CN111643612A

CN111643612A - Traditional Chinese medicine composition for preventing and treating new coronavirus pneumonia and preparation method and application thereof

Info

Publication number: CN111643612A
Application number: CN202010372164.2A
Authority: CN
Inventors: 杜飞
Original assignee: Yuanjian Biotechnology Shanghai Co ltd
Current assignee: Yuanjian Biotechnology Shanghai Co ltd
Priority date: 2020-05-06
Filing date: 2020-05-06
Publication date: 2020-09-11

Abstract

The application discloses a traditional Chinese medicine composition, wherein the active ingredients of the medicine comprise ephedra, coix seed, almond, aster, common coltsfoot flower, gypsum, magnolia officinalis, rhizoma anemarrhenae, white paeony root, baical skullcap root, honey-fried licorice root, white mustard seed, perilla seed, radish seed, wild buckwheat rhizome, membranous milkvetch root and lily. The invention also provides a method for preparing the traditional Chinese medicine composition, which comprises the steps of soaking the traditional Chinese medicine components in water, boiling with strong fire after soaking, maintaining micro-boiling with slow fire, and adding a pharmaceutically acceptable carrier to prepare the traditional Chinese medicines in various dosage forms. The invention also provides application of the traditional Chinese medicine composition, and application of the traditional Chinese medicine composition in preparing a medicine for preventing and treating new coronavirus pneumonia. The traditional Chinese medicine composition has the advantages that under the guidance of the theory of traditional Chinese medicine, the traditional Chinese medicine composition is strict from holistic concept and treatment based on syndrome differentiation, is used for preventing and treating new coronavirus, and improves the immunity of a human body. The invention adopts the ointment, has controllable preparation method, lasting drug effect, easy absorption, small toxic and side effect, and is convenient to carry and take.

Description

Traditional Chinese medicine composition for preventing and treating new coronavirus pneumonia and preparation method and application thereof

Technical Field

The application relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition for preventing and treating new coronavirus pneumonia and a preparation method and application thereof.

Background

Since the outbreak of new coronavirus, which has spread to the world and gradually enters a high-outbreak state, seriously impairs the life and health of human beings, the international committee on viral classification of this new coronavirus, named SARS-CoV-2, causes symptoms of different degrees of infection of people with the virus, some with fever or slight cough, some with pneumonia, and some with more serious or even death.

The treatment of the new coronavirus does not have a specific medicine at present, and the western medicine treatment mainly adopts general treatment, supportive treatment and antiviral treatment, but has higher cost and more side effects. Although western medicine treatment is still the main means for severe patients, the traditional Chinese medicine treatment has better effect on most mild and moderate patients.

The epidemiological characteristics, clinical symptom manifestations and disease development characteristics of the novel coronary virus pneumonia are in accordance with the characteristics of epidemic diseases or epidemic diseases of the traditional Chinese medicine, the traditional Chinese medicine does not focus on the diseases but regulates the disease resistance of the human body when treating the diseases, and the novel coronary virus pneumonia has unique advantages in the aspects of improving clinical symptoms, reducing complications, improving life quality and the like.

The traditional Chinese medicine is based on the principle that the existing disease is firstly prevented and the existing disease is prevented, whether the new coronary viral pneumonia develops into influenza is not clear, but the immunity of the organism is improved only by the aid of the new coronary viral pneumonia

Aiming at the problem of effective prevention and cure of the new coronavirus pneumonia in the related technology, no effective solution is provided at present.

Disclosure of Invention

The application mainly aims to provide a traditional Chinese medicine composition capable of effectively preventing and treating new coronavirus pneumonia in clinic and a preparation method thereof, so as to solve the problems in the related technology.

In order to achieve the above objects, in a first aspect, the present application provides a traditional Chinese medicine composition for preventing and treating new crown virus pneumonia, and a preparation method and a use thereof, wherein the pharmaceutical active ingredients of the pharmaceutical composition comprise a mixture of ephedra herb, coix seed, almond, aster, coltsfoot flower, gypsum, magnolia bark, anemarrhena, white peony root, scutellaria baicalensis, honey-fried licorice root, semen brassicae, perillaseed, radish seed, wild buckwheat rhizome, astragalus root and lily, wherein the weight portions of the components are respectively 1-10 parts of ephedra herb, 1-10 parts of coix seed, 1-10 parts of almond, 1-10 parts of aster, 1-10 parts of coltsfoot flower, 1-20 parts of gypsum, 1-15 parts of magnolia bark, 1-8 parts of anemarrhena, 1-15 parts of white peony root, 1-6 parts of scutellaria baicalensis, 1-10 parts of common coltsfoot flower, 1-10 parts of gypsum, 1-20 parts of honey-fried licorice root, 1-10 parts of white mustard seed, 1-10 parts of perillaseed, 1-10 parts of radish seed, 1-20 parts of wild buckwheat rhizome, 1-30 parts of astragalus root and 1-30 parts of lily.

The invention adopts components with high-grade comprehensive effects of nutrition, nourishing, treatment and prevention, has lasting drug effect, easy absorption and small toxic and side effect, and overcomes the defects of poor taking taste, inconvenient carrying and the like of other traditional Chinese medicine dosage forms. Has obvious effects of preserving health, preventing and treating diseases and improving the body resistance of people.

In a second aspect, the present application further provides a method for preparing the Chinese medicinal composition, comprising the steps of mixing and soaking ephedra herb, coix seed, almond, aster, coltsfoot flower, gypsum, magnolia officinalis, rhizoma anemarrhenae, white paeony root, baical skullcap root, honey-fried licorice root, white mustard seed, perilla fruit, radish seed, wild buckwheat rhizome, membranous milkvetch root and lily in water, the added water amount is 3-10 times of the total weight of the mixed traditional Chinese medicine components, after soaking for 24 hours, boiling with strong fire, maintaining slight boiling with slow fire, decocting for 2-4 times (each time for 10-60 min), filtering to obtain decoction, decocting the residue with cold water, mixing decoctions, standing for 1 day, adding pharmaceutically acceptable carrier, the carrier is 0.1-1.0 times of the total weight of the Chinese medicinal components, and the Chinese medicinal materials in various dosage forms can be prepared. Such as pills, powders, and ointments, using conventional methods and carriers required in the art for preparing such dosage forms.

Further, the pharmaceutically acceptable carrier is selected from any one or more of an emulsifier, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, a flavoring agent, a coloring agent and a cosolvent.

Preferably, the dosage form of the traditional Chinese medicine is a traditional Chinese medicine ointment formulation.

In a third aspect, the application also provides an application of the traditional Chinese medicine composition, and an application of the traditional Chinese medicine composition in preparing a medicine for preventing and treating the new coronavirus pneumonia.

The traditional Chinese medicine composition comprises the following components: herba ephedrae: pungent, slightly bitter and warm in nature. It enters lung and bladder meridians. Induce sweating and dispel cold, ventilate lung and relieve dyspnea, induce diuresis to alleviate edema.

Coix seed: sweet and bland in flavor and cool in nature. It enters spleen, stomach and lung meridians. Induce diuresis, drain dampness, invigorate spleen, check diarrhea, remove arthralgia, expel pus, remove toxicity and dissipate nodulation.

Almond: bitter taste and slightly warm nature; has little toxicity. It enters lung and large intestine meridians. Direct qi downward to relieve cough and dyspnea, moisten intestines to relieve constipation.

Radix asteris: bitter taste and warm nature. It enters lung meridian. Warm lung, descend qi, resolve phlegm and relieve cough.

And (3) common coltsfoot flower: pungent, slightly sweet and warm in nature. It enters lung meridian. Moisten lung and descend qi, resolve phlegm and stop cough.

Raw gypsum: sweet, pungent and cold in flavor. It enters lung and stomach meridians. Clear heat and purge fire, relieve restlessness and quench thirst.

Magnolia officinalis: bitter and pungent in flavor and warm in nature. It enters spleen, stomach and large intestine meridians. Move qi and remove food retention, dry dampness and remove fullness, check adverse rise of qi and relieve dyspnea.

Rhizoma anemarrhenae: bitter taste and cold nature. It enters lung, stomach and kidney meridians. Clear heat and purge fire, nourish yin and moisten dryness.

White peony root: bitter and sour in taste, slightly cold in nature. It enters liver and spleen meridians. Nourish blood and regulate menstruation, astringe yin and check sweating, soften liver and relieve pain, and calm liver yang.

Scutellaria baicalensis: bitter taste and cold nature. It enters lung, gallbladder, spleen, large intestine and small intestine meridians. Clear heat and dry dampness, purge fire and remove toxicity, stop bleeding, prevent abortion.

Honey-fried licorice root: sweet in flavor and neutral in nature. It enters heart, lung, spleen and stomach meridians. Tonify spleen and stomach, replenish qi and recover pulse.

White mustard seed: pungent flavor and warm nature. Enters the meridians of liver, spleen, lung, stomach, heart and uterus. Warm lung, eliminate phlegm and promote qi circulation, and calm five zang organs.

And (3) Perilla seed: pungent flavor and warm nature. It enters lung and large intestine meridians. Direct qi downward and resolve phlegm, stop cough and relieve dyspnea, moisten intestines and relieve constipation.

Radish seed: pungent and sweet in flavor and mild in nature. It enters lung, spleen and stomach meridians. Promote digestion, relieve distension, direct qi downward and resolve phlegm.

Wild buckwheat rhizome: sour, bitter and cold in nature. It enters lung, stomach and liver meridians. Clearing away heat and toxic materials; promoting blood circulation and removing carbuncle; wind dispelling device

Astragalus root: sweet in flavor and warm in nature. It enters lung and spleen meridians. Tonify qi, strengthen superficies, induce diuresis, expel pus, heal wound and promote tissue regeneration.

Lily: sweet in flavor and cold in nature. It enters heart and lung meridians. Nourish yin, moisten lung, relieve cough, clear heart and induce tranquilization.

In conclusion, the invention has the following beneficial effects: the invention is characterized in that: firstly, under the guidance of the theory of traditional Chinese medicine, from the overall concept and diagnosis and treatment based on syndrome differentiation, the formula is rigorous, and the human body function can be comprehensively and integrally regulated. Secondly, the immunity of the human body is improved, the metabolism of the human body is regulated, and the function of the human body is improved. The traditional Chinese medicine components adopted by the invention have the advantages of lasting drug effect, small toxic and side effects, convenient carrying and good taste, and have remarkable efficacies of leisure, health preservation, disease prevention and treatment and body resistance improvement for people.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, serve to provide a further understanding of the application and to enable other features, objects, and advantages of the application to be more apparent. The drawings of the illustrative embodiments of the present application and their description are for explanation of the application and do not constitute an undue limitation on the application. In the drawings:

FIG. 1 is a graph showing the change of the light microscope pictures and the relative cell numbers of BEAS-2B and 16HBE cells after 24 hours in the treatment group and the control group of the pharmaceutical composition of the present invention.

FIG. 2 shows that the pharmaceutical composition of the present invention can effectively reduce the concentration of viruses in cells by detecting the fluorescence intensity and measuring the virus titer in the cells after the pharmaceutical composition of the present invention is used for 72 hours after BEAS-2B and 16HBE stable transformants which over-express human ACE2 and are infected with COVID-19 pseudovirus liquid.

FIG. 3 shows the experiment of the traditional Chinese medicine composition for inhibiting the infection of the COVID-19 pseudovirus to lung tissues by GFP fluorescence.

FIG. 4 shows the inhibition effect of the Chinese medicinal composition on the expression of cell proliferation index Ki-67 and apoptosis index Bcl-2 through hematoxylin-eosin staining method (HE) and Immunohistochemistry (IHC).

FIG. 5 shows the result of testing the viral titer of lung tissue in a mouse animal model using the Chinese medicinal composition of the present invention.

Detailed Description

In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given in the present application without any inventive step, shall fall within the scope of protection of the present application.

It should be noted that the terms "first," "second," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It should be understood that the data so used may be interchanged under appropriate circumstances such that embodiments of the application described herein may be used. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

In this application, the terms "upper", "lower", "left", "right", "front", "rear", "top", "bottom", "inner", "outer", "middle", "vertical", "horizontal", "lateral", "longitudinal", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings. These terms are used primarily to better describe the present application and its embodiments, and are not intended to limit the indicated devices, elements or components to a particular orientation or to be constructed and operated in a particular orientation.

Moreover, some of the above terms may be used to indicate other meanings besides the orientation or positional relationship, for example, the term "on" may also be used to indicate some kind of attachment or connection relationship in some cases. The specific meaning of these terms in this application will be understood by those of ordinary skill in the art as appropriate.

In addition, the term "plurality" shall mean two as well as more than two.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.

The first embodiment is as follows: the invention provides a traditional Chinese medicine composition for preventing and treating new crown virus pneumonia, wherein the active ingredients of the medicine are a mixture consisting of ephedra, coix seed, almond, aster, coltsfoot flower, gypsum, magnolia officinalis, rhizoma anemarrhenae, white paeony root, scutellaria baicalensis, honey-fried licorice root, semen brassicae, perillaseed, radish seed, wild buckwheat rhizome, astragalus and lily, wherein the weight parts of the components are respectively 10 parts of ephedra, 10 parts of coix seed, 10 parts of almond, 10 parts of aster, 10 parts of coltsfoot flower, 20 parts of gypsum, 15 parts of magnolia officinalis, 8 parts of rhizoma anemarrhenae, 15 parts of white paeony root, 6 parts of scutellaria baicalensis, 20 parts of honey-fried licorice root, 10 parts of white mustard seed, 10 parts of perillaseed, 10 parts of radish seed, 20 parts of wild buckwheat, 30 parts of astragalus and 30 parts of lily.

The preparation method of the traditional Chinese medicine composition comprises the steps of mixing and soaking ephedra, coix seeds, almonds, aster tataricus, coltsfoot flowers, raw gypsum, mangnolia officinalis, rhizoma anemarrhenae, radix paeoniae alba, scutellaria baicalensis, honey-fried licorice roots, semen brassicae, perillaseeds, semen raphani, wild buckwheat rhizomes, astragalus mongholicus and lily in water, adding water in an amount which is 10 times of the total weight of the mixed traditional Chinese medicine components, soaking for 24 hours, boiling with strong fire, then maintaining slight boiling with slow fire, decocting for 2-4 times by adopting the decocting method, wherein the decocting time is 60min each time, filtering and remaining medicine juice after each decocting, continuously decocting cold water in medicine residues, finally combining all the medicine juice, standing for 1 day, and adding a pharmaceutically acceptable carrier which is 1.0 time of the total weight of the traditional Chinese medicine components to obtain the traditional Chinese medicine with various dosage forms.

The pharmaceutically acceptable carrier is selected from any one or more of emulsifier, excipient, filler, adhesive, humectant, disintegrant, absorption enhancer, flavoring agent, coloring agent and cosolvent. The preparation method is characterized by preparing corresponding preparation formulations by adopting the method in the prior art, and preferably, the preparation formulation of the traditional Chinese medicine is a traditional Chinese medicine ointment formula. During preparation, multiple layers of sterilized gauze may be used for filtering 2-5 times to reduce impurity, and the filtered liquid medicine is further decocted on slow fire for evaporation and concentration while stirring to avoid coking and form thick paste gradually. More preferably, the filtration is performed 3 times with sterile multi-layer gauze.

Example two: the invention provides a traditional Chinese medicine composition for preventing and treating new crown virus pneumonia, wherein the active ingredients of the medicine are a mixture consisting of ephedra, coix seed, almond, aster, coltsfoot flower, gypsum, magnolia officinalis, rhizoma anemarrhenae, radix paeoniae alba, scutellaria baicalensis, honey-fried licorice root, semen brassicae, perillaseed, radish seed, wild buckwheat rhizome, astragalus mongholicus and lily, wherein the weight parts of the components are respectively 1 part of ephedra, 1 part of coix seed, 1 part of almond, 1 part of aster, 1 part of coltsfoot flower, 1 part of gypsum, 1 part of magnolia officinalis, 1 part of rhizoma anemarrhenae, 1 part of radix paeoniae alba, 1 part of scutellaria baicalensis, 1 part of honey-fried licorice root, 1 part of white mustard seed, 1 part of perillaseed, 1 part of radish seed, 1 part of wild buckwheat rhizome, 1 part of astragalus mongholicus and 1 part of lily.

The preparation method of the traditional Chinese medicine composition comprises the steps of mixing and soaking ephedra, coix seeds, almonds, aster tataricus, coltsfoot flowers, raw gypsum, mangnolia officinalis, rhizoma anemarrhenae, radix paeoniae alba, scutellaria baicalensis, honey-fried licorice roots, semen brassicae, perillaseeds, semen raphani, wild buckwheat rhizomes, astragalus mongholicus and lily in water, adding water in an amount which is 3 times of the total weight of the mixed traditional Chinese medicine components, soaking for 24 hours, boiling with strong fire, then maintaining slight boiling with slow fire, decocting for 2-4 times by adopting the decocting method, wherein the time of each decocting is 10-20min, filtering and remaining medicine juice after each decocting, continuing to decoct the medicine residues with cold water, finally combining all the medicine juice, standing for 1 day, adding a carrier which can be accepted by medicines, wherein the carrier is 0.1 time of the total weight of the traditional Chinese medicine components, and preparing.

Example three: the invention provides a traditional Chinese medicine composition for preventing and treating new crown virus pneumonia, wherein the active ingredients of the medicine are a mixture consisting of ephedra, coix seed, almond, aster, coltsfoot flower, gypsum, mangnolia officinalis, rhizoma anemarrhenae, radix paeoniae alba, scutellaria baicalensis, honey-fried licorice root, semen brassicae, perillaseed, radish seed, wild buckwheat, astragalus mongholicus and lily, wherein the weight parts of the components are 5 parts of ephedra, 5 parts of coix seed, 5 parts of almond, 5 parts of aster, 5 parts of coltsfoot flower, 10 parts of gypsum, 10 parts of mangnolia officinalis, 5 parts of rhizoma anemarrhenae, 10 parts of radix paeoniae alba, 3 parts of scutellaria baicalensis, 10 parts of honey-fried licorice root, 5 parts of semen brassicae, 5 parts of perillaseed, 5 parts of radish seed, 10 parts of wild buckwheat, 20 parts of astragalus mongholicus and 20 parts of lily.

The preparation method of the traditional Chinese medicine composition comprises the steps of mixing and soaking ephedra, coix seeds, almonds, aster tataricus, coltsfoot flowers, raw gypsum, mangnolia officinalis, rhizoma anemarrhenae, radix paeoniae alba, scutellaria baicalensis, honey-fried licorice roots, semen brassicae, perillaseeds, semen raphani, wild buckwheat rhizomes, astragalus mongholicus and lily in water, adding water in an amount which is 6 times of the total weight of the mixed traditional Chinese medicine components, soaking for 24 hours, boiling with strong fire, maintaining slight boiling with slow fire, decocting for 2-4 times by adopting the decocting method, wherein the time of each decocting is 20-30min, filtering and remaining medicine juice after each decocting, continuing to decoct the medicine residues with cold water, finally combining all the medicine juice, standing for 1 day, adding a carrier which can be accepted by medicines, wherein the carrier is 0.5 times of the total weight of the traditional Chinese medicine components, and preparing the.

Example four: the invention provides a traditional Chinese medicine composition for preventing and treating new crown virus pneumonia, wherein the active ingredients of the medicine are a mixture consisting of ephedra, coix seed, almond, aster, coltsfoot flower, gypsum, magnolia officinalis, rhizoma anemarrhenae, white paeony root, scutellaria baicalensis, honey-fried licorice root, semen brassicae, perillaseed, radish seed, wild buckwheat rhizome, astragalus mongholicus and lily, wherein the weight parts of the components are respectively 3 parts of ephedra, 3 parts of coix seed, 3 parts of almond, 3 parts of aster, 3 parts of coltsfoot flower, 5 parts of gypsum, 5 parts of magnolia officinalis, 3 parts of rhizoma anemarrhenae, 5 parts of white paeony root, 2 parts of scutellaria baicalensis, 5 parts of honey-fried licorice root, 3 parts of white mustard seed, 3 parts of perillaseed, 3 parts of radish seed, 5 parts of wild buckwheat rhizome, 5 parts of astragalus mongholicus and 5 parts of lily.

The preparation method of the traditional Chinese medicine composition comprises the steps of mixing and soaking ephedra, coix seeds, almonds, aster tataricus, coltsfoot flowers, raw gypsum, mangnolia officinalis, rhizoma anemarrhenae, radix paeoniae alba, scutellaria baicalensis, honey-fried licorice roots, semen brassicae, perillaseeds, semen raphani, wild buckwheat rhizomes, astragalus mongholicus and lily in water, adding water in an amount which is 5 times of the total weight of the mixed traditional Chinese medicine components, soaking for 24 hours, boiling with strong fire, maintaining slight boiling with slow fire, decocting for 2-4 times by adopting the decocting method, wherein the time of each decocting is 30-40min, filtering and remaining medicine juice after each decocting, continuing to decoct the medicine residues with cold water, finally combining all the medicine juice, standing for 1 day, adding a carrier which can be accepted by medicines, wherein the carrier is 0.8 times of the total weight of the traditional Chinese medicine components, and preparing the.

Example five: the invention provides a traditional Chinese medicine composition for preventing and treating new crown virus pneumonia, wherein the active ingredients of the medicine are a mixture consisting of ephedra, coix seed, almond, aster, coltsfoot flower, gypsum, mangnolia officinalis, rhizoma anemarrhenae, radix paeoniae alba, scutellaria baicalensis, honey-fried licorice root, semen brassicae, perillaseed, radish seed, wild buckwheat, astragalus mongholicus and lily, wherein the weight parts of the components are respectively 8 parts of ephedra, 8 parts of coix seed, 8 parts of almond, 8 parts of aster, 8 parts of coltsfoot flower, 15 parts of gypsum, 13 parts of mangnolia officinalis, 6 parts of rhizoma anemarrhenae, 10 parts of radix paeoniae alba, 4 parts of scutellaria baicalensis, 15 parts of honey-fried licorice root, 8 parts of white mustard seed, 8 parts of perillaseed, 8 parts of radish seed, 15 parts of wild buckwheat, 25 parts of astragalus mongholicus and 25 parts of lily.

The preparation method of the traditional Chinese medicine composition comprises the steps of mixing and soaking ephedra, coix seeds, almonds, aster tataricus, coltsfoot flowers, raw gypsum, mangnolia officinalis, rhizoma anemarrhenae, radix paeoniae alba, scutellaria baicalensis, honey-fried licorice roots, semen brassicae, perillaseeds, semen raphani, wild buckwheat rhizomes, astragalus mongholicus and lily in water, adding water in an amount which is 8 times of the total weight of the mixed traditional Chinese medicine components, soaking for 24 hours, boiling with strong fire, maintaining slight boiling with slow fire, decocting for 2-4 times by adopting the decocting method, wherein the time of each decocting is 40-50min, filtering and remaining medicine juice after each decocting, continuing to decoct the medicine residues with cold water, finally combining all the medicine juice, standing for 1 day, adding a carrier which can be accepted by medicines, wherein the carrier is 0.3 times of the total weight of the traditional Chinese medicine components, and preparing the.

Example five: the invention provides a traditional Chinese medicine composition for preventing and treating new crown virus pneumonia, wherein the active ingredients of the medicine are a mixture consisting of ephedra, coix seed, almond, aster, coltsfoot flower, gypsum, mangnolia officinalis, rhizoma anemarrhenae, radix paeoniae alba, scutellaria baicalensis, honey-fried licorice root, semen brassicae, perillaseed, radish seed, wild buckwheat, astragalus mongholicus and lily, wherein the weight parts of the ingredients are 6 parts of ephedra, 6 parts of coix seed, 6 parts of almond, 6 parts of aster, 6 parts of coltsfoot flower, 13 parts of gypsum, 8 parts of mangnolia officinalis, 4 parts of rhizoma anemarrhenae, 13 parts of radix paeoniae alba, 2 parts of scutellaria baicalensis, 17 parts of honey-fried licorice root, 2 parts of semen brassicae, 2 parts of perillaseed, 9 parts of radish seed, 2 parts of wild buckwheat, 27 parts of astragalus mongholicus and 3 parts of lily.

The preparation method of the traditional Chinese medicine composition comprises the steps of mixing and soaking ephedra, coix seeds, almonds, aster tataricus, coltsfoot flowers, raw gypsum, mangnolia officinalis, rhizoma anemarrhenae, radix paeoniae alba, scutellaria baicalensis, honey-fried licorice roots, semen brassicae, perillaseeds, semen raphani, wild buckwheat rhizomes, astragalus mongholicus and lily in water, adding water in an amount which is 9 times of the total weight of the mixed traditional Chinese medicine components, soaking for 24 hours, boiling with strong fire, then maintaining slight boiling with slow fire, decocting for 2-4 times by adopting the decocting method, wherein the time of each decocting is 50-60min, filtering and remaining medicine juice after each decocting, continuing to decoct the medicine residues with cold water, finally combining all the medicine juice, standing for 1 day, adding a carrier which can be accepted by medicines, wherein the carrier is 0.9 times of the total weight of the traditional Chinese medicine components, and preparing.

The invention also provides application of the traditional Chinese medicine composition, and application of the traditional Chinese medicine composition in preparing a medicine for preventing and treating new coronavirus pneumonia. The use of the test is shown in the following experiments:

first, experiment method

1. Cell culture: human normal lung epithelial cells BEAS-2B and human bronchial epithelial cells 16HBE were cultured in a DMEM high-sugar culture medium containing 10% fetal bovine serum at 37 ℃ in an incubator with 5% CO2 and 95% saturation humidity, and when the cells reached the logarithmic phase, they were passed through 1 passage by conventional digestion and collected for experiments. Cell digestion was performed using trypsin-EDTA digest (0.25%).

CCK8 experiment 5 × 10³Inoculating lung epithelial cells in logarithmic growth phase into 96-well plate, adding 100 μ l complete culture medium into each well, setting 3 multiple wells for each sample, adding a blank well as blank control, placing at 37 deg.C and 5% CO₂Culturing for 6h in an incubator with 95% saturated humidity, and adding drugs after the cells adhere to the walls, wherein the drugs are specifically divided into a control group, a low dose group and a medium dose groupAnd (3) culturing the group and the high-dose group for 24 hours, absorbing the culture medium, washing the group with 1 × phosphate buffered saline solution for three times, replacing the group with a fresh culture medium containing 10% of CCK8 reagent, culturing for 1-4 hours, observing color change, taking out the group when the color of the culture medium is changed into yellow, reading the absorbance value at the wavelength of 450nm by using an enzyme labeling instrument, and calculating the inhibition rate of the pharmaceutical composition on the cell activity according to the absorbance value.

3. Establishment of stable transformants with lung epithelial cells overexpressing ACE 2:

a virus plasmid was prepared which overexpressed ACE2 from human sources (freely available from Guangzhou Korea Biotechnology Co., Ltd.).

(1) 293T cells are plated one day before transfection, and transfection is carried out when the cell density reaches 80%, taking a 6-well plate as an example;

(2) preparing a plasmid: 30min before preparation, 1.5ml of the 293T new culture medium is replaced. Adding 20 mu L of A reagent in a lentivirus packaging kit into 150 mu L of DMEM, then adding 2 mu g of overexpression ACE2 plasmid, reversing and mixing uniformly for 10s, and standing for 30min at room temperature;

(3) the mixture was added dropwise to 293T six-well plates, gently mixed, placed in an incubator, and incubated for 24 h.

(4) Collecting virus liquid, adding 1.5ml of new culture medium into the six-well plate, and culturing for 24 h.

(5) Collecting virus liquid again, mixing the virus liquid twice, and filtering with 0.45 μm filter.

(6) The filtrate was obtained to infect lung epithelial cell BEAS-2B and bronchial epithelial cell 16 BE.

(7) And after 24 hours, discarding cell supernatant, adding corresponding antibiotics to carry out cell screening, and finally obtaining the stable transformant.

4. Stable transformants overexpressing ACE2 were infected with COVID-19 pseudovirus:

a COVID-19 pseudovirus solution (purchased from Peyer Biotech, Inc.) was prepared.

Medium and pseudovirus solution 1: 1 mix and infect BEAS-2B and 16HBE stable transformants that overexpress ACE 2.

5. And (3) virus titer determination:

(1) preparation of the experiment: one 96-well plate, 10 μ L and 100 μ L pipette guns, one box for each small and medium gun head, and several 1.5mL EP tubes for complete culture medium (ph 7.2).

(2) Lung epithelial cells overexpressing ACE2 were dissociated and the cells counted.

(3) Virus dilution: taking 10 sterile EP tubes, adding 180 mu L of cell sap with the cell concentration of 5.56 multiplied by 104 cells/ml into each tube, then adding 20 mu L of virus stock solution into the first EP tube, sucking 20 mu L of virus stock solution from the first EP tube after mixing uniformly, and adding the virus stock solution into the second EP tube; by analogy, the virus stock solution was sequentially subjected to 10-fold dilution infection.

(4) The cell virus mixture of 10 EP tubes was pipetted into 10 wells of 96 wells in sequence. Thus, the virus fluid in the first well is 18. mu.L, and so on, the virus amount in the other wells is 1.8. mu.L, 1.8E-1. mu.L, 1.8E-2. mu.L, etc. Note that: each pipette must be replaced with a new tip or the virus outside the tip is carried into the next well, resulting in an overestimated titer endpoint.

(5) The virus-inoculated cell culture plate was returned to 37 ℃ with 5% CO₂An incubator.

(6) After 48 hours, the fluorescence expression was observed, and the number of fluorescent cells in the last well containing fluorescent cells was counted.

(7) Calculating infection titer:

titer value (TU/mL) of virus stock solution (number of fluorescent cells)/(volume of virus after dilution mL)

6. Experiment grouping

The control group and the experimental groups 1 to 5 (cells were treated with the ointments prepared in examples 1 to 5, respectively) were divided, the ointments prepared in examples 1 to 5 were prepared into 500mg/ml stock solutions, and the cells of the experimental groups were treated at a dose of 20 mg/ml.

Second, experimental results

1. The CCK8 experiment detects the inhibition rate of different drug doses on the activity of lung epithelial cells infected by the COVID-19 pseudovirus, and the results of the CCK8 experiment shown in the table 1 and the table 2 show that the pharmaceutical composition can effectively inhibit the activity of the infected cells and is drug dose-dependent.

2. FIG. 1 is an optical microscopic picture of cells and the change of relative cell number after 24 hours of treatment and control groups of the pharmaceutical composition of the present invention, and it can be seen that the pharmaceutical composition of the present invention has a significant inhibitory effect on the activity of lung epithelial cells infected with COVID-19 pseudovirus.

3. FIG. 2 shows the detection results of the assay of the viral titer in the BEAS-2B and 16HBE cells infected with the COVID-19 pseudovirus by fluorescence measurement, which shows that the pharmaceutical composition of the present invention can effectively reduce the viral titer.

TABLE 1 inhibition ratio (%)

TABLE 2 inhibition ratio (%)

The following is a test of the inhibition effect of the pharmaceutical composition of the invention on the mouse new coronavirus pneumonia:

experimental materials and methods

1. Laboratory animals and major reagents

Male C57 mice, aged 4 weeks, with a body mass of 20-30g, purchased from Shanghai Spikenard Limited department; AAV9-ACE2 virus, purchased from peyer biotechnology limited; COVID-19 pseudovirus liquid, purchased from Peizian Biotechnology, Inc. The reverse transcription kit and the fluorescent quantitative Realtime RT-PCR kit were purchased from Takara.

2. Experimental methods

2.1 preparation of mouse model of New coronavirus pneumonia

2.1.1A mouse model of overexpression of human ACE2 is first established, and nasal cavity instillation is adopted in the experiment.

(1) The mice were anesthetized by intraperitoneal injection of ketamine and xylazine;

(2) fixing the mouse on a rodent workbench in a dorsal decubitus posture;

(3) placing the incisor ring on the upper incisor of the mouse, and connecting the nylon fastener tape to the corresponding nylon fastener tape on the lower surface of the rodent workbench;

(4) AAV9-ACE2 virus suspension was slowly instilled through the inner walls of the mouse nostrils in a minimum volume of 50 μ L (25 μ L per nostril) and a maximum volume of 75 μ L (37.5 μ L per nostril) to ensure better lung infection efficiency. After the breathing of the mouse returns to normal, 75 mu L of AAV9-ACE2 virus suspension is dripped again;

(5) gently taking down the mouse from the workbench, and slightly heightening the head and the chest by using folded paper towels to ensure that the mouse breathes smoothly;

(6) after the mice recovered, they were returned to their cages.

(7) The mice are raised for 1-2 months, and the vital signs of the mice are noticed during the raising period.

2.1.2 establishing mouse model of new coronavirus pneumonia

(1) Obtaining mice with lung tissues over expressing ACE 2;

(2) the mice were anesthetized by intraperitoneal injection of ketamine and xylazine;

(3) fixing the mouse on a rodent workbench in a dorsal decubitus posture;

(4) the neck of the mouse is cut open to expose the trachea;

(5) the COVID-19 pseudovirus liquid is injected into the lung of the mouse from the trachea, and the movement is gentle and slow, so that the mouse is prevented from suffocating and dying.

(6) Suturing wound, sterilizing, and preventing and treating infection.

2.2 animal groups

Selecting mice with successful modeling, dividing the mice into 10 groups of a control group and an experimental group 1-5 (the traditional Chinese medicine paste prepared in the gavage example 1-5 respectively), diluting 10g of the traditional Chinese medicine paste prepared in the example 1-5 to 50ml with purified water, gavage the mice of the experimental group according to the dosage of 100 mu L/10g, and gavage the mice of the control group with physiological saline with the same dosage for 2 times a day for 15 days continuously. If no special requirement is required during the gavage period, the gavage can be carried out conventionally.

2.3 sample Collection and Virus detection

After 15 days of gavage, mice were decapped and sacrificed, lung tissue was dissected away, and virus titer was determined.

2.4 detection of HE, Ki-67, Bcl-2

After the mice were sacrificed by cervical dislocation, lung tissue was sectioned and examined by hematoxylin-eosin (HE) staining and Immunohistochemistry (IHC) experiments.

Immunohistochemistry assay (IHC):

(1) dewaxing the cut slices: placing the slices in dimethylbenzene I and dimethylbenzene II for 5min respectively; placing the slices in anhydrous alcohol I, anhydrous alcohol II, 95% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, and distilled water for 5min respectively;

(2) performing antigen retrieval for 2h by using an electric cooker, and naturally cooling;

(3) washing with PBS for 3 times, each for 2 min;

(4) dropwise adding 3% hydrogen peroxide for 10min to inactivate endogenous peroxidase;

(5) the subsequent steps were performed according to the Katy one-step immunohistochemical kit (purchased from Kyowa Katy Biotechnology Co., Ltd.).

HE staining:

(1) the steps of material taking, slicing, dewaxing and hydrating are the same as the steps (1) to (3) of the IHC experiment;

(2) placing the slices in hematoxylin staining jar, staining for 5min, and washing with clear water;

(3) placing the slices in an eosin dye vat, dyeing for 2min, and washing with clear water;

(4) removing excessive water, sequentially placing slices in 50% ethanol, 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol I and anhydrous ethanol II for 1min respectively;

(5) and (5) drying the gel in the air at room temperature, and sealing the gel with neutral resin.

2.5GFP fluorescence assay

(1) The steps of material taking, slicing, dewaxing and hydrating are the same as the steps (1) to (4) of the IHC experiment;

(2) tissue blocking for 1h, primary antibody incubation, 4 ℃, overnight.

(3) Washing with PBS for 3 times, each for 2 min;

(4) incubating 488 fluorescent secondary antibody corresponding to the species for 1h, washing for 3 times with PBS (phosphate buffer solution), and washing for 2min each time;

(5) incubating DAPI for 15min, washing with PBS for 3 times, each for 2 min;

(6) and adding an anti-fluorescence quencher seal, fixing with nail polish, and photographing.

2.6 data processing

Data are averaged. + -. standard deviation

Showing that SPSS 21.0 statistical software is used for carrying out statistical processing on data, and the statistical method adopts chi²Inspection when P is<A score of 0.05 was considered statistically significant.

Third, experimental results

FIG. 3 shows GFP fluorescence experiments, and the results show that the pharmaceutical composition of the present invention can effectively inhibit infection of the COVID-19 pseudovirus in lung tissue. It is also found through immunohistochemical experiments and HE staining that the pharmaceutical composition of the invention effectively inhibits the expression of cell proliferation index Ki-67 and apoptosis index Bcl-2 as shown in FIG. 4. FIG. 5 shows the result of the virus titer detection in the lung tissue of a mouse animal model, and it can be found that the concentration of COVID-19 pseudovirus in the lung tissue can be effectively reduced after the treatment by the pharmaceutical composition of the present invention.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A traditional Chinese medicine composition for preventing and treating new crown virus pneumonia is characterized in that the medicine active ingredients of the traditional Chinese medicine composition are a mixture consisting of ephedra, coix seed, almond, aster, coltsfoot flower, gypsum, mangnolia officinalis, rhizoma anemarrhenae, radix paeoniae alba, scutellaria baicalensis, honey-fried licorice root, semen brassicae alba, perillaseed, semen raphani, wild buckwheat, astragalus mongholicus and lily, wherein the weight parts of the components are respectively 1-10 parts of ephedra, 1-10 parts of coix seed, 1-10 parts of almond, 1-10 parts of aster, 1-10 parts of coltsfoot flower, 1-20 parts of gypsum, 1-15 parts of mangnolia officinalis, 1-8 parts of rhizoma anemarrhenae, 1-15 parts of white peony root, 1-6 parts of scutellaria baicalensis, 1-20 parts of honey-fried licorice root, 1-10 parts of semen brassicae, 1-10 parts of white mustard seed, 1-20, 1-10 parts of perillaseed, 1-10 parts of radish seed, 1-20 parts of wild buckwheat rhizome, 1-30 parts of astragalus and 1-30 parts of lily.

2. A method of preparing the Chinese medicinal composition of claim 1, comprising the steps of: mixing and soaking ephedra, coix seeds, almonds, aster, coltsfoot flowers, gypsum, mangnolia officinalis, rhizoma anemarrhenae, white paeony roots, scutellaria baicalensis, honey-fried licorice roots, semen brassicae, perillaseeds, semen raphani, wild buckwheat, astragalus membranaceus and lily in water, adding water in an amount which is 3-10 times of the total weight of the mixed traditional Chinese medicine components, soaking for 24 hours, boiling with strong fire, maintaining slight boiling with slow fire, decocting for 2-4 times by adopting the decocting method, wherein the time of each decocting is 10-60min, filtering and remaining medicinal juice after each decocting, continuing to decoct the medicinal residues with cold water, finally combining all the medicinal juice, standing for 1 day, and adding a pharmaceutically acceptable carrier which is 0.1-1.0 time of the total weight of the traditional Chinese medicine components to obtain the traditional Chinese medicine with various dosage forms.

3. The method according to claim 2, wherein the pharmaceutically acceptable carrier is selected from any one or more of an emulsifier, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption enhancer, a flavoring agent, a coloring agent and a solubilizing agent.

4. The preparation method according to claim 2, wherein the formulation of the Chinese medicine is a Chinese medicinal ointment formulation.

5. The use of the traditional Chinese medicine composition of claim 1, which is characterized in that the traditional Chinese medicine composition is used for preparing a medicine for preventing and treating new coronavirus pneumonia.