集尿囊液，传代2次，测定对ICR小鼠的半数致死量(LD50)。ICR小鼠，雌雄各半，按体重分层随机分为6组，每组14只，分别为正常对照组、模型对照组、达菲27.5mg/kg组、芳香水低剂量(12mL/kg)组、芳香水中剂量(24mL/kg)组和芳香水高剂量(40mL/kg)组。小鼠以乙醚轻度麻醉后，每只小鼠以20μl病毒液滴鼻，感染量为25倍LD50，正常对照组用等量无菌生理盐水滴鼻。芳香水各剂量组小鼠每日给药两次，每次给药间隔6h，每次给药剂量为总剂量的一半，分别为6mL/kg、12mL/kg和20mL/kg，连续7d，正常对照组和模型对照组灌胃等量的蒸馏水。
2.2指标检测
末次给药后1h，称取小鼠体重后处死，解剖摘取全肺称重，计算肺指数值和肺指数抑制率。将肺组织用生理盐水制备成10％肺组织匀浆，将制备好的匀浆液低温低速离心，采用ELISA法测定肺组织中的IL-8和TNF-α的含量。
肺指数＝[肺重量(g)/体重(g)]×100；
肺指数抑制率＝(模型对照组肺指数均值-试验组肺指数均值)/模型对照组肺指数均值。
2.3实验结果
与正常对照组比较，模型对照组小鼠的肺指数显著升高(P<0.01)；与模型对照组比较，芳香水中剂量组和高剂量组小鼠的肺指数显著降低(P<0.05或P<0.01)。结果见表22。
表22芳香水对流感病毒FM1感染小鼠肺炎肺指数的影响(
Figure PCTCN2021090137-appb-000036
n＝14)
Figure PCTCN2021090137-appb-000037
注：与正常对照组比较， ##P<0.01；与模型对照组比较，*P<0.05，**P<0.01。
与正常对照组比较，模型对照组小鼠肺组织匀浆中的IL-8和TNF-α的含量显著升高(P<0.01)；与模型对照组比较，芳香水各剂量组小鼠肺组织匀浆中的TNF-α的含量显著降低(P<0.05或P<0.01)，芳香水中剂量组和高剂量组小鼠肺组织匀浆中的IL-8的含量显著 降低(P<0.05或P<0.01)，结果见表23。
表23芳香水对流感病毒FM1感染小鼠肺炎IL-8和TNF-α的影响(
Figure PCTCN2021090137-appb-000038
n＝14)
Figure PCTCN2021090137-appb-000039
注：与正常对照组比较， ##P<0.01；与模型对照组比较，*P<0.05，**P<0.01。
综上，芳香水可以明显改善H1N1流感所致的病毒性肺炎。
3对肺炎双球菌感染小鼠的保护作用
3.1实验方法
ICR小鼠，雌雄各半，按照体重分层随机分为6组，每组20只，分别为正常对照组、模型对照组、双黄连20g/kg组、芳香水低剂量(12mL/kg)组、芳香水中剂量(24mL/kg)组和芳香水高剂量(40mL/kg)组。芳香水各剂量组小鼠每日给药两次，每次给药间隔6h，每次给药剂量为总剂量的一半，分别为6mL/kg、12mL/kg和20mL/kg，连续7d，正常对照组和模型对照组灌胃等量的蒸馏水。
3.2指标检测
第4天给药后1h，除正常对照组外，其余各组腹腔注射肺炎双球菌菌液0.5mL/只，继续给药3d，观察各组小鼠7日内死亡情况。
3.3实验结果
与正常对照组比较，肺炎双球菌感染的小鼠7日内死亡明显增加，存活率为20％(P<0.01)；与模型对照组比较，芳香水中剂量和高剂量组小鼠7日内死亡数明显降低(P<0.05或P<0.01)。结果见表24。
表24芳香水对肺炎双球菌感染小鼠生存率的影响(n＝20)
Figure PCTCN2021090137-appb-000040
Figure PCTCN2021090137-appb-000041
注：与正常对照组比较， ##P<0.01；与模型对照组比较，*P<0.05，**P<0.01。
由上表可知，芳香水可以明显提高肺炎双球菌感染小鼠的存活率，具有明显的抑菌作用。
实施例7
一种药用组合物，原料包括广藿香885g、苍术1869g、香薷1064g、艾叶2174g、丁香46g、薄荷532g、三氯蔗糖35g、苯甲酸钠35g。
称取广藿香、苍术、香薷、艾叶、丁香、薄荷，切段，混合，加入8倍质量的水浸泡过夜后，采用水蒸气蒸馏法蒸馏，收集馏出液，加入三氯蔗糖和苯甲酸钠，加热(不超过60℃)搅拌均匀；按照每瓶20mL的规格进行灌装，封口；灌装后采用湿热灭菌法121℃灭菌30min，即得露剂。
实施例8
按照实施例1方法制备药用挥发油，使用无水乙醇稀释药用挥发油调整总量至1000mL，搅匀，滤过，按照20mL/瓶的规格灌装如耐压铝罐中，压盖，充入一定量的氮气，使铝罐内压力为0.8Mpa，即得气雾剂。
实施例9
一种抗菌抗病毒药用组合物，原料包括广藿香960g、苍术1920g、香薷1200g、艾叶2160g、丁香240g、薄荷720g、天然冰片10g。
使用上述药用组合物制备药用挥发油，制备方法同实施例1。
药用挥发油滴丸的制备方法如下：
A.将乳化剂、玉米蛋白、葵花磷脂混合于上述制备的药用挥发油中，使其在药用挥发油中的质量分数分别为1％，1.5％，0.5％；
B.将辛烯基琥珀酸淀粉钠溶解到水中，配成质量分数20％的水溶液；
C、将A和B配置好的两种溶液以1:2重量比混合，使用12000r/min的超高速搅拌机搅拌乳化，并使用高压均质机均质3次；
D、对C中获得的均质溶液进行喷雾干燥，得到挥发油微囊粉；
E、按照挥发油微囊粉与滴丸基质质量比1:2的比例制备成滴丸，滴丸基质材料为聚乙二醇4000：聚乙二醇6000＝1:1。
每10丸重0.32g，每丸约含药用挥发油7mg。用于新冠肺炎的轻型、普通型、恢复期及感冒或流感引起的鼻塞、流涕、咳嗽和咽喉肿痛等治疗，一次14丸，早中晚各1次。
对本实施例药用挥发油滴丸进行药效实验，作为实验组，实验中小鼠剂量为1.10g/kg，大鼠剂量为0.56g/kg，豚鼠剂量为0.48g/kg。并设置对照组1、对照组2、对照组3。
其中，对照组1按照实验组方法制备滴丸，并且将艾叶替换为麻黄，薄荷替换为白芷，未添加丁香、天然冰片。
对照组2按照实验组方法制备滴丸，并且将广藿香替换为板蓝根，将苍术替换为防风。
对照组3按照实验组方法制备滴丸，并且将香薷替换为栀子，将艾叶替换为陈皮。
1小鼠浓氨水引咳实验(止咳作用)
1.1实验方法
ICR小鼠，雌雄各半。按照体重分层随机分为7组，每组14只，分别为：正常对照组、模型对照组、枸橼酸喷托维林20mg/kg组、药用挥发油滴丸组、对照组1、对照组2和对照组3，小鼠按20mL/kg灌胃给药，各给药组将滴丸研成粉末，使用0.5％CMC-Na混悬成相应浓度，每天1次，连续7d。正常对照组和模型对照组灌胃等量0.5％CMC-Na溶液。
1.2指标测定
末次给药后1h，将小鼠放入500mL透明密闭容器中，用超声雾化器向容器内均匀喷入雾化浓氨水，连续喷雾20s；以小鼠收缩腹部并张口作为咳嗽标准，记录小鼠咳嗽潜伏期和放入容器后2min内的咳嗽次数。
1.3实验结果
与模型对照组比较，药用挥发油滴丸组、对照组1组、对照组2组和对照组3组的小鼠咳嗽潜伏期均显著延长、小鼠咳嗽次数均显著减少(P<0.01)，与药用挥发油滴丸组比较，对照组1组、对照组2组和对照组3组的小鼠咳嗽潜伏期均显著降低、咳嗽次数均显著增加(P<0.05)。结果见表25。
表25药用挥发油滴丸及对照组对浓氨水致小鼠咳嗽的影响(
Figure PCTCN2021090137-appb-000042
n＝14)
Figure PCTCN2021090137-appb-000043
注：与模型对照组比较， *P<0.05， **P<0.01；与药用挥发油滴丸组比较， #P<0.05。
在本实验条件及所设计的剂量下，药用挥发油滴丸组、对照组1组、对照组2组和对照组3组具有明显的止咳作用，但药用挥发油滴丸组的止咳作用明显优于对照组1组、对照组2组和对照组3组。
2对乙酰胆碱合用磷酸组胺致哮喘豚鼠的影响(平喘作用)
2.1实验方法
普通级豚鼠，雌雄各半，将豚鼠逐只放入玻璃罩(容量4L)中，用超声雾化器喷入0.1％磷酸组胺和2％氯化乙酰胆碱等容积混合液15s；逐个记录豚鼠药前哮喘潜伏期(从喷雾开始至抽搐的时间)；哮喘潜伏期未超过120s者为合格豚鼠，超过者认为不敏感，弃除。
将筛选合格的豚鼠按照药前哮喘潜伏期分层随机分为6组，每组12只，分别为：模型对照组、氨茶碱60mg/kg组、药用挥发油滴丸组、对照组1、对照组2和对照组3，豚鼠按4mL/kg灌胃给药，各给药组将滴丸研成粉末，使用0.5％CMC-Na混悬成相应浓度，每天1次，连续7d。模型对照组灌胃等量的0.5％CMC-Na溶液。
2.2指标检测
末次给药后1h，同法记录豚鼠的药后哮喘潜伏期；若豚鼠在3min内仍未出现哮喘症状，则以180s计算。
2.3实验结果
与模型对照组比较，药用挥发油滴丸组、对照组1、对照组2和对照组3的药后哮喘潜伏期及药后哮喘潜伏期的延长值(药后潜伏期-药前潜伏期)均显著升高(P<0.01)，与药用挥发油滴丸组比较，对照组1、对照组2和对照组3的药后哮喘潜伏期及药后哮喘潜伏期的延长值(药后潜伏期-药前潜伏期)均显著降低(P<0.05)。结果见表26。
表26药用挥发油滴丸及对照组对乙酰胆碱和磷酸组胺致豚鼠哮喘潜伏期的影响(
Figure PCTCN2021090137-appb-000044
n＝12)
Figure PCTCN2021090137-appb-000045
注：与模型对照组比较， **P<0.01；与药用挥发油滴丸组比较， #P<0.05。
在本实验条件及所设计的剂量下，药用挥发油滴丸组、对照组1组、对照组2组和对照组3组具有明显的平喘作用，但药用挥发油滴丸组的平喘作用明显优于对照组1组、对照组 2组和对照组3组。
3对大鼠毛细管排痰量的影响(化痰作用)
3.1实验方法
SD大鼠，雌雄各半，按照体重分层随机分为6组，每组12只，分别为模型对照组、羧甲司坦0.2g/kg组、药用挥发油滴丸组、对照组1、对照组2和对照组3，大鼠按10mL/kg灌胃给药，各给药组将滴丸研成粉末，使用0.5％CMC-Na混悬成相应浓度，每天1次，连续7d。模型对照组灌胃等量的0.5％CMC-Na溶液。
3.2指标检测
末次给药后40min，麻醉，仰位固定，剪开颈中部皮肤，分离出气管，在甲状腺软骨下缘正中两软骨环之间用尖锐的注射针头扎一小孔，插入一根玻璃毛细管，使毛细管刚好接触气管底部表面，气管中痰液即被吸入毛细管，当毛细管被痰液充满后，立即换一根同直径的毛细管，连续收集2h，刻度尺测量毛细管内液柱长度，评价排痰量。
3.3实验结果
与模型对照组比较，药用挥发油滴丸组、对照组1、对照组2和对照组3的大鼠排痰量均显著增加(P<0.05或P<0.01)，与药用挥发油滴丸组比较，对照组1组、对照组2组和对照组3组的大鼠排痰量均显著降低(P<0.05)。结果见表27。
表27药用挥发油滴丸和对照组对大鼠毛细管排痰量的影响(
Figure PCTCN2021090137-appb-000046
n＝12)
Figure PCTCN2021090137-appb-000047
注：与模型对照组比较， *P<0.05， **P<0.01；与药用挥发油滴丸组比较， #P<0.05。
在本实验条件及所设计的剂量下，药用挥发油滴丸组、对照组1组、对照组2组和对照组3组具有明显的化痰作用，但药用挥发油滴丸组的化痰作用明显优于对照组1组、对照组2组和对照组3组。
4对肺炎双球菌感染小鼠的保护作用(抑菌作用)
4.1实验方法
ICR小鼠，雌雄各半，按照体重分层随机分为7组，每组20只，分别为正常对照组、模 型对照组、双黄连20g/kg组、药用挥发油滴丸组、对照组1、对照组2和对照组3，小鼠按20mL/kg灌胃给药，各给药组将滴丸研成粉末，使用0.5％CMC-Na混悬成相应浓度，每天1次，连续7d。模型对照组灌胃等量的0.5％CMC-Na溶液。
4.2指标检测
第4天给药后1h，除正常对照组外，其余各组腹腔注射肺炎双球菌菌液0.5mL/只，继续给药3d，观察各组小鼠7日内死亡情况。
4.3实验结果
与正常对照组比较，肺炎双球菌感染的小鼠7日内死亡明显增加，存活率为20％(P<0.01)；与模型对照组比较，药用挥发油滴丸组、对照组1、对照组2和对照组3组小鼠7日内死亡数明显降低(P<0.05)。与药用挥发油滴丸组比较，对照组1、对照组2和对照组3小鼠7日内死亡数有所增加，但无统计学差异(P>0.05)。结果见表28。
表28药用挥发油滴丸和对照组对肺炎双球菌感染小鼠生存率的影响(n＝20)
Figure PCTCN2021090137-appb-000048
注：与正常对照组比较， ##P<0.01；与模型对照组比较， *P<0.05。
在本实验条件及所设计的剂量下，药用挥发油滴丸组、对照组1、对照组2和对照组3组均可以明显提高肺炎双球菌感染小鼠的存活率，具有明显地抑菌作用。药用挥发油滴丸组的抑菌作用略好于对照组1、对照组2和对照组3组。
5对流感病毒致小鼠死亡的保护作用对比研究
5.1实验方法
流感病毒亚洲甲型鼠肺适应株A/FM/1/34(H1N1)(FM1)，使用前接种于9日龄鸡胚尿囊腔，常规培养，48h后新鲜无菌收集尿囊液，传代2次，测定对ICR小鼠的半数致死量(LD 50)。ICR小鼠，雌雄各半，按体重分层随机分为6组，每组24只，分别为正常对照组、模型对照组、利巴韦林100mg/kg组、药用挥发油滴丸1.10g/kg组、市售滴丸1.73g/kg组和市售喷雾20mL/kg组(最大给药量)。小鼠以乙醚轻度麻醉后，每只小鼠以25μL病毒液滴鼻，感染量为25倍LD 50，正常对照组用等量无菌生理盐水滴鼻。药用挥发油滴丸、市售滴丸及 市售喷雾组小鼠按20mL/kg灌胃给药，各给药组将滴丸研成粉末，使用0.5％CMC-Na混悬成相应浓度，利巴韦林组腹腔注射给药，每天1次，连续7d，正常对照组和模型对照组灌胃等量的0.5％CMC-Na溶液。
其中，市售滴丸中药用成分为：胆酸、珍珠母、猪去氧胆酸、栀子、水牛角、板蓝根、黄芩苷、金银花；市售喷雾中药用成分为艾纳香油，大果木姜子油，薄荷脑。
5.2指标检测
逐日观察小鼠的发病症状，记录死亡时间、死亡数以及体重，连续观察14d。第14d不死亡的，按14d计算，感染24h内死亡的视为非感染死亡。
10.3实验结果
正常对照组小鼠精神良好，行动敏捷，皮毛顺滑光亮，呼吸频率正常，进食及饮水量均正常，体重逐日增加。流感病毒感染小鼠，在感染24h后，出现甩头、呼吸频率加快急促，腹式呼吸为主，行动迟缓、卷缩、耸毛，饮食量和饮水量均明显减少。从第4d开始，小鼠开始出现死亡，第7d达到高峰，各给药治疗组小鼠状态均明显好于模型组。
正常对照组小鼠无死亡，模型对照组小鼠生存率仅为6.2％；与模型对照组比较，药用挥发油滴丸、市售滴丸和市售喷雾组的小鼠生存率分别为62.5％、41.7％和37.5％，生存率均明显升高(P<0.05或P<0.01)；与药用挥发油滴丸组比较，市售滴丸组和市售喷雾组小鼠生存率明显减少(P<0.05或P<0.01)。结果见表29。
表29药用挥发油滴丸、清开灵和金喉健对流感病毒FM1感染小鼠生存率的影响(n＝24)
Figure PCTCN2021090137-appb-000049
注：与正常对照组比较， ##P<0.01；与模型对照组比较， *P<0.05， **P<0.01；与药用挥发油滴丸组比较， ΔP<0.05， ΔΔP<0.01。
在本实验条件及所设计的剂量下，药用挥发油滴丸、市售滴丸和市售喷雾均可以明显提高流感病毒FM1感染小鼠的存活率，且药用挥发油滴丸的抗病毒作用明显优于市售滴丸和市售喷雾。
6对流感病毒感染小鼠病毒性肺炎的对比研究
6.1实验方法
流感病毒亚洲甲型鼠肺适应株A/FM/1/34(H1N1)(FM1)，使用前接种于9日龄鸡胚 尿囊腔，常规培养，48h后新鲜无菌收集尿囊液，传代2次，测定对ICR小鼠的半数致死量(LD 50)。ICR小鼠，雌雄各半，按体重分层随机分为6组，每组14只，分别为正常对照组、模型对照组、达菲27.5mg/kg组、药用挥发油滴丸1.10g/kg组、市售滴丸1.73g/kg组和市售喷雾20mL/kg组(最大给药量)。小鼠以乙醚轻度麻醉后，每只小鼠以20μl病毒液滴鼻，感染量为25倍LD 50，正常对照组用等量无菌生理盐水滴鼻。药用挥发油滴丸、市售滴丸及市售喷雾组小鼠按20mL/kg灌胃给药，利巴韦林组腹腔注射给药，每天1次，连续7d，正常对照组和模型对照组灌胃等量的0.5％CMC-Na溶液。
6.2指标检测
末次给药后1h，称取小鼠体重后处死，解剖摘取全肺称重，计算肺指数值和肺指数抑制率。将肺组织用生理盐水制备成10％肺组织匀浆，将制备好的匀浆液低温离心，采用ELISA法测定肺组织中的IL-8和TNF-α的含量。
肺指数＝[肺重量(g)/体重(g)]×100；
肺指数抑制率＝(模型对照组肺指数均值-试验组肺指数均值)/模型对照组肺指数均值。
6.3实验结果
与正常对照组比较，模型对照组小鼠的肺指数显著升高(P<0.01)；与模型对照组比较，药用挥发油滴丸组、市售滴丸组和市售喷雾组小鼠的肺指数均显著降低(P<0.05或P<0.01)；与药用挥发油滴丸组比较，市售滴丸组和市售喷雾组小鼠的肺指数显著升高(P<0.05)。结果见表30。
表30药用挥发油滴丸、清开灵和金喉健对小鼠肺炎肺指数的影响(
Figure PCTCN2021090137-appb-000050
n＝14)
Figure PCTCN2021090137-appb-000051
注：与正常对照组比较， ##P<0.01；与模型对照组比较， *P<0.05， **P<0.01；与药用挥发油滴丸组比较， ΔP<0.05。
与正常对照组比较，模型对照组小鼠肺组织匀浆中的IL-8和TNF-α的含量显著升高(P<0.01)；与模型对照组比较，药用挥发油滴丸、市售滴丸和市售喷雾组小鼠肺组织匀浆中的IL-8和TNF-α的含量显著降低(P<0.05或P<0.01)；与药用挥发油滴丸组比较，市售喷雾组小鼠肺组织匀浆中的IL-8和TNF-α的含量显著升高(P<0.05)。结果见表31。
表31药用挥发油滴丸、清开灵和金喉健对小鼠肺炎IL-8和TNF-α的影响(
Figure PCTCN2021090137-appb-000052
n＝14)
Figure PCTCN2021090137-appb-000053
注：与正常对照组比较， ##P<0.01；与模型对照组比较， *P<0.05， **P<0.01；与药用挥发油滴丸组比较， ΔP<0.05。
在本实验条件及所设计的剂量下，药用挥发油滴丸、市售滴丸和市售喷雾均可以明显改善H1N1流感所致的病毒性肺炎，且药用挥发油滴丸的抗病毒作用明显优于市售滴丸和市售喷雾。
实施例10
药用挥发油软胶囊制备方法如下：
(1)药用挥发油制备
按照实施例1方法分别制备广藿香挥发油、苍术挥发油、香薷挥发油、艾叶挥发油、丁香挥发油和薄荷挥发油。称取广藿香挥发油20g、苍术挥发油20g、香薷挥发油10g、艾叶挥发油10g、丁香挥发油5g、薄荷挥发油5g混合均匀后得到混合挥发油备用。
称取天然冰片10g置于适量容积器皿中，加入200ml中链甘油三酸酯，搅拌并适当加热(不高于60℃)加速天然冰片的溶解，待天然冰片完全溶解后备用。
将混合挥发油加入到天然冰片的中链甘油三酸酯溶液中，搅拌，再加入中链甘油三酸酯补足到1000mL，混合均匀，备用。
(2)囊材制备
将明胶：甘油：纯化水按质量比1:1:0.5的比例称取置于化胶桶中，于70℃下融化混合，真空脱气后备用。
(3)软胶囊制备
使用YWJ100-IP软胶囊生产线进行软降囊的制备，调节软胶囊机参数，使每粒软降囊的内容物为0.3g，即得药用挥发油软胶囊。
实施例11
药用挥发油滴鼻剂制备方法如下：
药用组合物原料配比同实施例1，按照实施例1方法制备广藿香挥发油、苍术挥发油、香薷挥发油、艾叶挥发油、丁香挥发油、薄荷挥发油，将其混合后得到混合挥发油。取上述混合挥发油7g，加入天然冰片1g，搅拌并适当加热(不高于60℃)使天然冰片完全溶解，得到药用挥发油，作为溶液A。
配置10％的乙基纤维素无水乙醇溶液100mL，作为溶液B。
将溶液A和溶液B混合，加入100mL中链甘油三酯，搅拌混合30min，减压干燥去除无水乙醇，得到浅黄色至黄色油状凝胶。
实施例12
药用挥发油雾化剂制备方法如下：
药用组合物原料配比同实施例1，按照实施例1方法制备广藿香挥发油、苍术挥发油、香薷挥发油、艾叶挥发油、丁香挥发油、薄荷挥发油，将其混合后得到混合挥发油。
取上述混合挥发油70g，加入天然冰片10g，适当加热(不高于60℃)搅拌使天然冰片完全溶解，得到药用挥发油。
使用中链甘油三酯稀释药用挥发油调整总量至1000mL，搅匀，滤过，获得浅黄色至黄色澄明油状溶液，气芳香，微苦，相对密度(药典通则0601)0.933～0.961；灌装，每瓶20mL，使用时将药液倒入雾化器中，雾化吸入使用。
上述所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。

Claims (11)

  1. 一种抗菌抗病毒药用组合物，其特征在于，以重量份数计，包括如下原料：
    广藿香4-14份、苍术9-28份、香薷5-18份、艾叶11-33份、丁香0.1-3份和薄荷2-10份。
  2. 根据权利要求1所述的一种抗菌抗病毒药用组合物，其特征在于，以重量份数计，还包括天然冰片0.01-0.2份。
  3. 根据权利要求1或2所述的一种抗菌抗病毒药用组合物，其特征在于，
    还包括药用辅料。
  4. 一种抗菌抗病毒药用挥发油，其特征在于，包括如下步骤：
    1)将权利要求2中广藿香、苍术、香薷、艾叶、丁香、薄荷分别加水蒸馏提取，得到各原料挥发油，将各原料挥发油混合得到混合挥发油；
    或，将权利要求2中广藿香、苍术、香薷、艾叶、丁香、薄荷混合，加水蒸馏提取得到总挥发油；
    或，将权利要求2中广藿香、苍术、香薷、艾叶、丁香、薄荷混合，置于超临界二氧化碳萃取装置中，通入液态二氧化碳萃取挥发油，进行超临界萃取，得到总挥发油；
    2)向混合挥发油或总挥发油中加入天然冰片，加热使天然冰片溶解，得到药用挥发油；
    或，将天然冰片加入到50-60℃溶剂中搅拌溶解，冷却至室温，再加入混合挥发油或总挥发油中，得到药用挥发油。
  5. 根据权利要求4所述的一种抗菌抗病毒药用挥发油，其特征在于，
    各原料挥发油制备方法如下：
    广藿香加5-8倍重量的水，浸泡过夜后水蒸气蒸馏法提取4-8h；
    苍术加5-8倍重量的水，浸泡20-60min后水蒸气蒸馏法提取4-6h；
    香薷加5-8倍重量的水，浸泡1-3h后水蒸气蒸馏法提取2-4h；
    艾叶加6-10倍重量的水，浸泡4-8h后水蒸气蒸馏法提取4-6h；
    丁香加5-8倍重量的水，浸泡过夜后水蒸气蒸馏法提取4-8h；
    薄荷加5-8倍重量的水，浸泡1-3h后水蒸气蒸馏法提取4-6h；
    总挥发油制备方法如下：
    广藿香、苍术、香薷、艾叶、丁香、薄荷混合后加入5-10倍重量的水，浸泡1-8h后水蒸气蒸馏法提取2-8h；或，广藿香、苍术、香薷、艾叶、丁香、薄荷混合，置于超临界二氧化碳萃取装置中，通入液态二氧化碳萃取挥发油，萃取压力20-40Mpa、萃取温度40-70℃、分离温度30-60℃，萃取时间0.5-10h。
  6. 一种抗菌抗病毒药用芳香水，其特征在于，
    将权利要求1中广藿香、苍术、香薷、艾叶、丁香、薄荷混合，加入5-12倍重量的水，浸泡过夜后蒸馏，得到药用芳香水。
  7. 权利要求1、2或3所述的药用组合物或权利要求4或5所述的药用挥发油或权利要求6所述的药用芳香水在制备抗菌、抗病毒、清热退烧和/或治疗呼吸系统疾病的药物中的应用。
  8. 根据权利要求7所述的应用，其特征在于，
    所述药物的剂型包括喷雾剂、露剂、气雾剂、滴丸、软胶囊、滴鼻剂或雾化剂。
  9. 根据权利要求8所述的应用，其特征在于，
    所述喷雾剂的制备方法如下：
    药用挥发油用稀释剂稀释后，搅拌均匀，过滤，得到喷雾剂；
    或，将天然冰片与药用辅料加入到50-60℃溶剂中搅拌溶解，冷却至室温，再加入混合挥发油或总挥发油，用稀释剂稀释后，搅拌均匀，过滤，得到喷雾剂；
    所述露剂的制备方法如下：
    将药用辅料加入到药用芳香水中搅拌均匀，得到露剂；
    所述气雾剂的制备方法如下：
    药用挥发油用稀释剂稀释后，搅拌均匀，过滤，灌装到耐压容器中，压盖，充入抛射剂，得到气雾剂；
    或，将天然冰片与药用辅料加入到50-60℃溶剂中搅拌溶解，冷却至室温，再加入混合挥发油或总挥发油，用稀释剂稀释后，搅拌均匀，过滤，灌装到耐压容器中，压盖，充入抛射剂，得到气雾剂；
    所述滴丸的制备方法如下：
    使用微囊包裹技术将药用挥发油制成挥发油微囊粉，与滴丸基质混合，制得滴丸；
    所述软胶囊的制备方法如下：
    使用软胶囊囊材包裹药用挥发油，制得软胶囊；
    所述滴鼻剂的制备方法如下：
    将药用挥发油溶于凝胶基质材料的无水乙醇溶液中，加入到稀释剂，搅拌均匀，减压干燥去除无水乙醇，制得油凝胶滴鼻剂；
    所述雾化剂的制备方法如下：
    将药用挥发油用稀释剂稀释后，搅拌均匀，过滤，灌装。
  10. 根据权利要求9所述的应用，其特征在于，
    所述溶剂为中链甘油三酯、1-98％酒精、无水乙醇、丙二醇、甘油或食用油中的任意一种；
    所述稀释剂为中链甘油三酯、1-98％酒精、无水乙醇、丙二醇、甘油或食用油中的任意一种；
    所述抛射剂为氮气、二氧化碳、七氟丙烷、四氟乙烷、1,3,3,3-四氟丙烯、2,3,3,3-四氟丙烯或压缩空气。
  11. 权利要求1、2或3所述的药用组合物或权利要求4或5所述的药用挥发油或权利要求6所述的药用芳香水在制备抗SARS-CoV-2药物中的应用。
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