Application of a Chinese medicine composition in medicines for resisting coronavirus, protecting organs and improving immunity

Abstract

The present invention discloses an application of a traditional Chinese medicine composition in the preparation of a drug for preventing and treating pneumonia caused by the novel coronavirus infection. The traditional Chinese medicine composition of the present invention is mainly composed of forsythia, honeysuckle, isatis root, rhubarb, patchouli, cyperus rotundus, rhodiola rosea and the like, and exerts the overall regulatory advantages of the compound traditional Chinese medicine, organically combines the elimination of pathogenic factors, relief of symptoms and immune regulation to achieve multi-target treatment. Clinical experiments have confirmed that the traditional Chinese medicine composition has a significant inhibitory effect on the prevention and treatment of wild-type and S protein D614G mutant novel coronaviruses, and has a protective effect on the heart and lungs.

Description

Technical Field

[0001] The present invention relates to the application of a traditional Chinese medicine composition in medicines for resisting novel coronavirus, protecting organs and improving immunity, and belongs to the field of traditional Chinese medicine. Background Art

[0002] The mutation of coronavirus is mainly determined by the biological and genetic characteristics of coronavirus. Through current genetic and proteomic determination, coronavirus is found to be a nucleic acid virus synthesized by RNA. The characteristic of RNA virus is that a gene chain is formed by a single-stranded nucleic acid. This chain is particularly prone to mutation. After mutation, its protein phenotype will change, just like many animals have close relatives, and many viruses have many different subtypes. For example, influenza has H5N1, H5N7, H7N9, etc. This is because the protein phenotype has changed after the nucleic acid mutation. Therefore, coronavirus mutation has been found. In the future, there may be a long way to go to prevent and control the new coronavirus, and it will be very difficult. Corona Virus Disease 2019 (COVID-19), referred to as "COVID-19", is characterized by fever, fatigue, and dry cough as the main clinical manifestations of patients. Upper respiratory tract symptoms such as nasal congestion and runny nose are rare. Hypoxia and hypoxia may occur. About half of the patients experience dyspnea after one week. Severe cases rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis that is difficult to correct, and coagulation dysfunction. It is worth noting that severe and critical patients may have a low or moderate fever or even no obvious fever during the course of the disease. Some patients have mild onset symptoms and may not have fever. Most patients recover after one week. Most patients have a good prognosis, while a few patients are in critical condition or even die. Since 2019, there have been more than 9 million confirmed cases and more than 400,000 deaths worldwide, posing a huge threat to human life, health, and economic development.

[0003] Coronavirus is one of the main pathogens of the common cold in adults. It can cause upper respiratory tract infections in children, but rarely affects the lower respiratory tract. The incubation period of coronavirus infection is generally 2 to 5 days, with an average of 3 days. Typical coronavirus infection presents cold symptoms such as runny nose and discomfort. There are 7 known coronaviruses that infect humans, of which four are more common in the population (HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1), and the other three are MERS virus (Middle East Respiratory Syndrome virus MERS-CoV), SARS virus (Severe Acute Respiratory Syndrome virus SARS-CoV) and 2019 novel coronavirus (2019-nCoV). HCoV-229E virus is a type of coronavirus. In 1965, medical experts used human embryo tracheal culture method to isolate a virus from the nasal mucus of patients with common cold, named B814 virus. Subsequently, researchers isolated similar viruses using human embryo kidney cells, and the representative strain was named 229E virus. CoV-229E infection is distributed globally and the population is generally susceptible. Several studies have shown that almost all children have been infected with HCoV-229E in their early childhood. The virus can cause common colds and upper respiratory tract infections.

[0004] The symptoms of coronavirus infection vary depending on the type. Common signs include respiratory symptoms, fever, cough, shortness of breath and difficulty breathing. In more serious cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death, and can cause certain damage to the heart and lungs.

[0005] This disease belongs to the category of "plague" in traditional Chinese medicine. The main pathogenic factor is the evil of epidemic toxins. Entering the interior and transforming into heat is an important pathogenesis and outcome. This is consistent with the clinical characteristics of pneumonia infected by the new coronavirus, which is mainly manifested by fever. The Chinese medicine composition of the present invention is mainly composed of Forsythia suspensa, honeysuckle, Isatis root, rhubarb, patchouli, Corydalis cyrtonema, Rhodiola rosea, etc. It is an innovative Chinese medicine composition developed by applying the theory of network disease in traditional Chinese medicine to reveal the law of transmission and change of respiratory infectious diseases caused by viruses, and taking "clearing plague, detoxifying, promoting lung function and relieving heat" as the treatment method. The whole prescription is based on the Ma Xing Shi Gan Tang in Zhang Zhongjing's Treatise on Febrile Diseases in the Han Dynasty and Yinqiao Powder in Wu Jutong's Treatise on Warm Diseases in the Qing Dynasty. It also draws on the experience of using rhubarb to treat epidemic symptoms in Wu Youke's Treatise on Warm Epidemics in the Ming Dynasty, and is combined with Rhodiola rosea to clear the lungs and remove blood stasis, regulate immunity, and the prescription reflects the experience of Chinese medicine in preventing and treating such epidemic diseases for two thousand years. In particular, the prescription and preparation method are adjusted for the pathogenesis and symptoms of pneumonia infected by the new coronavirus, and it is clinically applicable to respiratory infectious diseases accompanied by fever, chills, cough, muscle aches, etc. The present invention is an improved invention based on the patent ZL03143211, and the contents recorded in the patent document are quoted in full here. The above patent does not disclose the use of the Chinese medicine composition in the preparation of drugs against the new coronavirus, protecting organs, and improving immunity. Summary of the invention

[0006] The present invention provides an application of a traditional Chinese medicine composition in preparing a drug for preventing or treating coronavirus, wherein the coronavirus is a wild-type novel coronavirus and / or an S protein mutant novel coronavirus, and the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight:

[0007] Forsythia suspensa 150-260 Honeysuckle 150-260 Isatis root 150-260 Rhubarb 30-55

[0008] Patchouli 50-90, Cyperus rotundus 150-260, Rhodiola rosea 50-90, Menthol 5-8

[0009] Roasted Ephedra 50-90 Stir-fried Bitter Almond 50-90 Houttuynia Cordata 150-260

[0010] Licorice 50-90, Gypsum 150-260.

[0011] The weight ratio of the raw materials of the Chinese medicine composition of the present invention is preferably:

[0012] Forsythia suspensa 150 Honeysuckle 260 Isatis root 150 Rhubarb 55 Patchouli 50

[0013] Mianma Guanzhong 260 Rhodiola rosea 50 Menthol 8 Roasted Ephedra 50

[0014] Fried bitter almonds 90 grams, Houttuynia cordata 150 grams, licorice 90 grams, gypsum 150 grams.

[0015] The weight ratio of the raw materials of the Chinese medicine composition of the present invention is also preferably:

[0016] Forsythia suspensa 260 Honeysuckle 150 Isatis root 260 Rhubarb 30 Patchouli 90

[0017] Mianma Guanzhong 150 Rhodiola rosea 90 Menthol 5 Roasted Ephedra 90

[0018] Fried bitter almonds 50, Houttuynia cordata 260, licorice 50, gypsum 260.

[0019] The weight ratio of the raw materials of the Chinese medicine composition of the present invention is also preferably:

[0020] Forsythia suspensa 170 Honeysuckle 170 Isatis root 170 Rhubarb 34 Patchouli 57

[0021] Mianma Guanzhong 170 Rhodiola rosea 57 Menthol 5 Roasted Ephedra 57

[0022] Fried bitter almonds 57, Houttuynia cordata 170, licorice 57, gypsum 170.

[0023] The weight ratio of the raw materials of the Chinese medicine composition of the present invention is also preferably:

[0024] Forsythia suspensa 255 Honeysuckle 255 Isatis root 255 Rhubarb 51 Patchouli 85

[0025] Mianma Guanzhong 255 Rhodiola rosea 85 Menthol 7.5 Roasted Ephedra 85

[0026] Fried bitter almonds 85, Houttuynia cordata 255, licorice 85, gypsum 255.

[0027] The Chinese medicine of the present invention can be replaced by Chinese medicines with the same or similar effects, and these medicinal materials can be processed according to the "National Chinese Medicine Processing Standards" or the "Chinese Medicine Dictionary".

[0028] The active ingredients of the Chinese medicine composition of the present invention are prepared by the following steps:

[0029] (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them;

[0030] (2) Crush patchouli, add 5-8 times the amount of water to extract the volatile oil, extract the oil for 4 hours, collect the volatile oil and set aside; after filtering the extract, discard the residue and set aside the filtrate;

[0031] (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb were extracted twice with 6-10 times the amount of 50-90% ethanol, each time for 1-3 hours. The extracts were combined and filtered, and the ethanol was recovered and the filtrate was used for later use;

[0032] (4) Add 7-11 times the amount of water to honeysuckle, gypsum, isatis root, cyperus rotundus, liquorice, and rhodiola rosea, boil until boiling, add fried bitter almonds, boil twice, each time for 0.5-2.5 hours, combine the extracts and filter, combine the filtrate obtained with the filtrate after patchouli oil extraction in step (2), and concentrate into a clear paste with a relative density of 1.10-1.15 measured at 60° C., add ethanol, adjust the alcohol concentration to 70%, refrigerate, filter, and recover the ethanol until there is no alcohol taste, and obtain a clear paste for use;

[0033] (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating to a clear paste having a relative density of 1.15-1.20 measured at 60° C., drying to obtain a dry paste powder for later use;

[0034] The dry paste powder obtained in step (5), the volatile oil obtained in step (2) and menthol together constitute the active ingredients of the traditional Chinese medicine composition.

[0035] The dosage form of the medicine of the present invention is capsule, tablet, powder, granule, oral liquid, soft capsule, pill, tincture, syrup, suppository, gel, spray or injection.

[0036] In order to realize the above dosage forms, pharmaceutically acceptable excipients need to be added when preparing these dosage forms, such as fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases, etc. Fillers include starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants include magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include sodium saccharin, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, etc.; flavoring agents include sweeteners and various flavors; preservatives include parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, eucalyptus oil, etc.; matrices include PEG6000, PEG4000, insect wax, etc.

[0037] The capsule is prepared by the following steps:

[0038] (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them;

[0039] (2) Crush patchouli, add 5-8 times the amount of water to extract the volatile oil, extract the oil for 4 hours, collect the volatile oil and set aside; after filtering the extract, discard the residue and set aside the filtrate;

[0040] (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb were extracted twice with 6-10 times the amount of 50-90% ethanol, each time for 1-3 hours. The extracts were combined and filtered, and the ethanol was recovered and the filtrate was used for later use;

[0041] (4) Add 7-11 times the amount of water to honeysuckle, gypsum, isatis root, cyperus rotundus, liquorice, and rhodiola rosea, boil until boiling, add fried bitter almonds, boil twice, each time for 0.5-2.5 hours, combine the extracts and filter, combine the filtrate obtained with the filtrate after patchouli oil extraction in step (2), and concentrate into a clear paste with a relative density of 1.10-1.15 measured at 60° C., add ethanol, adjust the alcohol concentration to 70%, refrigerate, filter, and recover the ethanol until there is no alcohol taste, and obtain a clear paste for use;

[0042] (5) The clear paste obtained in step (4) is combined with the alcohol extract obtained in step (3), concentrated to a clear paste having a relative density of 1.15-1.20 measured at 60° C., dried to obtain a dry paste powder for later use;

[0043] (6) adding appropriate pharmaceutically acceptable excipients to the dry paste powder obtained in step (5) for granulation;

[0044] (7) Add ethanol to dissolve menthol and the volatile oil obtained in step (2), spray the granules obtained in step (6), seal, mix well, and put into capsules to obtain the product.

[0045] The preparation method of the granules is prepared by the following steps:

[0046] (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials, clean and select, and break them into pieces as appropriate;

[0047] (2) Crush patchouli, add 5-8 times the amount of water to extract the volatile oil, extract the oil for 4 hours, collect the volatile oil and set aside; after filtering the extract, discard the residue and set aside the filtrate;

[0048] (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb were extracted twice with 6-10 times the amount of 50-90% ethanol, each time for 1-3 hours. The extracts were combined and filtered, and the ethanol was recovered and the filtrate was used for later use;

[0049] (4) Add 7-11 times the amount of water to honeysuckle, gypsum, isatis root, cyperus rotundus, liquorice, and rhodiola rosea, boil until boiling, add fried bitter almonds, boil twice, each time for 0.5-2.5 hours, combine the extracts and filter, combine the filtrate obtained with the filtrate after patchouli oil extraction in step (2), concentrate into a clear paste with a relative density of 1.10-1.15 measured at 60°C, add ethanol, adjust to an alcohol concentration of 70%, refrigerate, filter, and recover ethanol until there is no alcohol taste, and obtain a clear paste for use;

[0050] (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating to a thick paste having a relative density of 1.25-1.35 measured at 60° C., and setting aside;

[0051] (6) adding appropriate pharmaceutically acceptable excipients to the thick paste obtained in step (5) for granulation;

[0052] (7) Add ethanol to dissolve menthol and the volatile oil obtained in step (2), spray the granules obtained in step (6), seal, mix well, and pack into bags.

[0053] The preferred method for preparing the granules is:

[0054] (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials, clean and select, and break them into pieces as appropriate;

[0055] (2) Chop patchouli into pieces and add 6 times the amount of water to extract the volatile oil. The extraction time is 4 hours. Collect the volatile oil and set aside. After filtering the extract, discard the residue and set aside the filtrate.

[0056] (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb were extracted twice with 8 times the amount of 70% ethanol, the first time for 2 hours and the second time for 1.5 hours. The extracts were combined and filtered, and the ethanol was recovered and the filtrate was used for later use;

[0057] (4) Add 9 times the amount of water to honeysuckle, gypsum, isatis root, cyperus rotundus, licorice, and rhodiola rosea, boil until boiling, add fried bitter almonds, and boil twice, the first time for 1.5 hours and the second time for 1 hour, combine the extracts and filter, combine the filtrate obtained with the water filtrate after patchouli oil extraction in step (2), and concentrate into a clear paste with a relative density of 1.10-1.15 measured at 60°C, add 95% ethanol, adjust the alcohol concentration to 70%, refrigerate, filter, and recover ethanol until there is no alcohol taste, to obtain a clear paste filtrate;

[0058] (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating to a thick paste having a relative density of 1.25-1.35 measured at 60° C., and setting aside;

[0059] (6) adding appropriate pharmaceutically acceptable excipients to the thick paste obtained in step (5) to granulate and shape the granules for later use;

[0060] (7) Screen the granules obtained in step (6) to obtain fine powder, add menthol and the volatile oil obtained in step (2) into ethanol to dissolve, spray the fine powder into the granules obtained in step (6), mix well, and then mix well with the granules obtained in step (6), seal for half an hour, and pack into a bag.

[0061] The preparation method of other dosage forms of the drug of the present invention is: weigh the raw materials according to the proportion, and prepare them by conventional preparation methods, for example, the preparation process recorded in Fan Biting's "Pharmaceutics of Chinese Materia Medica" (Shanghai Science Press, 1st edition, December 1997), to prepare conventional dosage forms acceptable to pharmacy.

[0062] The present invention also provides the use of the traditional Chinese medicine composition in medicines for preventing or treating respiratory diseases, wherein the respiratory diseases include pneumonia, bronchitis, fever, cough, cold and the like.

[0063] The present invention also provides the use of the traditional Chinese medicine composition in a drug for protecting heart damage, preferably heart damage caused by a virus, wherein the virus is preferably a coronavirus, and more preferably an HCoV-229E coronavirus.

[0064] The present invention also provides the use of the traditional Chinese medicine composition in a drug for protecting lung damage, more preferably lung damage caused by a virus, wherein the virus is preferably a coronavirus, more preferably an HCoV-229E coronavirus.

[0065] The invention also provides application of the Chinese medicine composition in medicine for improving human immunity. DETAILED DESCRIPTION

[0066] The following examples are used to illustrate the preparation of the drug of the present invention, but they are not intended to limit the scope of the present invention in any way.

[0067] Example 1

[0068] prescription:

[0069] Forsythia suspensa 255g Honeysuckle 255g Isatis root 255g Rhubarb 51g

[0070] Patchouli 85g Mianma Guanzhong 255g Rhodiola rosea 85g Menthol 7.5g

[0071] 85g roasted ephedra, 85g fried bitter almonds, 255g houttuynia

[0072] 85 grams of licorice and 255 grams of gypsum.

[0073] Preparation method:

[0074] (1) Weigh and select the Chinese medicinal materials according to the above prescription;

[0075] (2) Chop patchouli into pieces and add 6 times the amount of water to extract the volatile oil. The extraction time is 4 hours. Collect the volatile oil and set aside. After filtering the extract, discard the residue and set aside the filtrate.

[0076] (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb were extracted twice with 8 times the amount of 70% ethanol, the first time for 2 hours and the second time for 1.5 hours. The extracts were combined and filtered, and the ethanol was recovered and the filtrate was used for later use;

[0077] (4) Add 9 times the amount of water to honeysuckle, gypsum, isatis root, cyperus rotundus, licorice, and rhodiola rosea, boil until boiling, add fried bitter almonds, and boil twice, the first time for 1.5 hours and the second time for 1 hour, combine the extracts and filter, combine the filtrate obtained with the filtrate after patchouli oil extraction in step (2), and concentrate into a clear paste with a relative density of 1.10-1.15 measured at 60° C., add ethanol, adjust the alcohol concentration to 70%, refrigerate for 24 hours, filter, and recover the ethanol until there is no alcohol taste, and obtain a clear paste for use;

[0078] (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating to a clear paste having a relative density of 1.15-1.20 measured at 60° C., spray drying to obtain a dry paste powder for later use;

[0079] (6) Add 142 g of starch to the dry paste powder obtained in step (5) and granulate with 85% ethanol;

[0080] (7) Add ethanol to dissolve menthol and the volatile oil obtained in step (2), spray the granules obtained in step (6), seal the container, mix well, and put into 1000 capsules to obtain the product.

[0081] Example 2

[0082] prescription:

[0083] Forsythia suspensa 260g Honeysuckle 150g Isatis root 260g Rhubarb 30g

[0084] Patchouli 90g Mianma Guanzhong 150g Rhodiola rosea 90g Menthol 5g

[0085] 90g roasted ephedra, 50g fried bitter almonds, 260g houttuynia

[0086] 50 grams of licorice and 260 grams of gypsum.

[0087] Preparation method:

[0088] (1) Weigh and select the Chinese medicinal materials according to the above prescription;

[0089] (2) Chop patchouli into pieces and add 5 times the amount of water to extract the volatile oil. The extraction time is 4 hours. Collect the volatile oil and set aside. After filtering the extract, discard the residue and set aside the filtrate.

[0090] (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb were extracted twice with 6 times the amount of 50% ethanol, the first time for 1 hour and the second time for 2.5 hours. The extracts were combined and filtered, and the ethanol was recovered and the filtrate was used for later use;

[0091] (4) Add 7 times the amount of water to honeysuckle, gypsum, isatis root, cyperus rotundus, licorice, and rhodiola rosea, boil until boiling, add fried bitter almonds, and boil twice, the first time for 1.5 hours and the second time for 1 hour, combine the extracts and filter, combine the filtrate obtained with the filtrate after patchouli oil extraction in step (2), and concentrate into a clear paste with a relative density of 1.10-1.15 measured at 60°C, add ethanol, adjust the alcohol concentration to 70%, refrigerate for 24 hours, filter, and recover the ethanol until there is no alcohol taste, and obtain a clear paste for use;

[0092] (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating to a clear paste having a relative density of 1.15-1.20 measured at 60° C., spray drying to obtain a dry paste powder for later use;

[0093] (6) Add 151 g of starch to the dry paste powder obtained in step (5) and granulate with 85% ethanol;

[0094] (7) Menthol and the volatile oil obtained in step (2) were dissolved in ethanol, sprayed onto the granules obtained in step (6), sealed, mixed, and pressed into tablets to obtain 935 tablets.

[0095] Example 3

[0096] prescription:

[0097] 185g forsythia, 179g honeysuckle, 190g isatis root, 36g rhubarb

[0098] Patchouli 60g, Mianma Guanzhong 170g, Rhodiola rosea 57g, Menthol 5g

[0099] 60g roasted ephedra, 60g fried bitter almonds, 170g houttuynia

[0100] 60 grams of licorice and 170 grams of gypsum.

[0101] Preparation method:

[0102] (1) Weigh and select the Chinese medicinal materials according to the above prescription;

[0103] (2) Patchouli is broken into pieces, and volatile oil is extracted by adding 6 times the amount of water. The extraction time is 4 hours, and the volatile oil is collected. The oil yield is 0.33 and is set aside. After the extract is filtered, the residue is discarded and the water extract is set aside.

[0104] (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb were extracted twice with 8 times the amount of 70% ethanol, the first time for 2 hours and the second time for 1.5 hours. The extracts were combined and filtered, and the ethanol was recovered and the filtrate was used for later use;

[0105] (4) Add 9 times the amount of water to honeysuckle, gypsum, isatis root, cyperus rotundus, licorice, and rhodiola rosea, and boil until boiling. Add fried bitter almonds and boil twice, the first time for 1.5 hours and the second time for 1 hour. Combine the extracts and filter them. Combine the filtrate with the water extract after patchouli oil extraction in step (2), and concentrate it into a clear paste with a relative density of 1.10-1.15 measured at 60° C., add 95% ethanol, adjust the alcohol concentration to 70%, refrigerate for 24 hours, filter, and recover the ethanol until there is no alcohol taste, and obtain a clear paste for use;

[0106] (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating to a thick paste having a relative density of 1.25-1.30 measured at 60° C., spray drying to obtain a dry paste powder for later use;

[0107] (6) The dry paste powder obtained in step (5), the volatile oil obtained in step (2) and menthol were made into pills according to a conventional method to obtain 905 pills.

[0108] Embodiment 4:

[0109] The formula of the raw material is:

[0110] 170g forsythia suspensa, 170g honeysuckle, 57g roasted ephedra, 57g stir-fried bitter almonds

[0111] 170g gypsum, 170g isatis root, 170g mianma guanzhong, 170g houttuynia cordata

[0112] Patchouli 57g Rhubarb 34g Rhodiola rosea 57g Menthol 5.0g

[0113] Licorice 57g

[0114] Preparation method:

[0115] 1. Extraction process:

[0116] (1) Weigh the Chinese medicinal materials according to the above prescription, clean and select, and break them into pieces as appropriate;

[0117] (2) Add 6 times the amount of water to extract the volatile oil from patchouli for 4 hours. The volatile oil is collected with an oil yield of 0.33%. The extract is filtered and set aside. The residue is discarded and the water extract is set aside.

[0118] (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb were extracted twice with 8 times the amount of 70% ethanol, the first time for 2 hours and the second time for 1.5 hours. The extract was filtered, the filtrates were combined, and the ethanol was recovered until there was no alcohol taste, and then used for later use;

[0119] (4) Add 9 times the amount of water to honeysuckle, fried bitter almonds, gypsum, isatis root, cyperus rotundus, licorice, and rhodiola rosea, boil until boiling, add fried bitter almonds, boil twice, the first time for 1.5 hours, the second time for 1 hour, filter the extract, combine the filtrates, and add the aqueous solution obtained by extracting patchouli oil in step (2) at the same time, concentrate to a clear paste with a relative density of 1.10-1.15 at 60°C, add 95% ethanol, stir while adding, until the alcohol concentration reaches 70%, refrigerate for 24 hours, filter, recover ethanol from the filtrate until there is no alcohol taste, combine with the alcohol extract, and concentrate to a thick paste with a relative density of 1.25-1.35 at 60°C, set aside;

[0120] (II) Preparation process:

[0121] (5) The formulation is: 335.5 g of the thick paste obtained in step (4) 5 g of menthol

[0122] 0.2 ml of patchouli oil obtained in step (2) 342.5 g of powdered sugar 514.0 g of dextrin

[0123] (6) Granulation: Mix the powdered sugar and dextrin evenly, use the thick paste as a binder to make a soft material, granulate with a 14-mesh sieve, dry at 60-65°C, and granulate with a 10-mesh sieve;

[0124] (7) Packaging: sieve out an appropriate amount of fine powder, add an appropriate amount of ethanol to dissolve menthol and patchouli volatile oil, spray it into the fine powder, mix evenly, and mix it evenly with the granules. Seal it for half an hour and pack it into a bag. The above prescription can be used to make 1000g of granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 .EC 50 Calculation results, where A. is the EC of the drug of the present invention against Wt virus on Vero E6 cells50 B. EC of the drug of the present invention against D61G virus on Vero E6 cells 50

[0126] Figure 2 : Pathological effects of lung tissue (48h after administration) (×100), where A is the normal control group; B is the model control group; C is the drug group of the present invention

[0127] Figure 3 : Effect on lung tissue pathology (the day after the last administration) (×100), wherein A is the normal control group; B is the model control group; C is the drug group of the present invention.

[0128] Experimental example

[0129] In order to verify the efficacy of the Chinese medicine composition of the present invention in preventing and treating COVID-19, protecting organs, and improving immunity, the following clinical trial studies were conducted using the granules prepared according to the method of Example 4 (hereinafter referred to as the medicine of the present invention):

[0130] Test Example 1: Study on the inhibition of novel coronavirus by the drug of the present invention in vitro

[0131] 1. Test Materials

[0132] 1.1 Drugs

[0133] The drug of the present invention (batch number: 1804001), the concentration of the drug solution of the present invention: 12 mg / mL (1 mL DMSO dissolves 120 mg of the drug, then adds 9 mL DMEM culture medium (containing 100 U / mL double antibody (penicillin and streptomycin) and 2% FBS, vortexes for 5 min, places in a refrigerator at 4 ° C for 12 h, vortexes for another 5 min, and filters through a 0.22 µM filter);

[0134] 1.2 Cells: VERO-E6 cells

[0135] 1.3. Viruses

[0136] (1) Wild-type novel coronavirus [2019-nCoV, Wild-type (Wt)];

[0137] (2) S protein mutant coronavirus [2019-nCoV, D614G]

[0138] 1.4 Reagents

[0139] Virus diluent: DMEM medium, containing double antibodies (penicillin and streptomycin) at a final concentration of 100 U / mL, without serum;

[0140] Drug diluent: DMEM medium, containing double antibodies (penicillin and streptomycin) at a final concentration of 100 U / mL, without serum;

[0141] Cell culture medium: DMEM medium, containing double antibodies (penicillin and streptomycin) at a final concentration of 100 U / mL and 10% FBS;

[0142] Fetal bovine serum (FBS) (Cat. No.: 10270-044, Brand: GIBCO);

[0143] DMEM culture medium (catalog number: C11995500BT, brand: GIBCO);

[0144] 1× PBS (pH 7.4) (Cat. No.: C10010500BT, Brand: GIBCO);

[0145] 0.25% trypsin-EDTA (Trypsin 2.5g (Cat. No.: 0458-100g, Brand: Amresco) EDTA-Na2 0.34g (Cat. No.: 0105-100g, Brand: Amresco) dissolved in 1×PBS 1000mL, filtered with 0.22uM filter);

[0146] DMSO (Cat. No. D2650-100ML, Brand: Sigma);

[0147] 1.5 Consumables

[0148] 96-well flat-bottom cell culture plate (Cat. No.: 3599, Brand: costar); 15 mL centrifuge tube (Cat. No.: CFT-011-150, Brand: BIOFIL); 50 mL centrifuge tube (Cat. No.: CFT-011-150, Brand: BIOFIL); Pipette tip (Cat. No.: T-200-YRS, Brand: AXYGEN); 10 mL pipette (Cat. No.: 4488, Brand: costar);

[0149] 1.6 Instruments

[0150] Cell culture box; -80℃ refrigerator; centrifuge; inverted microscope; biological safety cabinet; electric pipette; pipette; 8-channel row pipette; water bath; oscillator

[0151] 2. Experimental Methods

[0152] 2.1 Cells: VERO-E6 cells were spread as monolayers for antiviral experiments of the drug of the present invention.

[0153] Virus: SARS-CoV-2 (Wt and D614G), 100 TCID 50 / well virus inoculated cells.

[0154] Drug: The test concentrations of the drug of the present invention are 75, 37.5, 18.75, 9.375, 4.69, 2.34, 1.17, 0.59, 0.29, and 0.15 µg / ml, for a total of 10 concentrations. The virus and the drug were incubated at 37 °C for 1 h before inoculation into the cells, and a normal cell control and a positive virus control were set up at the same time.

[0155] Observation: 72 hours after virus infection, observe CPE

[0156] 3. Results

[0157] 3.1 CPE observation: 72 hours after virus infection, there was no CPE in the normal cell control group; CPE was obvious in the virus control group; different drug concentrations showed different inhibitory effects on CPE, as shown in Tables 1 and 2.

[0158] The half inhibitory concentration (EC 50 ) and therapeutic index (TI) calculation: Calculate the half inhibitory concentration (EC) according to the results in Table 1 50 ),like Figure 1 As shown, on Vero E6 cells, the EC of the drug of the present invention against Wt virus 50 The EC value of the drug of the present invention for D614G virus is 17.77 μg / ml. 50 It is 35.85 μg / ml.

[0159] Based on the toxicity test results of the same batch of drugs of the present invention on cells (TD 50 264.8 μg / ml), and the therapeutic index (TI=TD) was calculated. 50 / EC 50 ). The results showed that the therapeutic index of the drug of the present invention for Wt virus was 14.90, and the therapeutic index for D614G virus was 7.39.

[0160] in conclusion

[0161] A certain concentration of the drug of the present invention can inhibit the formation of CPE by 2019 novel coronavirus (Wt and D614G mutant strains) on Vero E6 cells.

[0162]

[0163]

[0164] Test Example 2

[0165] 1. Test name: Experimental study on the drug of the present invention against coronavirus HCoV-229E

[0166] 2. Test Materials

[0167] 2.1 Test Articles

[0168] The drug batch number of the present invention is B2001013, specification: 0.35g / pill, valid until: June 2022, and is produced by Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0169] 2.2 Experimental animals

[0170] BALB / c mice, SPF grade, 120, half male and half female, weighing 10.0-14.0 g, animal quality certificate number: No.1107272011001651, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., experimental animal production license number: SCXK (Xiang) 2019-0004; kept in the Pathogenic Microbiological Safety Animal Laboratory (ABSL-Ⅱ) in the Barrier Environment Zone C of the Hunan Provincial Drug Safety Evaluation and Research Center, laboratory registration number: Changwei Jishibeizi (2019) No. B001, experimental animal use license number: SYXK (Xiang) 2015-0016.

[0171] 2.4 Virus strain: Coronavirus HCoV-229E, number: ATCC-VR-740, donated by Hunan Provincial Center for Disease Control and Prevention, cultured and amplified in the Pathogenic Microbiology Laboratory of this center (BSL-Ⅱ, laboratory registration number: Changwei Jishibeizi (2019) No. B001).

[0172] 2.5 Cell line: Human lung fibroblasts (MRC-5) were purchased from Wuhan Pronocell Life Science Co., Ltd. and subcultured in the cell room of Hunan Provincial Drug Safety Evaluation Research Center.

[0173] 2.6 Main Reagents

[0174] DMEM medium (lot number: AD22315267, product of HyClone); fetal bovine serum (lot number: M009-6, Sciencell, USA); interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor (TNF-α), brain natriuretic peptide (BNP), interleukin-2 (IL-2), interleukin-12 (IL-12), T cell immunoregulatory factor (TCITRG), interferon regulatory factor (IRF) enzyme-linked immunosorbent assay kits, all of which were provided by Jiangsu ELISA Company; IL-4 flow cytometry antibody (batch number: 2031934), CD4 flow cytometry antibody (batch number: 2115770), INF-γ flow cytometry antibody (batch number: 2161229), IL-17A flow cytometry antibody (batch number: 1995433), CD25 flow cytometry antibody (batch number: 2018381), FoxP3 flow cytometry antibody (batch number: 2105941), all of which were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0175] 2.7 Main instruments

[0176] BCR-MI02-72-C11-PPSU type mouse independent ventilation cage system (manufacturer: Shandong Xinhua Medical Instrument Co., Ltd., center number: 578), BSC1300-Ⅱ-B2 type biological safety cabinet (manufacturer: Shandong Xinhua Medical Instrument Co., Ltd., center number: 588), CJ-1F type medical clean bench (manufacturer: Suzhou Fengshi Experimental Animal Equipment Co., Ltd., center number: 176), BSCIIB2-1101 type biological safety cabinet (manufacturer: Shanghai Ruiyang Purification Equipment Co., Ltd., center number: 019), 3111 type CO 2 Incubator (manufacturer: Thermo Fisher, USA, center number: 147), DMIL inverted microscope (manufacturer: Leica, Germany, center number: 027), YXQ-50A vertical pressure sterilizer (manufacturer: Shanghai Boxun Medical Biological Instrument Co., Ltd., center number: 584), TD4 desktop low-speed centrifuge (manufacturer: Hunan Keda Industrial Development Co., Ltd., center number: 101).

[0177] 3 Experimental methods

[0178] 3.1 Virus amplification

[0179] MRC-5 cells were inoculated into culture flasks. When the cell density reached 70-80%, part of the culture medium was removed, leaving the remaining medium just enough to cover the cells. Appropriate amounts of HCoV-229E virus were added. After the virus was adsorbed on the cell surface (about 3 hours, the culture plate was gently shaken every 30 minutes to ensure uniform adsorption of the virus), fresh culture medium without FBS was replaced and placed at 37°C, 5% CO 2 After 96 hours (if the cells do not develop lesions, the culture time is extended), repeated freezing and thawing is performed, centrifugation is performed at 3000 rpm for 10 minutes to remove cell debris, and the supernatant is collected. The virus titer is determined by the cytopathic effect assay (CPE method), and the 50% tissue cell infection dose (TCID 50 ) is indicated. Then filter with a 0.22µm filter and dispense into cryopreservation tubes, label and store at -80℃ for short-term or liquid nitrogen for long-term for future use. Before infecting animals, use the hemagglutination method to detect the virus hemagglutination titer and adjust to the appropriate titer.

[0180] TCID 50 =Log(virus dilution at which CPE is less than 50%) + distance ratio × dilution interval

[0181] Where, distance ratio = (percentage above 50% - 50) / (percentage above 50% - percentage below 50%)

[0182] 3.2 Modeling, grouping and drug administration

[0183] 60 BALB / c mice, SPF grade, half male and half female, weighing 10.0-14.0g, were randomly divided into 6 groups according to gender and weight, namely normal control group, model control group, and drug group of the present invention, with 20 animals in each group. Each group of mice was slightly anesthetized with ether and nasally instilled with HCoV-229E coronavirus liquid (the hemagglutination titer of the virus liquid was 6Log2 by hemagglutination method, precooled at 4°C), 60μL / mouse, and inoculated for 2 consecutive days. The normal control group was nasally instilled with an equal volume of blank culture medium, and each group was first administered 2h after the second modeling. The drug of the present invention was prepared into a concentration of 0.156g crude drug / mL drug solution with pure water every day, and the drug of the present invention was administered orally at a concentration of 20mL / kg by oral gavage. The normal control group and the model control group were given an equal volume of pure water, once a day, and administered for 7 consecutive days.

[0184] 3.3 Dosage design

[0185] The clinical dosage of the drug of the present invention is 24g crude drug / day. The average adult weight is 60kg. The equivalent dose of the mouse is about 3.12g crude drug / kg according to the body surface area method. In this experiment, the equivalent dose of the drug of the present invention in mice is used as the dosage in the mouse experiment, that is, 3.12g crude drug / kg. The specific dosage design is shown in Table 3.

[0186]

[0187] 3.4 Observation indicators

[0188] 3.4.1 General physiological observations

[0189] The animals in each group were weighed and their physiological states were observed every day, and the changes in animal body weight during the experiment were compared.

[0190] 3.4.2 Determination of cytokine content in lung tissue

[0191] At 48h of administration and the day after the last administration, 8 animals were randomly selected from each group, half male and half female, and euthanized by cervical dislocation. The lungs were dissected and placed in a homogenizer. 0.9% sodium chloride injection was added according to the ratio of lung mass (g): 0.9% sodium chloride injection (mL) = 1:9, and the suspension was made into mouse lung suspension. The suspension was centrifuged at 3000rpm for 15min at 4℃, and the supernatant was taken for later use. The contents of IL-2, IL-6, IL-8, IL-12, and TNF-α in mouse lungs were detected by ELISA kits.

[0192] 3.4.3 Detection of cytokines in peripheral blood

[0193] At 48h after administration and the day after the last administration, 8 animals were randomly selected from each group, half of them were male and half were female, blood was collected from the eye sockets, centrifuged at 3000rpm for 15min at 4℃, and the supernatant was taken for later use. ELISA kits were used to detect the levels of BNP, CK-MB, Tn-I, TCITRG, and IRF in mouse serum.

[0194] 3.4.4 Detection of immune cells in lung tissue

[0195] At 48h of administration and the day after the last administration, 8 animals were randomly selected from each group, half male and half female, and euthanized by cervical dislocation. The lungs were dissected and then placed in a homogenizer. 0.9% sodium chloride injection was added at a ratio of lung mass (g): 0.9% sodium chloride injection (mL) = 1:9, and the lung suspension was prepared by grinding and homogenization. Flow cytometry was used to detect T cell subsets (Th0 / Th1 / Th2) and regulatory / helper T cells (Th17 / Treg) in the mouse lungs.

[0196] 3.4.5 Detection of virus titer in lung tissue

[0197] At 48h after administration and the day after the last administration, 10 animals were randomly selected from each group, half male and half female, and euthanized by cervical dislocation. The lungs were removed and placed in a homogenizer. 0.9% sodium chloride injection was added at a ratio of lung mass (g): 0.9% sodium chloride injection (mL) = 1:9, and the lung suspension was prepared by grinding and homogenization. The virus titer in the lung tissue was detected by hemagglutination.

[0198] 3.4.6 Lung tissue pathology examination

[0199] 48 hours after administration and the day after the last administration, the mice in each group were euthanized by cervical dislocation, and the lungs were dissected and fixed with formalin perfusion, embedded in paraffin, sectioned, and stained with HE. Pathological changes were observed under a light microscope, and scored based on four aspects: the degree of inflammatory and bloody exudation, the degree of alveolar epithelial destruction, the degree of lung scaffold collapse and alveolar epithelial hyperplasia, and the degree of chronic inflammatory cell infiltration around macrophages and small blood vessels.

[0200] 3.5 Statistical methods

[0201] The significant figures of the experimental data were rounded off and statistical analysis was performed in accordance with SOP regulations. The statistical software used was SPSS. The measurement data were expressed as mean ± standard deviation ( ) indicated that the normality and homogeneity of variance were tested by Leven's test. If there was no statistical significance (P>0.05), one-way analysis of variance (ANOVA) was used for statistical analysis. If ANOVA was statistically significant (P ≤ 0.05), LSD test (parametric method) was used for comparative analysis. If the variance was unequal (P ≤ 0.05), Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P ≤ 0.05), Dunnett's Test (non-parametric method) was used for comparative analysis. The statistical results were tested with α=0.05 as the test limit, where P ≤ 0.05 indicated statistical significance, and P ≤ 0.01 indicated that the difference tested was very significant.

[0202] 4 Experimental Results

[0203] 4.1 Half viral infection dose of HCoV-229E in MRC-5 cells

[0204] As shown in Table 4, the HCoV-229E stock solution was diluted 10 times and co-cultured with MRC-5 cells. The half viral infection dose (TCID 50 ) is 10 -4.62 / 0.1mL.

[0205]

[0206] 4.2 Effects on animal body weight

[0207] As shown in Table 5, Figure 1 As shown, compared with the normal control group, the body weight of the model control group was significantly reduced from D1 to D8 (P≤0.01), indicating that human coronavirus (HCoV-229E) can cause weight loss in mice. Compared with the model control group, the body weight of the drug group of the present invention increased significantly from D2 to D6 (P≤0.05 or P≤0.01) after administration. This indicates that infection of mice with coronavirus (HCoV-229E) will cause continuous weight loss, and oral administration of the drug of the present invention can alleviate the weight loss of infected mice.

[0208] 4.3 Effects on IL-6, TNF-α, IL-2, IL-8, and IL-12 levels in lung tissue

[0209] As shown in Table 6, after 48 h of drug administration, the levels of IL-6, TNF-α, IL-2, IL-8, and IL-12 in the model control group increased significantly compared with those in the normal control group ( P≤ 0.01). Compared with the model control group, the IL-6 in the drug group of the present invention decreased significantly ( P ≤ 0.05 or P ≤ 0.01); TNF-α and IL-8 in the drug group of the present invention were significantly decreased ( P ≤ 0.05 or P ≤ 0.01); IL-2 in the drug group of the present invention decreased significantly ( P ≤ 0.05 or P ≤ 0.01); IL-12 in the drug group of the present invention increased significantly ( P ≤ 0.05). On the second day after the last administration (D8), compared with the normal control group, the IL-6, TNF-α, IL-2, IL-8, and IL-12 in the model control group were significantly increased (P ≤ 0.01). Compared with the model control group, the IL-6, TNF-α, IL-2, and IL-8 in the drug group of the present invention were significantly decreased ( P ≤ 0.05 or P ≤ 0.01); IL-12 in the drug group of the present invention showed a downward trend, but there was no statistical difference. This suggests that infection of mice with coronavirus (HCoV-229E) will cause an increase in the content of inflammatory factors in the lungs and abnormal changes in immune factors. Oral administration of the drug of the present invention can regulate the content of inflammatory factors and immune factors in the lungs of infected mice.

[0210]

[0211] Effects on peripheral blood BNP, CK-MB, and Tn-I

[0212] As shown in Table 7, after 48 hours of administration, compared with the normal control group, the brain natriuretic peptide (BNP), creatine phosphoenzyme MB (CK-MB), and troponin I (Tn-I) in the model control group were significantly increased ( P ≤ 0.05 or P ≤ 0.01). Compared with the model control group, the BNP and Tn-I in the drug group of the present invention were significantly reduced ( P ≤ 0.05 or P ≤ 0.01); the drug of the present invention significantly reduced CK-MB ( P ≤ 0.05 or P ≤ 0.01). On the second day after the last administration (D8), compared with the normal control group, the BNP and CK-MB in the model control group increased significantly ( P ≤ 0.05 or P ≤ 0.01), Tn-I showed an increasing trend, but there was no statistical difference. Compared with the model control group, BNP in the drug group of the present invention was significantly reduced ( P ≤ 0.05 or P≤ 0.01); CK-MB in the drug group of the present invention decreased significantly ( P ≤0.05 or P ≤ 0.01); Tn-I in the drug group of the present invention showed a decreasing trend, but there was no statistical difference. It suggests that infection of mice with coronavirus (HCoV-229E) will cause an increase in cardiac-related functional enzymes in peripheral blood, and oral administration of the drug of the present invention can significantly reduce the content of cardiac-related functional enzymes in peripheral blood.

[0213]

[0214] Effects on IRF and TCITRG in peripheral blood

[0215] As shown in Table 8, after 48h of drug administration, the interferon regulatory factor (IRF) and cellular immune regulatory factor (TCITRG) in the model control group were significantly increased compared with the normal control group ( P ≤ 0.05 or P ≤ 0.01). Compared with the model control group, TCITRG in the drug group of the present invention was significantly reduced ( P ≤ 0.01); IRF in the drug group of the present invention was significantly reduced ( P ≤ 0.05). On the second day after the last administration (D8), compared with the normal control group, the interferon regulatory factor (IRF) and cellular immune regulatory factor (TCITRG) in the model control group were significantly increased ( P ≤ 0.05). Compared with the model control group, the IRF of the drug group of the present invention showed a downward trend, but there was no statistical difference. Compared with the model control group, the TCITRG of the drug group of the present invention showed a downward trend, but there was no statistical difference.

[0216]

[0217] Effects on Th0, Th1 / Th2, and Treg / Th17 in peripheral blood

[0218] As shown in Table 9, compared with the normal control group, the Th1 / Th2 ratio in the model control group was significantly increased, and the Treg / Th17 ratio was significantly decreased ( P ≤ 0.05), Th0 showed a decreasing trend, but there was no statistical difference. Compared with the model control group, the Th1 / Th2 ratio of the drug group of the present invention was significantly reduced ( P ≤ 0.05), the ratio of Treg / Th17 increased significantly ( P ≤ 0.05); compared with the model control group, there was no significant difference in Th0 in the drug group of the present invention.

[0219]

[0220] 4.7 Effect on the hemagglutination titer of coronavirus HCoV-229E in the lungs

[0221] As shown in Table 10, compared with the normal control group, the hemagglutination titer of the model control group increased significantly at 48h after administration and the day after the last administration ( P ≤ 0.01). Compared with the model control group, 48 hours after administration, the blood coagulation titer of the drug group of the present invention was significantly reduced ( P ≤ 0.05 or P ≤ 0.01); the day after the last administration, the hemagglutination titer of the drug group of the present invention was significantly reduced (P ≤ 0.01). The drug of the present invention can reduce the hemagglutination titer of coronavirus HCoV-229E in the lungs of mice.

[0222]

[0223] Effects on lung index and lung histopathology

[0224] As shown in Table 11, compared with the normal control group, the lung index of infected mice in the model control group was significantly increased 48 hours after administration and the day after the last administration ( P ≤ 0.01). Compared with the model control group, the lung index of the drug group of the present invention was significantly reduced 48 hours after administration ( P ≤ 0.05); the day after the last administration, the lung index of the drug group of the present invention was significantly reduced ( P ≤ 0.01).

[0225]

[0226] As shown in Table 12, Figure 2 , 3 As shown, the results of microscopic examination after HE staining showed that after 48 hours of administration, no obvious abnormal changes were observed in the lung tissue of the normal control group, and pathological changes such as inflammation, bloody exudation, alveolar epithelial destruction, lung stent collapse, alveolar epithelial hyperplasia, macrophage infiltration, and chronic inflammatory cell infiltration around small blood vessels appeared in the animals of the model control group. Compared with the model control group, the number of animals with lesions in the drug group of the present invention decreased, and the degree of lesions was alleviated. The next day after the last administration, inflammatory exudation, destruction of alveolar structure, and chronic inflammatory cell infiltration around small blood vessels were observed in the lungs of animals in the model control group. Compared with the model control group, the number of animals with lesions in the drug group of the present invention was significantly reduced, and the degree of lesions was significantly alleviated.

[0227]

[0228] 5. Summary

[0229] In this study, a BALB / c mouse model infected with coronavirus HCoV-229E was used to evaluate the therapeutic effect of the drug of the present invention by observing the physiological state of mice, inflammation of lung tissue, virus titer, level of inflammatory factors, and the proportion of cardiac-related functional enzymes, immune regulatory factors and peripheral immune cells in peripheral blood. The experimental results showed that compared with the normal control group, the mice in the model control group had a significant decrease in weight after virus infection, a significant increase in lung index, severe pathological changes in lung tissue, and a large increase in inflammatory factors in lung tissue, indicating that the model was successful. Compared with the model group, the weight of mice infected with the drug of the present invention (3.12g crude drug / kg) group first decreased but then recovered rapidly, indicating that the drug of the present invention can inhibit the weight loss of infected mice; 48h after administration and the next day after the last administration, the lung index of mice infected with the drug of the present invention (3.12g crude drug / kg) group was significantly reduced, the lung tissue lesions were improved, and the viral hemagglutination titer in the lungs was significantly reduced, indicating that the drug of the present invention has the effect of reducing lung inflammation damage in infected mice and inhibiting the damage of the virus to the lungs.

[0230] The proportion of peripheral blood lymphocyte subsets in mice in this study showed that compared with the normal control group, the Th1 / Th2 cell ratio in the model control group was significantly increased, and the Treg / Th17 cell ratio was significantly decreased, indicating that the T lymphocyte ratio in the coronavirus-infected mice was unbalanced and severe immune disorders occurred. Compared with the model control group, the Th1 / Th2 cell ratio in the drug group of the present invention was significantly reduced, and the Treg / Th17 cell ratio was significantly increased, indicating that the drug of the present invention has the effect of regulating mouse immune T lymphocytes.

[0231] The phenomenon of a large amount of cytokines (TNF-α, IL-1, IL-6, IL-12, IFN-α, IFN-β, IFN-γ and IL-8, etc.) produced after pathogenic microorganisms infect the body is called cytokine storm, which can cause acute respiratory distress syndrome and multiple organ failure. The results of this study found that after infection with coronavirus HCoV-229E, the content of inflammatory factors IL-6, TNF-α, IL-2, IL-8, and IL-12 in the lung tissue of mice in the model control group was significantly higher than that in the normal control group; compared with the model control group, the drug of the present invention can significantly reduce the production of IL-6, TNF-α, IL-2, IL-8, and IL-12. The results show that the drug of the present invention can effectively reduce the cytokine storm, which is consistent with the results of lung tissue pathological observation.

[0232] In addition, compared with the normal control group, the BNP, CK-MB, and Tn-I in the peripheral blood of mice infected in the model control group were significantly increased, indicating that the heart of mice infected with coronavirus HCoV-229E was also damaged. Compared with the model control group, the BNP, CK-MB, and Tn-I levels in the peripheral blood of mice in the drug group of the present invention showed a downward trend, indicating that the drug of the present invention has the effect of protecting the heart of mice infected with coronavirus.

[0233] In summary, the drug of the present invention (3.12g crude drug / kg) can significantly inhibit the increase of lung index of mice infected with coronavirus HCoV-229E, alleviate pathological changes in lung tissue, inhibit the replication of coronavirus HCoV-229E in the lungs of infected mice, and regulate the proportion of peripheral blood lymphocyte subsets of infected mice, improve immune function, and reduce the production of inflammatory factors in the lungs. At the same time, it has a certain protective effect on the heart of mice infected with coronavirus HCoV-229E.

Claims

1. Use of a Chinese medicine composition in the preparation of a drug for preventing or treating coronavirus, characterized in that: The coronavirus is a S protein D614G mutant coronavirus, and the Chinese medicine composition is made of the following raw materials in parts by weight: Forsythia suspensa 150-260 Honeysuckle 150-260 Isatis root 150-260 Rhubarb 30-55 Patchouli 50-90, Cyperus rotundus 150-260, Rhodiola rosea 50-90, Menthol 5-8 Roasted Ephedra 50-90 Stir-fried Bitter Almond 50-90 Houttuynia Cordata 150-260 Licorice 50-90, Gypsum 150-260.

2. The use according to claim 1, characterized in that Made from the following raw materials in parts by weight: Forsythia suspensa 150 Honeysuckle 260 Isatis root 150 Rhubarb 55 Patchouli 50 Mianma Guanzhong 260 Rhodiola rosea 50 Menthol 8 Roasted Ephedra 50 Fried bitter almonds 90 grams, Houttuynia cordata 150 grams, licorice 90 grams, gypsum 150 grams.

3. The use according to claim 1, characterized in that Made from the following raw materials in parts by weight: Forsythia suspensa 260 Honeysuckle 150 Isatis root 260 Rhubarb 30 Patchouli 90 Mianma Guanzhong 150 Rhodiola rosea 90 Menthol 5 Roasted Ephedra 90 Fried bitter almonds 50, Houttuynia cordata 260, licorice 50, gypsum 260.

4. The use according to claim 1, characterized in that Made from the following raw materials in parts by weight: Forsythia suspensa 170 Honeysuckle 170 Isatis root 170 Rhubarb 34 Patchouli 57 Mianma Guanzhong 170 Rhodiola rosea 57 Menthol 5 Roasted Ephedra 57 Fried bitter almonds 57, Houttuynia cordata 170, licorice 57, gypsum 170.

5. The use according to claim 1, characterized in that Made from the following raw materials in parts by weight: Forsythia suspensa 255 Honeysuckle 255 Isatis root 255 Rhubarb 51 Patchouli 85 Mianma Guanzhong 255 Rhodiola rosea 85 Menthol 7.5 Roasted Ephedra 85 Fried bitter almonds 85, Houttuynia cordata 255, licorice 85, gypsum 255.

6. The use according to any one of claims 1 to 5, characterized in that The active ingredients of the Chinese medicine composition are prepared by the following steps: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; (2) Crush patchouli, add 5-8 times the amount of water to extract the volatile oil, extract the oil for 4 hours, collect the volatile oil and set aside; after filtering the extract, discard the residue and set aside the filtrate; (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb are extracted twice with 6-10 times the amount of 50-90% ethanol, each time for 1-3 hours. The extracts are combined and filtered, and the ethanol is recovered and the alcohol extract is reserved; (4) Add 7-11 times the amount of water to honeysuckle, gypsum, isatis root, cyperus rotundus, liquorice, and rhodiola rosea, boil until boiling, add fried bitter almonds, boil twice, each time for 0.5-2.5 hours, combine the extracts and filter, combine the filtrate obtained with the filtrate after patchouli oil extraction in step (2), and concentrate into a clear paste with a relative density of 1.10-1.15 measured at 60° C., add ethanol, adjust the alcohol concentration to 70%, refrigerate, filter, and recover the ethanol until there is no alcohol taste, and obtain a clear paste for use; (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating to a clear paste having a relative density of 1.15-1.20 measured at 60° C., drying to obtain a dry paste powder for later use; The dry paste powder obtained in step (5), the volatile oil obtained in step (2) and menthol together constitute the active ingredients of the traditional Chinese medicine composition.

7. The use according to any one of claims 1 to 5, characterized in that The drug dosage form is capsule, tablet, powder, granule, oral liquid, pill, tincture, syrup, suppository, gel, spray or injection.

8. The use according to claim 7, characterized in that The capsules are made by the following steps: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; (2) Crush patchouli, add 5-8 times the amount of water to extract the volatile oil, extract the oil for 4 hours, collect the volatile oil and set aside; after filtering the extract, discard the residue and set aside the filtrate; (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb were extracted twice with 6-10 times the amount of 50-90% ethanol, each time for 1-3 hours. The extracts were combined and filtered, and the ethanol was recovered and the filtrate was used for later use; (4) Add 7-11 times the amount of water to honeysuckle, gypsum, isatis root, cyperus rotundus, liquorice, and rhodiola rosea, boil until boiling, add fried bitter almonds, boil twice, each time for 0.5-2.5 hours, combine the extracts and filter, combine the filtrate obtained with the filtrate after patchouli oil extraction in step (2), and concentrate into a clear paste with a relative density of 1.10-1.15 measured at 60° C., add ethanol, adjust the alcohol concentration to 70%, refrigerate, filter, and recover the ethanol until there is no alcohol taste, and obtain a clear paste for use; (5) The clear paste obtained in step (4) is combined with the alcohol extract obtained in step (3), concentrated to a clear paste having a relative density of 1.15-1.20 measured at 60° C., dried to obtain a dry paste powder for later use; (6) adding appropriate pharmaceutically acceptable excipients to the dry paste powder obtained in step (5) for granulation; (7) Add ethanol to dissolve menthol and the volatile oil obtained in step (2), spray the granules obtained in step (6), seal, mix well, and put into capsules to obtain the product.

9. The use according to claim 7, characterized in that Granules are made by the following steps: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials, clean and select, and break them into pieces as appropriate; (2) Crush patchouli, add 5-8 times the amount of water to extract the volatile oil, extract the oil for 4 hours, collect the volatile oil and set aside; after filtering the extract, discard the residue and set aside the filtrate; (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb were extracted twice with 6-10 times the amount of 50-90% ethanol, each time for 1-3 hours. The extracts were combined and filtered, and the ethanol was recovered and the filtrate was used for later use; (4) Add 7-11 times the amount of water to honeysuckle, gypsum, isatis root, cyperus rotundus, liquorice, and rhodiola rosea, boil until boiling, add fried bitter almonds, boil twice, each time for 0.5-2.5 hours, combine the extracts and filter, combine the filtrate obtained with the filtrate after patchouli oil extraction in step (2), and concentrate into a clear paste with a relative density of 1.10-1.15 measured at 60° C., add ethanol, adjust the alcohol concentration to 70%, refrigerate, filter, and recover the ethanol until there is no alcohol taste, and obtain a clear paste for use; (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating to a clear paste having a relative density of 1.15-1.20 measured at 60° C., drying to obtain a dry paste powder for later use; (6) adding appropriate pharmaceutically acceptable excipients to the dry paste powder obtained in step (5) for granulation; (7) Add ethanol to dissolve menthol and the volatile oil obtained in step (2), spray the granules obtained in step (6), seal, mix well, and bag to obtain the product.

10. The use according to claim 7, characterized in that The preparation method of the granules is prepared by the following steps: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials, select them, and break them into pieces as appropriate; (2) Patchouli was broken into pieces and volatile oil was extracted by adding 6 times the amount of water for 4 hours. The volatile oil was collected and the oil yield was 0.33±0.05%. The volatile oil was set aside. After filtering the extract, the residue was discarded and the water extract was set aside. (3) Forsythia suspensa, roasted ephedra, houttuynia cordata, and rhubarb were extracted twice with 8 times the amount of 70% ethanol, the first time for 2 hours and the second time for 1.5 hours. The extracts were combined and filtered, and the ethanol was recovered and the filtrate was used for later use; (4) Add 9 times the amount of water to honeysuckle, gypsum, isatis root, cyperus rotundus, licorice, and rhodiola rosea, boil until boiling, add fried bitter almonds, and boil twice, the first time for 1.5 hours and the second time for 1 hour, combine the extracts and filter, combine the filtrate obtained with the water extract after patchouli oil extraction in step (2), and concentrate into a clear paste with a relative density of 1.10-1.15 measured at 60° C., add 95% ethanol, adjust the alcohol concentration to 70%, refrigerate, filter, and recover the ethanol until there is no alcohol taste, to obtain a clear paste; (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating to a thick paste having a relative density of 1.25-1.35 measured at 60° C., and setting aside; (6) adding appropriate pharmaceutically acceptable excipients to the thick paste obtained in step (5) to granulate and shape the granules for later use; (7) Screen the granules obtained in step (6) to obtain fine powder, add menthol and the volatile oil obtained in step (2) into ethanol to dissolve, spray the fine powder into the granules obtained in step (6), mix well, and then mix well with the granules obtained in step (6), seal for half an hour, and pack into a bag.

11. Use of the Chinese medicine composition according to any one of claims 1 to 5 in the preparation of a drug for protecting against cardiac damage caused by coronavirus HCoV-229E, wherein the cardiac damage is manifested by increased levels of BNP, CK-MB, and Tn-I in peripheral blood.

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