CN111643621A

CN111643621A - Antiviral traditional Chinese medicine composition, preparation method and application thereof

Info

Publication number: CN111643621A
Application number: CN202010526700.XA
Authority: CN
Inventors: 万天石
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-06-09
Filing date: 2020-06-09
Publication date: 2020-09-11

Abstract

The invention relates to the technical field of medicine research, and particularly discloses an antiviral traditional Chinese medicine composition which comprises the following components in parts by weight: 20-160 parts of astragalus membranaceus, 10-100 parts of bighead atractylodes rhizome, 10-100 parts of divaricate saposhnikovia root, 10-100 parts of fructus forsythiae, 10-100 parts of wrinkled gianthyssop herb, 10-100 parts of honeysuckle, 10-100 parts of baical skullcap root, 10-100 parts of rhizoma anemarrhenae, 3-80 parts of coptis chinensis, 10-100 parts of great burdock achene, 3-80 parts of rhizoma atractylodis, 3-30 parts of betel nut, 10-100 parts of radix stemonae, 5-20 parts of liquorice, 10-100 parts of cyrtomium rhizome, 3-20 parts of ginger and 3-80 parts of buffalo. The antiviral traditional Chinese medicine composition has the characteristics of strengthening body resistance and invigorating yang, eliminating dampness by aroma, removing dirt and eliminating evil, warming lung and expelling toxin, tonifying qi and strengthening body resistance and enhancing immunity.

Description

Antiviral traditional Chinese medicine composition, preparation method and application thereof

Technical Field

The invention relates to the technical field of medicine research, in particular to an antiviral traditional Chinese medicine composition, a preparation method and application thereof.

Background

At present, the number of patients with pneumonia caused by the novel coronavirus is continuously increased in global diagnosis, and the medication requirement is increasingly urgent. For potential therapeutic drugs, western medicines include interferon, lopitavir, ritonavir, redexivir, bartinib and lopinavir, and traditional Chinese medicines include lotus antipyretic capsule. The western medicines have potential curative effects on severe pneumonia patients caused by the novel coronavirus, but for asymptomatic infectors and mild patients, the symptoms cannot be completely relieved by using the expensive western medicines, and various adverse reactions and side effects also exist.

Chinese herbs have been used for thousands of years in China. Traditional Chinese medicine is mainly based on multiple chemical components in plant preparations, which interact and act simultaneously through multiple molecular targets and cellular mechanisms. The various components have different functions, some of which may exert therapeutic effectiveness, while others may reduce toxicity or increase bioavailability. At present, mixtures of plant extracts have been widely used worldwide for disease control and are increasingly accepted by western countries.

Disclosure of Invention

Therefore, the invention provides a traditional Chinese medicine composition with antiviral activity, which meets the practical requirements.

In order to realize the purpose, the invention is realized by the following technical scheme:

an antiviral traditional Chinese medicine composition comprises the following components in parts by weight:

20-160 parts of astragalus membranaceus, 10-100 parts of bighead atractylodes rhizome, 10-100 parts of divaricate saposhnikovia root, 10-100 parts of fructus forsythiae, 10-100 parts of wrinkled gianthyssop herb, 10-100 parts of honeysuckle, 10-100 parts of baical skullcap root, 10-100 parts of rhizoma anemarrhenae, 3-80 parts of Sichuan coptis root, 10-100 parts of great burdock achene, 3-80 parts of rhizoma atractylodis, 3-30 parts of betel nut, 10-100 parts of radix stemonae and 5-20 parts of liquorice.

A preparation method of an antiviral traditional Chinese medicine composition comprises the following steps:

s1: extracting fructus forsythiae with water, and filtering to obtain volatile oil;

s2: extracting Arecae semen and herba Agastaches with ethanol to obtain ethanol extractive solution;

s3: decocting radix astragali, Atractylodis rhizoma, Scutellariae radix, Coptidis rhizoma, fructus Arctii, rhizoma Atractylodis, radix Stemonae, and Glycyrrhrizae radix with water to boil, adding flos Lonicerae and rhizoma anemarrhenae, decocting, filtering, adding the oil extract obtained in step S1 into the filtrate to obtain water phase liquid, and concentrating into fluid extract;

s4: adding an alcohol solution into the clear paste obtained in the step S3, mixing, concentrating, treating at 1-8 ℃ for 24h, filtering, removing alcohol from the filtrate, mixing with the alcohol extract obtained in the step S2, concentrating into clear paste, and drying to obtain dry paste powder;

s5: mixing the dry paste powder and the auxiliary material (1) (0.3-0.5) according to the mass ratio, and adding an alcohol solution to be used as a raw material for preparing the medicinal preparation.

An antiviral medicinal preparation comprises the Chinese medicinal composition and a pharmaceutically acceptable carrier or auxiliary agent.

The application of the traditional Chinese medicine composition in preparing a medicine for treating or preventing a novel coronavirus 2019-nCoV infectious disease or disorder.

Compared with the prior art, the invention has the beneficial effects that:

1. the antiviral traditional Chinese medicine composition has obvious inhibition effect on the proliferation of coronavirus, and the inhibition effect tends to increase along with the increase of the administration concentration.

2. The invention discovers through in vitro direct virus killing tests that: the traditional Chinese medicine composition prepared by taking astragalus, bighead atractylodes rhizome, divaricate saposhnikovia root and weeping forsythia as monarch drugs, taking wrinkled gianthyssop, honeysuckle, baical skullcap root, common anemarrhena rhizome and Sichuan coptis root as ministerial drugs, taking burdock, swordlike atractylodes rhizome, betelnut and sessile stemona root as adjuvant drugs and taking liquorice as a conductant drug has obvious antiviral activity against the novel coronavirus 2019-nCoV.

3. The traditional Chinese medicine composition preparation provided by the invention can prevent 2019-nCoV virus from adsorbing Vero cells, so that the combination of the virus and a receptor is reduced, and the virus is prevented from invading the Vero cells. The traditional Chinese medicine preparation obtained by the preparation process can better play a role in preventing viruses from infecting cells.

4. The antiviral traditional Chinese medicine composition provided by the invention has an obvious effect of inhibiting the replication of 2019-nCoV. The antiviral traditional Chinese medicine preparation prepared by the process provided by the invention has a treatment effect obviously superior to that of the traditional Chinese medicine preparation obtained by decocting by a traditional method.

Drawings

FIG. 1 is a graph of pre-treatment CT results for a new crown patient case provided by an embodiment of the present invention;

fig. 2 is a graph of CT results after a new coronary patient case provided by the embodiment of the present invention is treated with the traditional Chinese medicine composition provided by the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Traditional Chinese medicine composition formula

The invention provides an antiviral traditional Chinese medicine composition which comprises the following components in parts by weight: 20-160 parts of astragalus membranaceus, 10-100 parts of bighead atractylodes rhizome, 10-100 parts of divaricate saposhnikovia root, 10-100 parts of fructus forsythiae, 10-100 parts of wrinkled gianthyssop herb, 10-100 parts of honeysuckle, 10-100 parts of radix scutellariae, 10-100 parts of rhizoma anemarrhenae, 3-80 parts of coptis chinensis, 10-100 parts of burdock, 3-80 parts of rhizoma atractylodis, 3-30 parts of betel nut, 10-100 parts of radix stemonae and 5-20 parts of liquorice; the virus is a novel coronavirus 2019-nCoV.

The invention discovers through in vitro direct virus killing tests that: the traditional Chinese medicine composition prepared by taking astragalus, bighead atractylodes rhizome, divaricate saposhnikovia root and weeping forsythia as monarch drugs, taking wrinkled gianthyssop, honeysuckle, baical skullcap root, common anemarrhena rhizome and Sichuan coptis root as ministerial drugs, taking burdock, swordlike atractylodes rhizome, betelnut and sessile stemona root as adjuvant drugs and taking liquorice as a conductant drug has obvious antiviral activity against the novel coronavirus 2019-nCoV.

Further, the antiviral traditional Chinese medicine composition also comprises the following components in parts by weight: 10-100 parts of cyrtomium rhizome, 3-20 parts of ginger and 3-80 parts of buffalo horn powder.

The monarch drug of the invention is astragalus root, white atractylodes rhizome, divaricate saposhnikovia root and weeping forsythia for clearing away heat and toxic material and dispelling wind-cold-dampness arthralgia, and simultaneously has the main functions of anti-inflammation, anti-infection and enhancing the immunity of the organism; the ministerial drugs are herba Agastaches, flos Lonicerae, Scutellariae radix, Coptidis rhizoma, rhizoma anemarrhenae, cornu Bubali powder, radix astragali, Atractylodis rhizoma, fructus forsythiae, and have effects of clearing heat and toxic substances, eliminating cold and dampness, relieving epigastric distention and sweating, invigorating spleen and invigorating qi; the functions of dispersing lung qi, relieving sore throat, dispelling phlegm, relieving asthma, relieving cough, expelling pus and enhancing detoxification function; cornu Bubali powder can be used for treating pestilence, hyperpyrexia, unconsciousness , inflammation, and infection; rhizoma Osmundae has effects of clearing pneumonia, relieving cough, relieving hemoptysis, and sore throat, relieving pain, and expelling pus; the ginger is heated to dispel dampness and warm yang to build middle energizer; the adjuvant drugs are fructus Arctii, rhizoma Atractylodis, Arecae semen, and radix Stemonae for dispersing lung qi, removing toxic substance, and inhibiting influenza and viral cold; leading the liquorice to detoxify and harmonize the effects of the other drugs in the recipe.

In some embodiments, the astragalus root is selected from any of 20 to 160 parts, such as 30 parts, 35 parts, 45 parts, 55 parts, 65 parts, 75 parts, 85 parts, 95 parts, 105 parts, 115 parts, 125 parts, 135 parts, 145 parts, 155 parts, more preferably 80 to 155 parts, and most preferably 95 to 135 parts.

In some specific embodiments, any part of 10 to 100 parts of the atractylodes macrocephala koidz is selected, such as 20 parts, 30 parts, 35 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts and 90 parts, more preferably 50 parts to 100 parts, and most preferably 70 parts to 90 parts.

In some embodiments, ledebouriella root is optionally present in any of 10 to 100 parts, such as 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts, more preferably 50 to 100 parts, and most preferably 70 to 90 parts.

In some embodiments, the forsythia suspensa is optionally selected from any of 10 to 100 parts, such as 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts, more preferably 50 parts to 100 parts, and most preferably 70 parts to 90 parts.

In some embodiments, the Agastache rugosa is optionally present in any of 10-100 parts, such as 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts, more preferably 50 parts to 100 parts, and most preferably 70 parts to 90 parts.

In some embodiments, the honeysuckle is optionally any one of 10-100 parts, such as 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts, more preferably 50 parts to 100 parts, and most preferably 70 parts to 90 parts.

In some embodiments, the scutellaria baicalensis is optionally any of 10 to 100 parts, such as 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts, more preferably 50 parts to 100 parts, and most preferably 70 parts to 90 parts.

In some embodiments, the amount of the anemarrhena asphodeloides is optionally any amount of 10 to 100 parts, such as 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts, more preferably 50 parts to 100 parts, and most preferably 70 parts to 90 parts.

In some specific embodiments, the coptis chinensis is optionally selected from any of 3 to 80 parts, such as 5 parts, 7 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, 35 parts, 40 parts, 45 parts, 50 parts, 55 parts, 60 parts, 65 parts, 70 parts and 75 parts, more preferably 25 to 60 parts, and most preferably 25 to 30 parts.

In some specific embodiments, the burdock is optionally any of 10-100 parts, such as 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts, more preferably 50 parts to 100 parts, and most preferably 70 parts to 90 parts.

In some embodiments, the amount of atractylodes rhizome is optionally any of 3 to 80 parts, such as 5 parts, 7 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, 35 parts, 40 parts, 45 parts, 50 parts, 55 parts, 60 parts, 65 parts, 70 parts, 75 parts, more preferably 25 to 60 parts, and most preferably 25 to 30 parts.

In some embodiments, the betel nut is optionally any of 3 to 30 parts, such as 5 parts, 7 parts, 10 parts, 13 parts, 15 parts, 17 parts, 20 parts, 23 parts, 25 parts, 27 parts, 30 parts, more preferably 25 to 60 parts, and most preferably 25 to 30 parts.

In some specific embodiments, the stemona root is selected from any of 10 to 100 parts, such as 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts and 90 parts, more preferably 50 to 100 parts, and most preferably 70 to 90 parts.

In some embodiments, any of 5 to 20 parts of licorice root, such as 6 parts, 8 parts, 10 parts, 12 parts, 14 parts, 16 parts, 18 parts, 20 parts, more preferably 8 to 15 parts, and most preferably 10 to 14 parts.

In some embodiments, any part of Cyrtomium fortunei 10-100 parts, such as 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts, more preferably 50 parts to 100 parts, and most preferably 70 parts to 90 parts.

In some embodiments, any of 3 to 20 parts of ginger are used, such as 4 parts, 5 parts, 6 parts, 8 parts, 10 parts, 12 parts, 14 parts, 16 parts, 18 parts, 19 parts, more preferably 12 to 18 parts, and most preferably 12 to 15 parts.

In some specific embodiments, the buffalo horn powder is optionally any of 3 to 80 parts, such as 5 parts, 7 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, 35 parts, 40 parts, 45 parts, 50 parts, 55 parts, 60 parts, 65 parts, 70 parts, 75 parts, more preferably 25 parts to 60 parts, and most preferably 25 parts to 30 parts.

The term "part" in the present invention means only part by weight, and specifically any weight may be defined as 1 part, such as 0.001g, 0.01g, 0.05g, 0.1g, 0.5g, 1g, 10g, 100g, 1000g, 5000g, 10000g, etc.

The traditional Chinese medicine composition prepared from the traditional Chinese medicine raw materials has remarkable antiviral efficacy, and particularly aims at the novel coronavirus 2019-nCoV.

The raw materials of the composition are 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 some embodiments, the present invention provides an antiviral Chinese medicinal composition, wherein the dosage of each raw material drug is more than that described in each specific example provided in the present specification, and the dosage of each drug may vary within about ± 10% from the specific dosage in the examples, and more preferably within about ± 5%, 3%, 2% or 1% from the specific dosage in the examples. And it has been found that the antiviral activity of the Chinese medicinal composition according to the present invention is particularly significant when the weight ratio of the respective medicinal materials is within the above preferred range. Specifically, as shown in table 1, different formulations for processing each raw material are listed.

TABLE 1

In vitro pharmacodynamic assay

1. Test materials

A test article: according to the formula of the embodiments 1-6, the Chinese medicinal composition is prepared by directly adding water for decoction according to the traditional Chinese medicine method, concentrating the liquid medicine, and freeze-drying to obtain the freeze-dried powder.

Positive control 1: lianhua Qingwen granules (Beijing manufactured by Shaling pharmaceutical Co., Ltd., approved No.: national Standard Z20100040, specification: 6g × 9 bags).

Positive control 2: lopinavir (provided by Shijiazhuang four drug group).

The test particles, the lotus antipyretic particles and the lopinavir tablets are dissolved in DMSO in a vortex manner before use, are prepared into 500mg/mL solutions respectively, are 3300r/min, are centrifuged for 10min, and then supernatant is taken as stock solution for use and is stored at 4 ℃.

Cell and virus: MDCK cells are provided by the shanghai cell bank of the chinese academy of sciences. The novel coronavirus 2019-nCoV Chinese strain was provided by the Wuhan virus institute of Chinese academy of sciences.

Culture: the cell culture medium is MEM containing 5% bovine serum (FBS), and is placed in 37C, 5% CO₂Culturing in an incubator. Modified Eagle's Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum were purchased from GIBCO, USA.

2. Test main instrument and reagent

Dissolving dimethyl sulfoxide to prepare 1mg/mL mother solution, subpackaging and storing at 4 ℃; 3- (4, 5-Dimethylthiazole-2, 5-Diphenyltetrazolium bromide (MTT), DMSO, formaldehyde were obtained from Sigma, USA, methylcellulose and Crystal Violet were obtained from Jidbis Biotech, Guangzhou.

MEM powder (Gibco), newborn bovine serum (Hangzhou Sijiqing biologicals Co., Ltd.), MTT (Sigma), dimethyl sulfoxide (Amresco), inverted biomicroscope (OLYMPUS Co., Japan), clean bench (HFsafe-1200), carbon dioxide incubator (HF90), microplate reader (KHBST-360), and shaker (MX 100-4P).

3. Test method

3.1 cytotoxicity assay

After digesting the cells with pancreatin, the cells were pressed to 5 × 10⁵MDCK cells were seeded in 96-well plates at a density of one ml, and 100. mu.L of MEM medium was added thereto at 37 ℃ with 5% CO₂Culturing until a monolayer of cells is formed. Dosing was started after 1 h.

The experimental groups are: a virus group, a normal cell group, a test article group, a positive control 1 group and a positive control 2 group. No drug and no cells were added to the virus group. The normal cell group is a MDCK normal cell culture group without virus. The drug in the test group was diluted to 7 dose groups (10mg/ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml, 0.625mg/ml, 0.312mg/ml, 0.156mg/ml) by a 2-fold dilution method. The drug of the positive control group 1 was diluted into 7 dose groups (10mg/ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml, 0.625mg/ml, 0.312mg/ml, 0.156mg/ml) by a 2-fold dilution method. The drugs in the positive control group 2 were diluted into 7 dose groups (80mg/ml, 40mg/ml, 20mg/ml, 10mg/ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml) by a 2-fold dilution method.

Adding the prepared medicines into cell culture wells in sequence, making 4 multiple wells for each dosage, placing at 37 deg.C and 5% CO₂After 48h incubation in an incubator, 20. mu. of LMTT solution (5mg/mL) was added to each well at 37 ℃ with 5% CO₂After further incubation for 4h, the supernatant was removed, 150. mu.L DMSO/well was added, shaken at 150r/min for 5min and then incubated at 37C, 5% CO₂Incubation was continued for 4 h. In Thermo Scientific^TMThe absorbance (OD) at 490nm of each well was measured on a Varioskan Flash full-wavelength scanning multifunction reader, and the half-toxic concentration TC was calculated by the Reed-Muench method₅₀. The rate of cell pathology is [ (A-B)/A ═]× 100%, wherein A is the OD value of the normal cell group, and B is the OD value of the test sample group, the positive control 1 group or the positive control 2 group.

3.2 drug inhibition of Virus assay

After digesting the cells with pancreatin, the cells were pressed to 5 × 10⁵MDCK cells were seeded in 96-well plates at a density of one ml, and 100. mu.L of MEM medium was added thereto at 37 ℃ with 5% CO₂Culturing until a monolayer of cells is formed.

The experimental groups are: a virus group, a normal cell group, a test article group, a positive control 1 group and a positive control 2 group. No drug and no cells were added to the virus group. The normal cell group is a MDCK normal cell culture group without virus. The test group was diluted 2-fold into 7 dose groups (20mg/ml, 10mg/ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml, 0.625mg/ml, 0.312 mg/ml). The drug of the positive control group 1 was diluted into 7 dose groups (10mg/ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml, 0.625mg/ml, 0.312mg/ml, 0.156mg/ml) by a 2-fold dilution method. The drugs in the positive control group 2 were diluted into 7 dose groups (80mg/ml, 40mg/ml, 20mg/ml, 10mg/ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml) by a 2-fold dilution method.

Adding the prepared medicines into cells in sequence respectively, culturing for 24 hr, making 4 multiple holes per dosage, placing at 37 deg.C and 5% CO₂Culturing in incubator, observing cell morphology change under inverted microscope, detecting relative amount of cell death by MTT method, and calculating half effective concentration (IC) of medicine₅₀) And a drug Therapeutic Index (TI). according to the following formula, the viral inhibition rate (%) is [ (A-B)/(C-B) × 100%]Wherein A is the OD value of the test sample group, the positive control group 1 or the positive control group 2, B is the OD value of the virus group, and C is the OD value of the normal cell group, and the inhibition rate of the drug to the influenza virus is calculated according to the formula.

3.3 statistical treatment

The data obtained in the text are statistically processed, drugs TC50 and IC50 are calculated by a Reed-Muench method to obtain drug therapy index (TI is TC50/IC50), SPSS17.0 software is used for analysis, the data are expressed by mean number standard deviation (x +/-s), metering data are tested by t, counting data are tested by x2, and p <0.05 is statistically significant.

4. Results

Virus titer TCID of virus was calculated according to Reed-Muench method₅₀Is 10^-3.50.1 ml. The virus titer for the test is 100TCID₅₀. The results of the drug toxicity test on cells and the test results of the drug inhibition of viruses are shown in Table 2.

TABLE 2

Comparing the results of the cytotoxicity test, it can be seen from Table 2 that TC is contained in the test substance group₅₀TC higher than the Positive control 1 group₅₀And TC of the test article₅₀TC of positive control 1 group₅₀Are all obviously higher than the positive control group 2, which shows that the toxicity of the test sample group to the cells is equivalent to that of the traditional Chinese medicine lotus antipyretic, and the toxicity to the cellsIs smaller than the lopinavir tablets. To compare IC₅₀The test sample group is obviously higher than the positive control group 2 and is equivalent to the positive control group 1; the test sample group has the effect of resisting 2019-nCoV virus which is equivalent to the antipyretic effect of the traditional Chinese medicine lotus, but is not as good as the lopinavir tablet, and the antiviral effect can be achieved only by the need of higher medicine concentration. In contrast to TI, the test group was equivalent to the positive control 1 group and the positive control 2 group, indicating that the drug of the test was effective for treating the disease caused by 2019-nCoV virus. Further comparing the test article groups, for different formulations, the test articles of examples 1-3 are superior to the test articles of examples 4-6 in toxicity to cells, inhibitory action against 2019-nCoV virus and medicinal effect in the safe concentration range of the medicament, which further shows the effect of basket fern, ginger and buffalo horn powder as ministerial medicaments in the whole antiviral Chinese medicinal composition.

In conclusion, the antiviral traditional Chinese medicine composition has the beneficial effects that:

the research result shows that the antiviral traditional Chinese medicine composition has obvious inhibition effect on the proliferation of coronavirus, and the inhibition effect tends to increase along with the increase of the administration concentration. Although the research proves that the antiviral traditional Chinese medicine composition has the effect of resisting the coronavirus on a cellular level, the effective components of the antiviral traditional Chinese medicine composition are not clear as a compound preparation of the traditional Chinese medicine, the specific action mechanism of the antiviral traditional Chinese medicine composition is still needed to be researched, and in addition, the safety of the clinical treatment of the antiviral traditional Chinese medicine composition provided by the invention is further evaluated.

Preparation method of antiviral traditional Chinese medicine composition

The antiviral traditional Chinese medicine composition can be directly provided in a combination mode (such as a medicine bag) of various raw material medicines, and patients can directly decoct with water according to the traditional Chinese medicine method after taking the medicine. However, preferably, the antiviral traditional Chinese medicine composition of the invention can be prepared into a traditional Chinese medicine extract composition (such as a premix) form by adopting a modern traditional Chinese medicine processing mode, so that the administration of a patient is more convenient and quicker, and the drug effect utilization rate is high.

Accordingly, in some embodiments, the present invention provides a method for preparing an antiviral Chinese medicinal composition, comprising the steps of:

In the present specification, the term "extract" refers to an extract obtained by extracting a Chinese medicinal material raw material using water or an organic solvent. Preferably, an extract obtained by using an extraction solvent such as water, C1-C6 alcohol, hexane, chloroform, methyl acetate, diethyl ether, etc., 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 water extract and ethanol extract of each raw material medicine. 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, in the step S1, the raw material added is buffalo horn powder, the mass of the added water is 8-12 times of the sum of the mass of the buffalo horn powder and the mass of the forsythia, and the treatment time after the water is added is 4 hours.

Preferably, in step S2, the alcohol-extracted solution is an aqueous solution of ethanol, such as 50-90% aqueous solution of ethanol, or 50-80% aqueous solution of ethanol, and depending on the specific embodiment, various concentrations of aqueous solutions of ethanol, such as 60%, 65%, 70%, 75%, etc., can be used. The mass of the alcohol extraction solution is 4-12 times of the sum of the masses of areca and agastache rugosus, and can be 5 times, 6 times, 7 times, 8 times, 9 times, 10 times and 11 times of the sum of the masses of the areca and agastache rugosus, depending on the specific implementation mode.

Preferably, in step S3, rhizoma Osmundae and rhizoma Zingiberis recens are added. The mass ratio of the added astragalus, the bighead atractylodes rhizome, the scutellaria baicalensis, the coptis chinensis, the great burdock achene, the rhizoma atractylodis, the radix stemonae and the liquorice to the added water is 1 (8-12), and the mass ratio can be 9 times, 10 times and 11 times of the sum of the mass ratio of the added astragalus, the large atractylodes rhizome, the scutellaria baicalensis, the coptis chinensis, the great burdock achene, the rhizoma atractylodis, the radix stem. Decocting in water until boiling, which means boiling under normal pressure and the temperature reaches 100 ℃. Adding flos Lonicerae and rhizoma anemarrhenae immediately after boiling at normal pressure, decocting for 2 hr, and filtering to obtain filtrate; and adding 8-12 times of water by mass, and decocting for 1.5h again. And filtering again to obtain filtrate, combining the two filtrates, adding the water phase liquid obtained after the oil extraction in the step S1 into the combined filtrate, mixing to obtain homogeneous phase, and concentrating to obtain the clear paste with the density of 1.0-1.15.

Preferably, in step S4, ethanol water solution with alcohol concentration of 95% is added to the fluid extract obtained in step S3, and the ethanol concentration after concentration is 50%.

Preferably, in step S5, the alcohol solution is 70% ethanol aqueous solution.

As shown in Table 3, the different processes in the steps S1-S4 are listed. The concrete step of S1 is listed as adding water with the mass being multiple of the sum of the mass of the buffalo horn powder and the mass of the forsythia, and adding water for post-treatment time; the column of the step S2 is an alcohol extraction solution (which is an aqueous solution of ethanol), and the mass of the alcohol extraction solution is a multiple of the sum of the mass of areca and ageratum; the column in the step S3 is that the water adding quality is multiple times of the sum of the qualities of astragalus, bighead atractylodes rhizome, cyrtomium rhizome, scutellaria, coptis, great burdock achene, rhizoma atractylodis, radix stemonae, ginger and liquorice, the decocting times and the decocting time each time; the column in the step S4 is an ethanol water solution with the added alcohol solution concentration of 95%, and the concentration of the concentrated ethanol is 50%; wherein, the antiviral traditional Chinese medicine composition is prepared by adopting the formula of the embodiment 1 according to the steps of S1-S4 in the embodiments 7-21, and a medicinal carrier is added after the step of S5 to prepare powder; comparative example 1 the raw materials are prepared according to the formula of example 1, and then are directly decocted with water according to the traditional method of traditional Chinese medicine to obtain the liquid medicine of the antiviral traditional Chinese medicine composition, and a medicinal carrier is added to prepare powder.

TABLE 3

The traditional Chinese medicine composition obtained by the preparation method can be used as a medicine active substance, and can be added with a pharmaceutically acceptable carrier or an auxiliary agent as required to prepare a required preparation according to the conventional technology of pharmaceutics. The pharmaceutically active substance in the preparation can be 0.1-99.9% (e.g. 10-99% or 20-80% or 50-75%), and the rest can be used as pharmaceutically acceptable carrier or adjuvant.

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 formulation of the invention is an oral dosage form, such as: granules, tablets, capsules, pills and the like.

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

The unit dosage form preferably contains 1-99.9% (e.g., 10-99%, or 20-80%, or 50-75%) of the active ingredient. For example, unit dosage forms for single administration, such as capsules, tablets, or dragees, can contain from about 1mg to about 100g (e.g., 10mg to 99g, 20mg to 80g, 50g to 75g, etc.) of the active ingredient.

The Chinese medicinal composition of the present invention as a pharmaceutically active substance may be optionally mixed or combined with inorganic or organic, solid or liquid pharmaceutically acceptable carriers or adjuvants suitable for administration in order to be formulated into a suitable dosage form. 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 the use of a traditional Chinese medicine composition according to any embodiment of the present invention for the treatment of a disease or condition caused by a virus, in particular an infectious disease caused by a novel coronavirus 2019-nCoV, 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 viral infectious diseases or disorders, in particular infectious diseases caused by the novel coronavirus 2019-nCoV. 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 viral infectious disease or disorder. In the present specification, the terms "viral infectious disease or disorder" and "disease or condition caused by a virus" have the same meaning, and are used interchangeably to refer to a disease or disorder caused by the spread and infection of a virus.

Particularly suitable for treatment with the Chinese medicinal composition of the present invention are diseases or disorders caused by viruses of the family coronaviridae, in particular of the genus coronavirus, especially 2019-nCoV. Examples of the symptoms include influenza, atypical pneumonia, fever, watery nasal discharge, asthenia, dry cough, shortness of breath, respiratory distress, respiratory failure, infectious viral pneumonia, etc.

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 of the mammal is in the range of about 1 to 20000 mg/kg. For example, for preferred treatment of human patients, suitable dosages may range from 1 to 2,000mg/kg, 2 to 1000mg/kg, 5 to 500mg/kg, or 10 to 100mg/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 dose may be administered repeatedly at intervals, for example, three times per day, once per week, and the like.

In the present specification, "mg/kg" means milligrams per kilogram of body weight of the mammal (including human) to be treated, "g/kg" means grams per kilogram of body weight of the mammal (including human) to be treated.

Method for preparing Chinese medicinal composition with antiviral effect

1. Test materials and reagents

Test article group: the resulting pharmaceutical powders were prepared separately according to the procedures of examples 7 to 21 and comparative example 1, and used as drugs for test groups.

Positive control group 1: lianhua Qingwen granules (approved by Beijing Shilingyao pharmaceutical Co., Ltd., national drug Standard Z20100040, specification: 6g × 9 bags) were administered as the positive control group 1.

Positive control 2: lopinavir (provided by Shijiazhuang four drug group) was administered as the positive control group 2.

The test powder, the lotus antipyretic granules and the lopinavir tablets are dispersed in 0.85% of physiological saline to prepare suspension before use. The concentration of the test product suspension is 10.52mg/ml, and the concentration of the lotus antipyretic granules is 15.21 mg/ml.

Test animals: SPF-grade BALB/c female mice 6-8 weeks old are provided by Experimental animal center of university of agriculture in Huazhong.

Cell and virus: MDCK cells, Vero cells and recombinant adenovirus vectors expressing T cell surface antigen CD26 receptors are provided by Shanghai cell banks of Chinese academy of sciences. The novel coronavirus 2019-nCoV Chinese strain was provided by the Wuhan virus institute of Chinese academy of sciences.

2. Test of

2.1 in vitro inhibition of viral adsorption assay

Test powder of various concentrations (10mg/ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml, 0.625mg/ml, 0.312mg/ml, 0.156mg/ml) was added to the cell plate of Vero cells grown in a monolayer, 200. mu.l per well, 4 wells per concentration, at 37 ℃ with 5% CO₂Incubate for 3 h. Discard the solution, wash 2 times with PBS, add 100. mu.l of a suitable diluent virus (10)⁵PFU/ml), adsorbing at 37 ℃ for 2 hours, aspirating each well of virus solution, and titrating the virus by PFU method. The non-dosed well plates were set up as virus control. Similarly, the positive control group 1 was dosed at 10mg/ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml, 0.625mg/ml, 0.312mg/ml, 0.156 mg/ml. The positive control group 2 was dosed at 80mg/ml, 40mg/ml, 20mg/ml, 10mg/ml, 5mg/ml, 2.5mg/ml, 1.25 mg/ml.

2.2 in vivo inhibition assay

2.5 × 10⁸Recombinant adenovirus vector for PFU expression of hCD26 was nasally transfected into BALB/c mice, 2019-nCoV (1 × 10) on day 4 after transfection⁵PFU) nasal drip infection expressing hCD26 mice, the mice were randomly divided into 4 groups of virus, test article, positive control 1 and positive control 2, each group containing 6 females, and the administration was by gavage on day 3 after infection. The virus group used 0.85% physiological saline in amounts equal to the test article group, positive control 1 group and positive control 2 group, respectively, so as to serve as controls for the test article group, positive control 1 group and positive control 2 group, respectively. The test group was separately gavaged at 4500mg/(kgd), and the corresponding drug solutions prepared in examples 6 to 20 and comparative example 1 were administered. The administration concentration of the positive control group 1 for the antipyretic lotus is 1300mg/(kg d), and the administration concentration of the positive control group 2 for the lopinavir is 60mg/(kg d). 3-16 days after infection (i.e. 14 days total treatment, starting from day 3 after infection) 3 mice per group were randomly selected for post-anesthesia CO₂Sacrifice, transfer mouse lung into PBS, use hand homogenizer on ice homogenate, 4 degrees C12000 r/min centrifugal 5min, collect the supernatant for virus titer detection.

3. Statistical treatment

The data obtained in the text were statistically processed and analyzed using the software SPSS17.0, the data are expressed as mean squared standard deviation (x ± s), the metrology data are tested using t, the count data are tested using x2, and p <0.05 is statistically significant.

4. Results

The results of the test article for inhibiting the adsorption of the virus are shown in Table 4, the results show that the virus particles in the extracellular virus liquid in the experimental group are obviously increased, the average virus residual quantity of the culture solution of the experimental group of the test article is respectively 5.1 × 10 by 10mg/ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml, 0.625mg/ml, 0.312mg/ml and 0.156mg/ml⁵pfu/ml，3.7×10⁵pfu/ml，1.2×10⁵pfu/ml，9.3×10⁴pfu/ml，6.1×10⁴pfu/ml， 5.5×10⁴pfu/ml and 4.2 × 10⁴pfu/ml was significantly higher than the virus control (1.7 × 10)⁴pfu/ml), so that the virus residual quantity of a culture solution after virus is adsorbed and inoculated can be obviously increased by pretreating cells with a test article, the adsorption of the virus to Vero cells is obviously inhibited, and the virus is influenced to enter the cells.

The results of inhibiting virus adsorption in the positive control group 1 are shown in Table 5. The result shows that the virus residual quantity of the culture solution is not obviously increased after virus is adsorbed and inoculated after the cells are treated by the antipyretic lotus flowers.

Results of inhibition of virus adsorption in the positive control 2 group are shown in table 6. The results show that after the lopinavir treats the cells, the virus residual quantity of the culture solution after virus is adsorbed and inoculated is obviously increased, the adsorption of the virus to Vero cells is obviously inhibited, and the virus is influenced to enter the cells.

TABLE 4 Virus yield after treatment of cells with test substances obtained by different preparation methods (10)⁵PFU/ml)

TABLE 5 viral yield following antipyretic treatment of cells with Lotus flowers (10)⁵PFU/ml)

TABLE 6 Virus yield following lopinavir treatment of cells (10)⁵PFU/ml)

The results of tables 4-6 show that the traditional Chinese medicine composition preparation prepared by the preparation process provided by the invention can obviously increase the virus residual amount of the culture solution after virus is adsorbed and inoculated by pretreating cells, obviously inhibit the adsorption of the virus on Vero cells and influence the virus to enter the cells. The traditional Chinese medicine composition preparation provided by the invention can prevent the adsorption of viruses on Vero cells, so that the combination of the viruses and receptors is reduced, the viruses are prevented from invading the Vero cells, and the lotus antipyretic has no obvious effect of preventing the viruses from infecting the cells. Moreover, the effect of preventing virus infection of the traditional Chinese medicine preparation prepared by the processes of examples 7-21 in the test article is obviously better than that of the traditional Chinese medicine preparation obtained by decoction, which shows that the traditional Chinese medicine preparation obtained by the preparation process of the invention can better play the role of preventing virus from infecting cells.

Table 7 lists the mean viral titers at day 1, day 7 and day 14 after treatment for each group tested

1g PFU/ml). On day 1 of treatment after infection of BALB/c mice with 2019-nCoV, the mean virus titers of the test group, the positive control 1 group and the positive control 2 group were not significantly different from those of the virus group.

On day 7 of treatment, the mean viral titers of the test group, the positive control 1 group and the positive control 2 group were all decreased relative to the viral group, and the results after treatment were comparable to those of the positive control 1 group and higher than those of the positive control 2 group. In the test group, examples 7 to 21 had better therapeutic effects than comparative example 1, while examples 7 to 20 had less therapeutic effects than example 21.

On the 14 th day of treatment, the average virus titer of the test sample group, the positive control group 1 and the positive control group 2 is obviously reduced compared with that of the virus group, and the virus inhibition effect is obvious. And the average titer of the tested group is lower than that of the positive control group 1 and higher than that of the positive control group 2 after the virus is reduced. In the test group, the average virus titer of the examples 7 to 21 is lower than that of the comparative example 1, and the average virus titer of the example 21 is lower than that of the examples 7 to 20, which shows that the preparation process of the antiviral traditional Chinese medicine composition of the invention is superior to the traditional decoction process, the antiviral traditional Chinese medicine composition has better antiviral effect, and the traditional Chinese medicine composition prepared according to the steps of the example 21 has the best antiviral effect.

In vivo animal experiments show that the antiviral traditional Chinese medicine composition provided by the invention has an obvious effect of inhibiting the replication of 2019-nCoV. The antiviral traditional Chinese medicine preparation prepared by the process provided by the invention has a treatment effect obviously superior to that of the traditional Chinese medicine preparation prepared by decocting by a traditional method.

TABLE 7

Clinical cases

In order to further verify the effect of the traditional Chinese medicine composition against 2019-nCoV, the technical effect of the traditional Chinese medicine composition is described in combination with cases.

Case one: chen is a female, aged 59 years, and is hospitalized in the 20 th month 1 in 2020. The main complaints are: cough for 2 days, fever for 1 day at 38.5 ℃. The medical history: the patient can start cough without phlegm and fever 2 days before, the muscles of the whole body are weak and limp, the sodium food is reduced, and the night sleep is uneasy. Checking: cough without phlegm, chest CT shows the speckle shadow of the upper left lung and the lower two lungs, and the middle right lung and the lower left lung consider little chronic inflammation, and bilateral pleura is sticky (as shown in figure 1). In order to relieve symptoms, the powder of the traditional Chinese medicine composition provided by the invention is taken 3 times a day, 2g for each time, and is taken with 200-250ml of warm water; after a patient takes the medicine continuously for one week, fever symptoms are eliminated, cough is weakened after one month, and CT in the chest shows that the change is absorbed by compound lung infection after three months, and the bilateral local pleura is hypertrophic and aged (as shown in figure 2); and the result of the nucleic acid detection of the novel coronavirus is negative, which indicates that the nucleic acid result of a patient can be changed to negative by treating or preventing the novel coronavirus 2019-nCoV infectious disease or disorder by taking the traditional Chinese medicine composition provided by the invention.

Case two: li Shi, 50 years old, came to the hospital in 3 months of 2020. The main complaints are: cough for 2 weeks. The medical history: the patients are weak, have inappetence, and have emaciation, and cough begins to be coughed due to cold before 2 months, so the cough is lingering and difficult to cure. Checking: mental fatigue, white complexion, cough with profuse sputum, white and fresh xi, lung smelling dry and humid Luo Yin, and red finger print. For cough caused by deficiency of lung and spleen qi and excessive phlegm-dampness, the powder of the traditional Chinese medicine composition provided by the invention is taken 3 times a day, 2g each time, with 200-250ml warm water, after continuous taking for one week, the symptoms disappear and are cured, and after 2 weeks, the return visit is carried out without relapse.

Case three: wangzhi, male, 62 years old, comes to a hospital for treatment in 2 months in 2020. The main complaints are: fever was 38.7 ℃ for 2 days and two days, cough was severe, and dyspnea was observed. Checking: the nucleic acid detection result of a patient is positive, the chest CT shows that the lung consolidation is changed, pleural effusion is rarely seen, the respiration frequency is obviously more than 30 times/minute, the blood oxygen saturation is reduced by less than or equal to 93 percent in the resting state, the blood oxygen partial pressure is obviously reduced in arterial blood gas analysis, and the severe coronavirus pneumonia can be diagnosed. The powder of the traditional Chinese medicine composition provided by the invention is taken 3 times a day, 2g each time, with 200-250ml warm water, after 2 days of continuous taking, the fever symptom is eliminated, after one week of continuous taking, the symptoms are completely disappeared and cured, and after 2 weeks, the return visit is carried out without relapse.

The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An antiviral traditional Chinese medicine composition is characterized by comprising the following components in parts by weight:

2. The antiviral traditional Chinese medicine composition according to claim 1, further comprising the following components by weight: 10-100 parts of cyrtomium rhizome, 3-20 parts of ginger and 3-80 parts of buffalo horn powder.

3. The method for preparing an antiviral Chinese medicinal composition as claimed in claim 1, comprising the steps of:

4. The preparation method according to claim 3, wherein in the step S1, the raw material is buffalo horn powder, the mass of the added water is 8-12 times of the sum of the mass of the buffalo horn powder and the mass of the forsythia suspensa, and the post-treatment time after the water is added is 4 hours.

5. The preparation method according to claim 3, wherein in the step S2, the alcohol extraction solution is 50-90% ethanol, and the mass of the alcohol extraction solution is 4-12 times of the sum of the mass of the areca catechu and the agastache rugosus.

6. The preparation method according to claim 3, wherein in step S3, the raw materials include rhizoma Osmundae and rhizoma Zingiberis recens, the mass ratio of the added radix astragali, rhizoma Atractylodis Macrocephalae, rhizoma Osmundae, Scutellariae radix, rhizoma Coptidis, fructus Arctii, rhizoma Atractylodis, radix Stemonae, rhizoma Zingiberis recens and radix Glycyrrhizae to the added water is 1 (8-12), and the density of the concentrated fluid extract is 1.0-1.15.

7. The method according to claim 3, wherein in step S4, an aqueous ethanol solution with an alcohol solution concentration of 95% is added, and the concentration of ethanol after concentration is 50%.

8. An antiviral pharmaceutical formulation comprising the Chinese medicinal composition of claim 1 and a pharmaceutically acceptable carrier or adjuvant.

9. The antiviral pharmaceutical preparation according to claim 8, wherein the dosage form is granules, tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, drop pills, granules, pills, powders, suspensions or powders.

10. Use of the antiviral traditional Chinese medicine composition of claim 1 in the preparation of a medicament for treating or preventing a novel coronavirus 2019-nCoV infectious disease or disorder.