CN113456767B

CN113456767B - Anti-coronavirus traditional Chinese medicine composition and application thereof

Info

Publication number: CN113456767B
Application number: CN202010243905.7A
Authority: CN
Inventors: 贾洪章
Original assignee: Individual
Current assignee: Beijing Hong Tianli Pharmaceutical Co ltd
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2022-11-01
Anticipated expiration: 2040-03-31
Also published as: CN113456767A

Abstract

The invention belongs to the field of medicine research and development, and relates to an anti-coronavirus traditional Chinese medicine composition and application thereof. The composition takes dandelion, radix stemonae, edible tulip, kudzuvine root, largehead atractylodes rhizome and cynanchum atratum as main raw material medicines, and can be prepared into a proper pharmaceutically acceptable preparation according to the requirement, wherein the optional additional raw material medicines comprise cortex lycii radicis and cortex mori radicis. The composition of the invention has the anti-coronavirus activities of SARS-CoV, MERS-CoV, SARS-CoV-2 and the like, has good drug safety, can be used for treating new coronary pneumonia, and is an innovative breakthrough of clinical treatment in the disease field.

Description

Anti-coronavirus traditional Chinese medicine composition and application thereof

Technical Field

The invention belongs to the field of research and development of medicines, and particularly relates to an anti-coronavirus traditional Chinese medicine composition and application thereof.

Background

Since the 21 st century, humans have been faced with serious threats to coronaviruses, including Severe Acute Respiratory Syndrome (SARS), also known as infectious atypical pneumonia, caused by SARS-CoV virus, which was developed in 2002-2003, mideast respiratory syndrome, which was initiated in 2012, caused by Middle East respiratory syndrome coronavirus (MERS _ respiratory syndrome), and recently developed new coronary pneumonia (NCP, world health organization known as covi-19), caused by SARS-CoV-2 virus. SARS-CoV, MERS-CoV and SARS-CoV-2 are all of these coronaviruses that pose a great threat to human society and are of global concern.

Although coronaviruses pose a great threat to human society, specific drugs against coronaviruses have not been found so far, and there is no satisfactory clinical treatment. Therefore, research and development of effective drugs for preventing and treating coronavirus diseases are urgent tasks faced by the medical field.

Traditional Chinese Medicine (TCM) has been used for thousands of years in China. Traditional Chinese medicine is mainly based on various chemical components in plant preparations, which interact and act simultaneously through various molecular targets and cellular mechanisms. The various components have different functions: some may exert therapeutic efficacy while others may reduce toxicity or improve bioavailability. At present, mixtures of plant extracts have been widely used worldwide for disease control and are increasingly accepted by western countries. Attempts have been made to develop antiviral drugs based on a combination of traditional Chinese medicines, but the current research is still in the preliminary exploration stage and lacks convincing experimental data. Therefore, anti-coronavirus Chinese medicinal compositions are also very desirable.

Disclosure of Invention

Therefore, it would be desirable to provide a medicament having anti-coronavirus activity. In particular, there is an urgent need for a drug effective against coronaviruses such as SARS-CoV, MERS-CoV and SARS-CoV-2, or a drug effective for the treatment and prevention of diseases caused by these coronaviruses.

In a first aspect, the present invention provides an anti-coronavirus traditional Chinese medicine composition (i.e., a traditional Chinese medicine composition capable of being used for preventing or treating coronavirus infectious diseases or disorders), the traditional Chinese medicine composition is an extract of raw material medicines of dandelion, stemona root, edible tulip, kudzuvine root, white atractylodes rhizome, cynanchum atratum, optionally cortex lycii radicis, and optionally cortex mori radicis, wherein the relative proportions of the raw material medicines are as follows: 30-70 parts of dandelion, 20-40 parts of radix stemonae, 20-40 parts of edible tulip, 20-50 parts of cynanchum atratum, 20-50 parts of kudzu root, 20-60 parts of bighead atractylodes rhizome, 20-60 parts of cortex lycii radicis and 20-60 parts of cortex mori radicis.

In a second aspect, the present invention provides a use of a traditional Chinese medicine composition in the preparation of a medicament for preventing or treating a disease caused by a coronavirus, wherein the traditional Chinese medicine composition is the traditional Chinese medicine composition according to the first aspect of the present invention.

In a third aspect, the present invention provides a use of a Chinese medicinal composition for preventing or treating a disease caused by a coronary virus, wherein the Chinese medicinal composition is the Chinese medicinal composition according to the first aspect of the present invention.

In a fourth aspect, the present invention provides a method of treating or preventing a coronavirus infectious disease or condition in a patient in need of such treatment, the method comprising administering to the patient an effective amount of a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition is a traditional Chinese medicine composition according to the first aspect of the present invention.

Detailed Description

In PCT application WO2018/210228A1 (the patent document is incorporated in the specification by reference in its entirety) published on 11/22/2018, an antiviral traditional Chinese medicine composition is disclosed, which is an extract of raw material medicines of dandelion, stemona root, edible tulip, kudzuvine root, bighead atractylodes rhizome, cynanchum atratum, optional cortex lycii radicis and optional cortex mori radicis, wherein the relative proportions of the raw material medicines are as follows: 30-70 parts of dandelion, 20-40 parts of radix stemonae, 20-40 parts of edible tulip, 20-50 parts of cynanchum atratum, 20-50 parts of kudzu root, 20-60 parts of bighead atractylodes rhizome, 20-60 parts of cortex lycii radicis and 20-60 parts of cortex mori radicis. According to WO2018/210228A1, in vitro and in vivo experiments show that the traditional Chinese medicine composition has remarkable antiviral activity on influenza A viruses (including H1N1, H7N7, H9N2 viruses and the like), zika viruses, dengue viruses (including dengue I viruses and dengue II viruses), chikungunya viruses and the like; and the medicine does not generate toxic or side effect after continuously administering the medicine to the rhesus monkey for 30 days under extremely high dosage, and has good safety.

However, WO2018/210228A1 does not disclose whether the traditional Chinese medicine composition is effective against coronaviruses. In view of the huge structural difference between influenza virus and coronavirus, and the fact that various anti-influenza virus drugs published and reported during the outbreak of new coronary pneumonia prove to be ineffective against coronavirus, it is difficult for those skilled in the art to predict whether the traditional Chinese medicine composition described in WO2018/210228A1 is effective against coronavirus.

The inventor surprisingly finds that the traditional Chinese medicine composition disclosed in WO2018/210228A1 has obvious effect of resisting coronavirus, especially has high-efficiency antiviral activity on various acute infectious disease viruses such as SARS-CoV, MERS-CoV and SARS-CoV-2, and shows obvious curative effect on new coronary pneumonia in clinical experiments.

Therefore, in some embodiments, the present invention provides an anti-coronavirus traditional Chinese medicine composition, which is an extract of the following raw materials, namely dandelion, radix stemonae, edible tulip, radix puerariae, rhizoma atractylodis macrocephalae, cynanchum atratum, optional cortex lycii radicis, and optional cortex mori radicis, wherein the relative ratio of the raw materials is as follows: 30-70 parts of dandelion, 20-40 parts of stemona root, 20-40 parts of edible tulip, 20-50 parts of cynanchum atratum, 20-50 parts of kudzuvine root, 20-60 parts of bighead atractylodes rhizome, 20-60 parts of cortex lycii radicis and 20-60 parts of cortex mori radicis.

In some embodiments, the preferred relative proportions of each drug substance are as follows:

dandelion: preferably 30 to 60 parts by weight, more preferably 35 to 55 parts by weight, most preferably 40 to 50 parts by weight;

radix stemonae: preferably 25 to 35 parts by weight, most preferably 25 to 30 parts by weight;

and (3) edible tulip: preferably 25 to 35 parts by weight, most preferably 25 to 30 parts by weight;

radix cynanchi atrati: preferably 20 to 45 parts by weight, more preferably 20 to 40 parts by weight, most preferably 25 to 35 parts by weight;

kudzu root: preferably 20 to 45 parts by weight, more preferably 20 to 40 parts by weight, most preferably 25 to 35 parts by weight;

white atractylodes rhizome: preferably 20 to 55 parts by weight, more preferably 20 to 50 parts by weight, most preferably 30 to 40 parts by weight.

When the weight ratio of each medicinal material is within the above preferred range, the anti-coronavirus activity of the Chinese medicinal composition according to the present invention is particularly significant.

After the cortex lycii radicis and the cortex mori radicis are introduced into the anti-coronavirus traditional Chinese medicine composition, the traditional Chinese medicine composition is more suitable for in-vivo application. For example, the Chinese medicinal composition for resisting coronavirus has excellent antipyretic effect after cortex lycii radicis and cortex mori radicis are additionally added, and the antipyretic effect is helpful for enhancing the resistance of a patient to the virus, so that the antiviral effect of the Chinese medicinal composition is further enhanced.

Therefore, in some preferred embodiments, the anti-coronavirus traditional Chinese medicine composition of the present invention additionally comprises cortex lycii radicis and cortex mori radicis, wherein the cortex mori radicis is 20 to 60 parts by weight (preferably 20 to 55 parts by weight, more preferably 20 to 50 parts by weight, and most preferably 25 to 40 parts by weight), and the cortex lycii radicis 20 to 60 parts by weight (preferably 20 to 55 parts by weight, more preferably 20 to 50 parts by weight, and most preferably 30 to 40 parts by weight).

In some embodiments, the present invention provides an anti-coronavirus traditional Chinese medicine composition, which comprises extracts of raw material medicines of dandelion, stemona root, edible tulip, kudzuvine root, white atractylodes rhizome, cynanchum atratum, cortex lycii radicis and cortex mori radicis, wherein the relative proportions of the raw material medicines are as follows:

dandelion: 30 to 70 parts by weight, preferably 30 to 60 parts by weight, more preferably 35 to 55 parts by weight, most preferably 40 to 50 parts by weight;

radix stemonae: 20 to 40 parts by weight, preferably 25 to 35 parts by weight, most preferably 25 to 30 parts by weight;

and (3) edible tulip: 20 to 40 parts by weight, preferably 25 to 35 parts by weight, most preferably 25 to 30 parts by weight;

radix cynanchi atrati: 20 to 50 parts by weight, preferably 20 to 45 parts by weight, more preferably 20 to 40 parts by weight, most preferably 25 to 35 parts by weight;

kudzu root: 20 to 50 parts by weight, preferably 20 to 45 parts by weight, more preferably 20 to 40 parts by weight, most preferably 25 to 35 parts by weight;

white atractylodes rhizome: 20-60 parts by weight, preferably 20-55 parts by weight, more preferably 20-50 parts by weight, most preferably 30-40 parts by weight;

white mulberry root-bark: 20 to 60 parts by weight, preferably 20 to 55 parts by weight, more preferably 20 to 50 parts by weight, most preferably 25 to 40 parts by weight;

cortex lycii radicis: 20 to 60 parts by weight, preferably 20 to 55 parts by weight, more preferably 20 to 50 parts by weight, most preferably 30 to 40 parts by weight.

In some embodiments, the Chinese medicinal composition of the present invention consists of only the extracts of the raw materials of dandelion, stemona root, edible tulip, pueraria root, atractylodes macrocephala, cynanchum atratum, cortex lycii radicis and cortex mori radicis in the above-specified ratio.

Those skilled in the art will understand that: any preferred dosage range for each of the above-described drug substances can be combined with any preferred dosage range for other drug substances at will, and the resulting combinations constitute particular embodiments of the present invention.

In other preferred embodiments, the present invention provides an anti-coronavirus traditional Chinese medicine composition, wherein the dosage of each raw material medicine is more than that described in each specific example provided in the specification, and the dosage of each medicine can be within about ± 16% relative to the specific dosage in the examples, and more preferably the dosage of each medicine can be within about ± 15%, 12%, 10%, 8%, 5%, 4%, 3% or 2% relative to the specific dosage in the examples.

The raw materials of the composition are all common traditional Chinese medicines, are recorded in detail in Chinese pharmacopoeia, chinese materia medica and the like, and can be obtained easily through commercial approaches. The origin and the like of the traditional Chinese medicinal materials are not particularly limited as long as the traditional Chinese medicinal materials meet the relevant national standards or regulations.

In the present specification, the term "dandelion" (Herba Taraxaci) refers to dried whole herb of Taraxacum mongolicum hand-Mazz, taraxacum sinicum Kitag, a plant of the Compositae family, or several plants of the same genus.

<xnotran> , "" (Radix Stemonae) Stemona sessilifolia (Miq.) Miq., Stemona japonica (Bl.) Miq. Stemona tuberosa Lour. . </xnotran>

In the present specification, the term "iphigenia indica" refers to dried pseudobulb of Makino, cremastra apenta (d.don) of arethusa, also known as "pleione indica".

In this specification, the term "kudzu root" (Radix Puerariae) refers to the dried root of Pueraria lobata (Willd.) Ohwi or Pueraria thomsonii Benth.

In this specification, the term "white Atractylodes rhizome" (Rhizoma Atractylodis macrocephala) refers to the dried rhizome of the asteraceae plant white Atractylodes rhizome, macrocephala koidz.

In this specification, the term "Cynanchum atratum" (Radix Cynanchi Atrati) refers to dried roots and rhizomes of Cynanchum atratum Bge. Or Cynanchum versicolor Bge. Of Asclepiadaceae.

In this specification, the term "Cortex Lycii" (Cortex Lycii) refers to the dried root bark of the solanaceae plant Lycium barbarum Chinese mill or Lycium barbarum l.

In the present specification, the term "Cortex Mori radicis" (Cortex Mori) refers to the dried root bark of Morus alba l.

In the present specification, the term "extract" refers to an extract obtained by extracting a raw material of a Chinese medicinal material using water or an organic solvent. Preferably, an extraction solvent such as water, a C1-C6 alcohol, hexane, chloroform, methyl acetate, diethyl ether, and the like can be used, more preferably, the extraction solvent is water, methanol, ethanol, propanol, butanol, pentanol, hexanol, or a combination thereof, and most preferably, the extraction solvent is selected from water, ethanol, or a combination thereof.

Most preferably, the extract in the traditional Chinese medicine composition is a combination of an aqueous extract and an ethanol extract of each crude drug. Herein, the "water extract" refers to an extract obtained by extracting a Chinese medicinal material with water (including cold water, warm water or hot water). Here, the "ethanol extract" refers to an extract obtained by extracting a Chinese medicinal material with pure ethanol or an aqueous ethanol solution.

Preferably, the extract in the traditional Chinese medicine composition is dried. Most preferably, the extract in the Chinese medicinal composition of the present invention is in the form of dried powder or granules.

In order to obtain the Chinese medicinal composition of the present invention in the form of an extract composition, for example, each of the Chinese medicinal materials may be extracted with water to obtain an aqueous extract; or extracting with ethanol water solution with various concentrations (such as 40-95%) to obtain ethanol extract; the aqueous extract and the ethanol extract may also be combined together to obtain a combination of the aqueous extract and the ethanol extract. In some preferred embodiments, the Chinese medicinal composition of the present invention comprises a combination of aqueous and ethanol extracts. Most preferably, the traditional Chinese medicine composition of the invention is prepared by a method comprising the following steps:

1) Extracting herba Taraxaci, radix Stemonae, radix Cynanchi Atrati, optional cortex Mori and optional other materials with ethanol to obtain ethanol extract;

2) Mixing the dregs generated in the preparation of the alcohol extract in the step 1) with kudzu root, largehead atractylodes rhizome, indian iphigenia bulb, optional cortex lycii radicis and optional other medicinal materials, adding water for extraction, concentrating the obtained water extract, adding alcohol for precipitation, and taking supernatant fluid to obtain a water extract;

3) Combining the alcohol extract obtained in the step 1) and the water extract obtained in the step 2), and carrying out optional post-treatment to obtain the anti-coronavirus traditional Chinese medicine composition.

Preferably, the alcohol in the above step is an aqueous solution of ethanol, such as 40-95% aqueous solution of ethanol, or 40-80% aqueous solution of ethanol. Depending on the particular embodiment, various concentrations of aqueous ethanol solutions of 60%, 65%, 70%, 75%, 90%, etc. may be used.

The "post-treatment" as described in step 3) is a conventional post-treatment step well known in the art, and the desired post-treatment operation can be appropriately determined by those skilled in the art according to the finally desired form of the composition. For example, if only the composition in the form of a solution is required as an intermediate product, no post-treatment step is required. On the other hand, if a composition in solid form is desired, the post-treatment step may comprise concentration followed by drying to obtain the composition in solid form. The drying may be carried out by any drying method commonly used in the pharmaceutical field, such as spray drying, microwave drying, vacuum drying, etc.

The above step 1), step 2), before, after or in the middle of step 3) may include other additional operations, for example, for convenience of operation, the alcohol extract obtained in step 1) may be concentrated, and the water extract obtained in step 2) may be concentrated. If necessary, a sterilization operation may be performed.

In the present specification, a drug, component, etc., modified with "optional" means that the drug or component may be present in certain embodiments, and may be absent in certain other embodiments, as desired. Similarly, an operation or step modified by "optional" or "optionally" means that the step or operation may be present in the methods of certain embodiments, and may not be present in the methods of other certain embodiments, as desired.

The specific extraction time, repetition times, concentration of the ethanol solution, amount of the extraction solvent, and other operating parameters in each step can be appropriately adjusted as needed.

In some embodiments, the alcoholic extract of step 1) is prepared by: reflux-extracting herba Taraxaci, radix Stemonae, radix Cynanchi Atrati, and optionally cortex Mori with 6-10 times of ethanol for 1-3 times (each for 1-2 hr), filtering, mixing extractive solutions, and concentrating under reduced pressure to obtain the above alcoholic extract. For example, 8 times of 40-95% ethanol (such as 60%, 75% or 90% ethanol aqueous solution) can be added for 1 and half hours, and then filtered; then adding 8 times of 40-95% ethanol (such as 60%, 75% or 90% ethanol water solution) for extraction for 1 half hour, and filtering; mixing the filtrates to obtain the alcoholic extract.

In some embodiments, the water extract of step 2) is prepared by: mixing the residues generated in the preparation of the alcohol extract with radix Puerariae, pseudobulbus Cremastrae seu pleiones, atractylodis rhizoma, and optionally cortex Lycii, adding 6-12 times of water, reflux-extracting for 1-3 times (1-4 hr each time), filtering, mixing the filtrates, and concentrating into soft extract; adding ethanol water solution (such as 70-95% ethanol water solution) into the soft extract until ethanol concentration is 60-80%, standing for precipitation for 12-48 hr, and filtering to remove residue to obtain the water extract. For example, the dregs generated in the preparation of the alcohol extract are taken and combined with the kudzu root, the white atractylodes rhizome and the Indian iphigenia bulb, 10 times of water is added for extracting for 2 hours, and then the filtration is carried out; then adding 10 times of water for extraction for 2 hours, filtering, and combining the two obtained filtrates to obtain water extract; concentrating to obtain soft extract, adding 75% or 90% ethanol water solution to ethanol final concentration of about 70%, standing for 36 hr, filtering to remove residue to obtain water extract;

in some embodiments, the step 3) operates to: combining the alcohol extract obtained in the step 1) and the water extract obtained in the step 2), concentrating and spray-drying to obtain the anti-coronavirus traditional Chinese medicine composition.

The anti-coronavirus traditional Chinese medicine composition is prepared from medicinal materials such as dandelion, radix stemonae, edible tulip, kudzu root, white atractylodes rhizome, cynanchum atratum and the like. If necessary, the traditional Chinese medicine composition of the invention can comprise other traditional Chinese medicines (or traditional Chinese medicine extracts).

In other embodiments, the Chinese medicinal composition of the present invention may further comprise other Chinese medicinal materials as needed or for other purposes in addition to dandelion, stemona root, bulb of edible tulip, pueraria root, atractylodis macrocephala, cynanchum atratum, optionally cortex lycii radicis, and optionally cortex mori radicis.

The traditional Chinese medicine composition can be used as a pharmaceutical active substance, and can be added with pharmaceutically acceptable carriers or auxiliary agents as required to prepare a required preparation according to the conventional technology of pharmaceutics. The pharmaceutically active substance in the formulation may be 0.1-99.9% (e.g. 1-99% or 50-98% or 50-95%, etc.), the remainder being pharmaceutically acceptable carriers or adjuvants.

The formulation of the invention may be in any pharmaceutically acceptable dosage form, including: granules, tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, dropping pills, granules, pills, powders, suspensions, powders and the like.

Preferably, the formulations of the invention are oral dosage forms, such as: granules, tablets, capsules, pills and the like.

Preferably, the pharmaceutical preparation of the present invention is in unit dosage form, which means the unit of the preparation, such as each tablet of the tablet, each bag of the granule, each capsule of the capsule, etc.

The formulations in unit dosage form preferably contain from about 1% to about 90% (e.g., 20% -80% or 30-60%) of the active ingredient. For example, a unit dosage form for single administration, such as a capsule, tablet or dragee, can contain from about 1mg to about 100g (e.g., 10mg to 80g, 50mg to 50g, 1g-20g, etc.) of the active ingredient.

For the preparation of suitable dosage forms, the Chinese medicinal composition of the present invention as a pharmaceutically active substance may be optionally mixed or associated with inorganic or organic, solid or liquid pharmaceutically acceptable carriers or adjuvants suitable for administration. Suitable carriers include, for example, fillers such as sugars (e.g. lactose), mannitol or sorbitol, cellulose preparations and/or calcium phosphates (tricalcium phosphate or calcium hydrogen phosphate); binders such as starch paste, gelatin, methylcellulose and/or polyvinyl pyrrolidone; disintegrating agents such as starch, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (e.g., sodium alginate).

In some embodiments, the present invention also provides a method of treating a coronavirus infectious disease or condition in a patient in need thereof, the method comprising administering to the patient an effective amount of a traditional Chinese medicine composition according to any embodiment of the present invention. Wherein the patient is preferably a mammal, particularly preferably a human. The administration is preferably oral.

In some embodiments, the present invention also provides the use of a traditional Chinese medicine composition according to any one of the embodiments of the present invention for treating a disease or condition caused by a coronavirus in a patient. Wherein the patient is preferably a mammal, particularly preferably a human.

In some embodiments, the present invention also provides the use of a traditional Chinese medicine composition according to any embodiment of the present invention for the preparation of a medicament for the treatment of a coronavirus infectious disease or disorder. Wherein the disease or condition is preferably a disease or condition in a mammal, especially a human. The medicament is preferably an oral medicament.

In the present specification, the term "treatment" refers to both prophylactic or preventative treatment as well as curative or palliative treatment of a coronavirus infectious disease or disorder.

In the present specification, the term "Coronavirus" refers to a virus of the genus Coronavir (Coronavir) of the family Coronaviridae (Nidovirales) of the order Nidovirales. Coronaviruses are generally enveloped (envelope) RNA viruses with a linear single-stranded positive strand genome, and are a large group of viruses widely found in nature.

The 7 coronaviruses currently known to infect humans are HCoV-229E, HCoV-OC 43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2 (also known as 2019-nCoV), respectively, where SARS-CoV, MERS-CoV and SARS-CoV-2 cause severe disease. The Chinese medicinal composition is particularly suitable for resisting SARS-CoV, MERS-CoV and SARS-CoV-2.

In the present specification, the term "coronavirus infectious disease or condition" refers to a disease or condition caused or contributed to by a coronavirus.

Particularly suitable for prevention or treatment with the Chinese medicinal composition of the present application are SARS (Severe acute respiratory syndrome), MERS (middle east respiratory syndrome), covid-19 (New crown pneumonia, also known as NCP) and their related diseases or complications.

The particular mode and dosage of administration in the treatment can be selected by the attending physician, taking into account the particular circumstances of the patient, especially the age, weight, lifestyle, activity level, severity of the disease, and the like.

Generally, the single dose in mammals is in the range of about 1-20,000mg/kg. For example, for preferred treatment of human patients, suitable dosages may range from 1 to 2,000mg/kg, 2 to 1,000mg/kg, 5 to 500mg/kg or 10 to 100 mg/kg when administered as a premix of herbal extracts. If desired, the amount can also be divided into several portions, optionally averaged. The above-mentioned doses may be administered repeatedly at intervals, for example, three times a day, once a week, and the like.

In the present specification, "mg/kg" or "mg/kg^-1"means the milligrams given per kilogram of body weight of the mammal (including man) to be treated," g/kg "or" g.kg^-1By "is meant the grams per kilogram body weight of the mammal (including a human) to be treated.

The above-mentioned preferred embodiments of the invention and preferred aspects and preferences of the individual features can be combined in any desired manner, and all combinations of these embodiments are intended to be within the scope of the invention.

The beneficial effects of the invention are as follows:

the results of in vitro pharmacodynamic tests prove that the traditional Chinese medicine composition has obvious effect of resisting coronavirus, especially SARS-CoV, MERS-CoV and SARS-CoV-2 virus, and clinical experiments show that the traditional Chinese medicine composition has exact curative effect on new crown pneumonia and is safe for clinical use.

Therefore, the traditional Chinese medicine composition can be used for preventing and treating various coronavirus infectious diseases, has good safety and convenient use, and is an innovative and important breakthrough in the clinical treatment of the disease field.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The above-mentioned aspects of the present invention will be further described in detail by the following specific embodiments of examples. It should be understood that the specific examples described herein are intended to be illustrative only and are not intended to be limiting. Any modification made without departing from the spirit and principle of the present invention and equivalent replacement or improvement made according to the common knowledge and conventional means in the art should be included in the protection scope of the present invention.

The numbers reported in the following examples are as precise as possible, but it is understood by those skilled in the art that each number should be understood as a divisor, not an absolutely exact number, due to inevitable measurement errors and experimental operating problems. For example, due to errors in the weighing apparatus, it should be understood that the weight values for each drug in the compositions of the examples may have errors of ± 2% or ± 1%.

Example 1: preparation of Chinese medicinal composition

Example 6 in WO2018/210228A1 was repeated to obtain the traditional Chinese medicine composition of example 1 of the present application. Specifically, the raw material medicaments used in the traditional Chinese medicine composition of the embodiment 1 are as follows:

dandelion: 480g

Stemona root: 290g

And (3) edible tulip: 290g

Radix cynanchi atrati: 320g

Kudzu root: 320g

White atractylodes rhizome: 385g

White mulberry root-bark: 320g

Cortex lycii radicis: 385g

Among the raw materials used, dandelion is collected from north China Hebei province, cynanchum atratum is collected from south China Henan province, kudzuvine root is collected from north China Hebei province, sessile stemona root is collected from Guangxi province, pseudobulbus cremastrae seu pleiones is collected from Sichuan province, largehead atractylodes rhizome is collected from Zhejiang province, white mulberry root-bark is collected from Shandong province, and cortex lycii radicis is collected from autonomous region of Ningxia Hui nationality.

The specific preparation process of the traditional Chinese medicine composition of the embodiment 1 is as follows:

(1) Extracting 480g of dandelion, 290g of radix stemonae, 320g of cynanchum atratum and 320g of cortex mori radicis with 8 times of 60% ethanol for 1 hour and half, and filtering; then 8 times of 60% ethanol is added for extraction for 1 half hour, and filtration is carried out; mixing the two filtrates to obtain an alcohol extract;

(2) Taking the medicine residues generated in the preparation of the alcohol extract, combining 320g of kudzu root, 290g of edible tulip, 385g of bighead atractylodes rhizome and 385g of cortex lycii radicis, adding 10 times of water for extracting for 2 hours, then adding 10 times of water for extracting for 2 hours, filtering, combining the obtained filtrates of the two times, concentrating into thick paste, adding 90% ethanol and stirring until the final concentration of the ethanol is about 70%, standing for 36 hours, and taking the supernatant to obtain water extract;

(3) And (3) combining the alcohol extract obtained in the step (1) and the water extract obtained in the step (2), concentrating, and performing spray drying to obtain the traditional Chinese medicine composition in the embodiment 1. The resulting composition was a brown powder weighing about 0.48Kg and was used directly in the following in vitro and clinical experiments.

Various pharmacodynamic studies were performed on the Chinese medicinal composition described in example 1 (the composition is referred to as "test drug" or "test substance" in the experiment) to verify the drug efficacy.

Example 2: experiment of Chinese medicinal composition for inhibiting SARS-CoV in vitro

1. Experimental Material

1) The tested drugs are: example 1 Chinese medicinal composition

2) Cell: vero-E6, provided by the institute for microbiology and logistics disease, institute molecular biology, institute of military medical sciences, china.

3) Virus: SARS virus (SARS-CoV) BJ-01 strain is provided by institute of microbioepidemics research institute of Chinese academy of military medical sciences.

CPE method for determining half Toxicity Concentration (TCID) of virus to Vero-E6 cells₅₀)

Vero-E6 cells were seeded in 96-well plates at 100. Mu.L/well containing 2X 10 cells⁴Cell/well, 5% 37 ℃,5% CO₂After 24 hours of culture, the cells grew into a monolayer. Diluting the virus stock solution with the culture solution 10 times in sequence to give 9 concentrations, 4 wells per concentration, 37 ℃ and 5% CO₂And (5) incubator culture. Cytopathic effect (CPE) was observed daily with an inverted microscope. The cytopathic effect is + 25%, 26-50% are +++, 51-75% are +++, and 76-100% are ++++. The degree of cytopathic effect (CPE) was recorded. Calculation of half the viral infectious dose (TCID) by Reed-Muench method₅₀)。

Determination of TCID of SARS virus BJ-01 strain on Vero-E6 cell by CPE method₅₀Is 10^-7。

Determination of half inhibitory concentration (TC) of test drug on Vero-E6 cells by MTT method₅₀)

Vero-E6 cells were seeded in 96-well plates at 100. Mu.L/well containing 2X 10 cells⁴Cells were cultured at 37 ℃ for 24 hours per well, and the cells grew into a monolayer. The supernatant was aspirated off, and 5000, 2500, 1250, 625, 312.5. Mu.g/ml test drugs were added, 4 wells per concentration, respectively, with cell controls (cells + media). Observing for 5 days, adding MTT for staining for 4 hours, sucking out supernatant, adding DMSO for dissolving for 0.5 hour, measuring OD570nm absorption value with enzyme-linked detector, and calculating TC by Reed-Muench method₅₀。

Experiments show that the maximum nontoxic concentration of the tested drug (the traditional Chinese medicine composition in the example 1) is 2500 mug/ml, and the cell morphology is the same as that of the control cell at the concentration; calculating the half inhibitory concentration TC of the tested drug to the cell by using a Reed-Muench method₅₀3869 +/-125 mu g/ml (the measurement values of three experiments are 3750, 4000 and 3856 mu g/ml respectively).

Determination of antiviral Activity of test drug by MTT method

Vero-E6 cells were seeded in 96-well plates at 100. Mu.L/well containing 2X 10 cells⁴Cells were cultured at 37 ℃ for 24 hours per well, and the cells grew into a monolayer. Add 100. Mu.L SARS virus (100 TCID) per well₅₀I.e. 10^-5Dilution), after 2 hours of adsorption, the virus-containing supernatant was discarded. Then 2500, 1250, 625, 312.5, 156.25. Mu.g/ml of test drug per 4 wells of concentration were added to the different wells, respectively. At the same time, a virus control (cell + virus + culture medium), a cell control (cell + culture medium), and a drug control (drug + cell + culture medium at 2500. Mu.g/ml) were set. When the virus control well cells became ++++ the cytopathic results were recorded (cytopathic effect was +++ at 25% or less, between 26% and 50% lesions, between 51% and 75% lesions, between 76% and 100% lesions, and ++++ at normal cells), and the MTT staining method measured the cell activity. The test was repeated 3 times. Calculating half inhibitory concentration IC of drug to virus by Reed-Muench method₅₀。

Of three experimentsIC₅₀The values were 1327, 1127, 1357. Mu.g/ml, respectively, with a mean of 1270. + -. 125. Mu.g/ml. The therapeutic index TI = TC of the test drug₅₀/IC₅₀About 3.0.

The test results show that the traditional Chinese medicine composition in the example 1 has obvious effect of inhibiting SARS-CoV in vitro.

Example 3: experiment of Chinese medicinal composition for inhibiting SARS-CoV-2 in vitro

1. Experimental materials

1) The tested medicine is as follows: example 1 Chinese medicinal composition

2) Cell: vero-E6, provided by the institute for animal research and medicine, academy of Chinese medical sciences.

3) Virus: new coronavirus (SARS-CoV-2) is preserved at-80 deg.C by the institute of animal research and medicine of Chinese academy of medical sciences.

2. Test drug cytotoxicity test

(1) Taking sterile 96-well culture plate, adding 100 μ L of 5 × 10⁴Vero-E6 cells per ml, 37 ℃ C., 5% CO₂Culturing for 24 hours;

(2) The test drug is serially diluted in DMEM medium (containing 2% penicillin-streptomycin mixture and 16 μ g/ml trypsin) in equal proportion, and the stock solution, 1;

(3) Discarding cell culture solution in 96-well culture plate, adding diluted medicine respectively, each well is 100 μ L, each concentration is 4 multiple wells, each well is added with 100 μ L DMEM culture medium (containing 2% penicillin-streptomycin mixed solution and 16 μ g/ml trypsin);

(4) Cell controls were also set: adding 200 mu L of DMEM culture medium (containing 2% penicillin-streptomycin mixed solution and 16 mu g/ml trypsin) into a Vero-E6 cell 96-well culture plate growing into a single layer for 4 multiple wells;

(5)37℃、5％CO₂incubating in an incubator for 3 days;

(6) Cytopathic effect (CPE) was observed under an optical microscope, and changes in CPE in the cells were noted as "+", and changes in CPE-free or normal cell morphology were noted as "-".

Half-cell toxic concentration (CC) of drug₅₀) The calculation formula of (2) is as follows:

wherein A is the percentage of more than 50% of cytopathic rate, B is the percentage of less than 50% of cytopathic rate, C is log (dilution multiple), and D is log (drug concentration corresponding to less than 50% of cytopathic rate).

The tested drugs are respectively set at 8 concentrations for testing, the test results show that the tested drugs have toxicity to cells at the concentration of 5000 mug/ml or more, the results are shown in table 1 according to CC₅₀Calculating formula to obtain CC₅₀＝3548.13μg/ml。

TABLE 1 test results for toxicity of test drugs

3. Antiviral test of test drug

(1) Taking a sterile 96-well culture plate, adding 100 μ l of 5 × 10 concentration into each well⁴Vero-E6 cells per ml, 37 ℃,5% CO₂Culturing for 24 hours;

(2) Diluting the tested medicine to 5 concentrations, adding 100TCID with equal volume into each concentration hole₅₀Mixing the virus at 37 deg.C and 5% CO₂Hatching for lh;

(3) Discarding cell culture solution in 96-well culture plate, adding the above mixed solution, and repeating the wells with concentration of 4 for each tested drug, wherein each well is 100 μ l;

(4) Simultaneously setting cell contrast and virus contrast;

cell control: adding 200 mul DMEM culture medium (containing 2% penicillin-streptomycin mixed solution and 16 mu g/ml trypsin) into a Vero-E6 cell 96-well culture plate growing into a single layer for 4 multiple wells;

virus control: mu.l of DMEM medium (containing 2% penicillin-streptomycin mixture and 16. Mu.g/ml trypsin) per well was added with an equal volume of 100TCID₅₀And (3) viruses are added into 4 multiple wells, the mixture is acted at 37 ℃ for lh, after lh, cell culture fluid in a 96-well culture plate is discarded, and the virus fluid is added.

(5) Cells at 37 deg.C, 5%₂Incubating in an incubator for 4-5 days;

The percent virus inhibition by the drug was defined as:

drug-inhibited virus percentage% = (% (viral control morbidity-test cellular morbidity%)/viral control morbidity%) × 100%

Drug inhibits the half Effective Concentration (EC) of virus₅₀) The calculation formula is as follows:

wherein, A is the percentage of more than 50 percent of inhibition rate, B is the percentage of less than 50 percent of inhibition rate, C is log (dilution multiple), and D is log (drug concentration corresponding to less than 50 percent of inhibition rate).

The test results are shown in Table 2, in terms of EC₅₀Calculating formula to obtain the half effective concentration EC of the drug for inhibiting virus₅₀=1766.04 μ g/ml. In addition, the selection index of the test drug SI = CC₅₀/EC₅₀Is about 2.

TABLE 2 anti-SARS-CoV-2 Effect of the test drugs

The test results show that the traditional Chinese medicine composition in the embodiment 1 has obvious effect of inhibiting SARS-CoV-2 in vitro.

Example 4: research on clinical treatment of new coronary pneumonia (Covid-19) by using traditional Chinese medicine composition

In order to verify whether the traditional Chinese medicine composition has a treatment effect on the new coronary pneumonia (caused by SARS-CoV-2 virus infection), 61 new coronary pneumonia patients (including asymptomatic infectors) accepted by a fixed-point hospital in a certain city of Hubei province of China are taken as research objects and are researched in a parallel control and non-double-blind mode.

1. Research method

1.1 Experimental period

From 1/2/2020 to 20/2/2020.

1.2 general case and packet case

61 patients were from the New crown pneumonia fixed-point admission agency in Hubei, a city. All patients were investigated for their medical history and performed for routine and biochemical tests, CT, nucleic acid tests, etc. Among them, 21 cases in group A were asymptomatic infected patients who had no symptoms such as fever and cough, but had positive nucleic acid test. Both group B and group C were patients with neocoronary pneumonia, with 20 patients in group B being at the mean age (45 + -15.56) years and 20 patients in group C being at the mean age (50.4 + -18.26) years, with no statistical difference (P > 0.05).

The groups of intervention measures are as follows:

observation group (group a): for group A asymptomatic infected subjects, the composition of example 1 was administered alone at a dose of 1.5 g/dose, 3 times/day.

Experimental group (group B): the composition of example 1 was administered at a dose of 1.5 g/dose, 3 times/day; meanwhile, according to the scheme of diagnosis and treatment scheme for novel coronavirus pneumonia (fifth edition), the corresponding standard treatment scheme is given for treatment, including oxygen therapy, virus resistance, bacteria resistance and the like.

Conventional control group (group C): according to the scheme of the diagnosis and treatment scheme for novel coronavirus pneumonia (fifth edition), the corresponding standard treatment scheme is given for treatment, including oxygen therapy, antivirus, antibiosis and the like.

The effect of the composition of example 1 was observed alone in 21 asymptomatic infectors of group a; group B20 and group C20 were used as concurrent controls.

1.3 inclusion and exclusion criteria

Inclusion criteria were: 1) According to the diagnosis standards of the diagnosis and treatment scheme for the novel coronavirus pneumonia (fifth edition) and the prevention and control scheme for the novel coronavirus pneumonia (fourth edition), the new coronavirus pneumonia is diagnosed (Covid-19); 2) Age between 18 and 80 years; 3) The standard of treatment is light or common type, or patients without symptom infection.

Exclusion criteria: 1) Those who cannot be matched with a treatment regimen; 2) Patients with serious basic diseases of other organs such as heart, liver and kidney; 3) Pregnant women.

1.4 statistical analysis method

Statistical analysis was performed using SPSS24.0, the measured data was compared using the t-test, and the counted data was compared using the chi-square test. The significance is based on the P value less than 0.05.

2. Results of the experiment

2.1 Total negative conversion ratio of Observation group (group A)

In 21 cases in group A, 18 cases turned negative after 5 days, and the total negative conversion rate was 85.71%. No adverse reactions were observed during treatment for all patients, and no new symptoms appeared.

2.2 symptom improvement:

the following table 3 shows the comparison of the symptom improvement of the experimental group and the control group.

TABLE 3 comparison of two groups of symptoms

The overall symptom improvement rate (97.06%/vs 38.71%) was significantly superior in the experimental group compared to the control group, and P <0.001, which was statistically significantly different. Wherein the effect on heat generation is particularly significant. The symptoms of weakness common to new coronary pneumonia were not statistically analyzed since 2 cases were counted in group B.

2.2 average annealing time:

the average defervescence time of the group B is 2.35 +/-0.72 hours, and the average defervescence time of the group C is 7.36 +/-

1.52 hours, P =0.0000. This indicates that the mean time to defervesce was significantly shorter in the experimental group compared to the control group, with statistically significant differences.

2.3 curative discharge rate

Table 4 below shows the comparison of the discharge rate of the cured products of the experimental group and the control group.

TABLE 4 comparison of the discharge rates of the two groups for cure

The experimental group had a higher cure rate (70% vs 35%) compared to the control group, and the difference was statistically significant (P < 0.05); the experimental group was more effective (90% vs 60%) than the control group, and the difference was statistically significant (P < 0.05).

2.4 Severe conversion

Severe transformation was compared between the two groups of patients and the results were as follows:

TABLE 5 comparison of the discharge rates of the two groups for cure

The severe transformation of the experimental group is less than that of the control group, and the difference has significance (P < 0.05).

3. Conclusion of the study

In 21 out of the observation group, 18 turned negative, the overall negative conversion rate was 85.71%, and no adverse reaction was observed in all patients during the treatment period.

The experimental group treated with the Chinese medicinal composition of example 1 is obviously superior to the control group in terms of the overall symptom improvement rate (97.06%/vs 38.71%, P < 0.001), the average defervescence time (2.35 + -0.72vs 7.36 + -1.52, P < 0.001), the effective rate (90%/vs 60%, P < 0.05), the cure rate (70%/vs 35%, P < 0.05), the severe transformation rate (0%/vs 20%), etc., the effect on improving the main symptoms related to the new crown pneumonia such as fever, hypodynamia, cough, etc. is obvious, and the total effective rate and the cure rate are also obviously higher than the control group; the effect on the aspect of fever reduction is very remarkable, the average fever reduction time is obviously shortened compared with the conventional treatment, the effect is totally achieved on 14 observed patients (100 percent), and rebound is avoided after the fever reduction.

Therefore, the traditional Chinese medicine composition is safe for clinical use, has definite curative effect on the Xinguan pneumonia, has obvious effects in aspects of improving the negative turning rate, improving symptoms, improving the effective rate, the cure rate and the like, particularly has an antipyretic effect, and has quick response, stability and no rebound.

Although specific embodiments have been described above, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. It will be appreciated that various changes can be made in adapting the description of different embodiments without departing from the broader scope disclosed herein and as interpreted in accordance with the following claims.

Claims

1. The application of a traditional Chinese medicine composition in preparing a medicine for preventing or treating diseases caused by coronavirus is characterized in that the traditional Chinese medicine composition is an extract of raw material medicines of dandelion, radix stemonae, edible tulip, radix puerariae, rhizoma atractylodis macrocephalae, cynanchum atratum, cortex lycii radicis and cortex mori radicis, wherein the relative proportion of the raw material medicines is as follows: 30-70 parts of dandelion, 20-40 parts of radix stemonae, 20-40 parts of edible tulip, 20-50 parts of cynanchum atratum, 20-50 parts of kudzu root, 20-60 parts of bighead atractylodes rhizome, 20-60 parts of cortex lycii radicis and 20-60 parts of cortex mori radicis, wherein the coronavirus is selected from SARS-CoV BJ-01 strain and SARS-CoV-2.

2. The use of claim 1, wherein the extract is an aqueous and/or ethanol extract of a drug substance.

3. The use of claim 1, wherein the medicament is an oral medicament.

4. The use of claim 1, wherein: 30-60 parts of dandelion; 25-35 parts of radix stemonae; 25-35 parts of edible tulip; 20-45 parts of cynanchum atratum; 20-45 parts of kudzu root; 20-55 parts of bighead atractylodes rhizome; 20-55 parts of cortex lycii radicis; 20-55 parts of cortex mori radicis.

5. The use according to any one of claims 1 to 4, wherein the Chinese medicinal composition is prepared by a preparation method comprising the steps of:

1) Extracting herba Taraxaci, radix Stemonae, radix Cynanchi Atrati, and cortex Mori with alcohol to obtain alcohol extract;

2) Mixing the dregs generated in the preparation of the alcohol extract in the step 1) with the kudzu root, the white atractylodes rhizome, the Indian iphigenia bulb and the cortex lycii radicis, adding water for extraction, concentrating the obtained water extract, adding alcohol for precipitation, and taking supernatant fluid to obtain a water extract;

3) Mixing the alcohol extract obtained in the step 1) and the water extract obtained in the step 2), and performing post-treatment to obtain the traditional Chinese medicine composition;

wherein the alcohol in the above step is 40-95% ethanol water solution.

6. The use of any one of claims 1 to 4, wherein the traditional Chinese medicine composition is in a dry powder or granular form.

7. The use of claim 4, wherein: 40-50 parts of dandelion; 25-30 parts of radix stemonae; 25-30 parts of edible tulip; 25-35 parts of cynanchum atratum; 25-35 parts of kudzu root; 30-40 parts of bighead atractylodes rhizome; cortex lycii radicis 30-40 parts by weight; 25-40 parts of cortex mori radicis.