CN111840374A

CN111840374A - Application of traditional Chinese medicine preparation in preparation of medicine for treating pneumonia caused by coronavirus

Info

Publication number: CN111840374A
Application number: CN202010955379.7A
Authority: CN
Inventors: 杜成强
Original assignee: Tsing Hua De Ren Xi'an Happiness Pharmaceutical Co ltd
Current assignee: Beijing happy future health technology Co.,Ltd.
Priority date: 2020-09-11
Filing date: 2020-09-11
Publication date: 2020-10-30

Abstract

The invention discloses application of a traditional Chinese medicine preparation in preparing a medicine for treating pneumonia caused by coronavirus. The Chinese medicinal composition comprises the following raw materials of effective components: 300-350 parts of prepared polygonum multiflorum, 400-440 parts of glossy privet fruit, 400-440 parts of mulberry, 400-440 parts of eclipta, 300-350 parts of white paeony root, 300-350 parts of astragalus mongholicus and 300-350 parts of rhizoma cibotii. The traditional Chinese medicine preparation can be used for treating pneumonia caused by coronavirus, the treatment effect of the traditional Chinese medicine preparation is comprehensively evaluated by detecting indexes such as lung tissue lung index of mice, peripheral blood immune cell percentage change and the like through in-vivo experiments, and the results show that the traditional Chinese medicine preparation can obviously reduce the lung index of mice infected by coronavirus, and effectively improve the lung index of the mice infected by the coronavirusMouse peripheral blood CD4⁺T lymphocytes, CD8⁺The percentage of T lymphocytes and the percentage of total B lymphocytes show that the Chinese medicinal preparation has remarkable treatment effect.

Description

Application of traditional Chinese medicine preparation in preparation of medicine for treating pneumonia caused by coronavirus

Technical Field

The invention relates to the technical field of medicines, in particular to application of a traditional Chinese medicine preparation in preparation of a medicine for treating pneumonia caused by coronavirus.

Background

Acute upper respiratory infections are caused by 70% to 80% of the viruses, including rhinoviruses, coronaviruses, adenoviruses, influenza and parainfluenza viruses, respiratory syncytial viruses, echoviruses, coxsackie viruses, and the like. Another 20-30% is caused by bacteria. Bacterial infections may be either direct or secondary to viral infections, with hemolytic streptococci being the most common, followed by haemophilus influenzae, pneumococcus, staphylococci, etc., and occasionally gram-negative bacteria.

The new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is called new coronavirus pneumonia for short, and is pneumonia caused by 2019 new coronavirus infection, and specific medicines for treating the new coronavirus pneumonia are in urgent need, so that research and development of new coronavirus pneumonia medicines and scientific research and development are not in short time.

Disclosure of Invention

The invention aims to provide application of a traditional Chinese medicine preparation in preparing a medicine for treating pneumonia caused by coronavirus, and provides a medicine capable of preparing the medicine for treating the pneumonia caused by coronavirus.

In order to achieve the above object, according to one aspect of the present invention, there is provided a use of a Chinese medicinal preparation in the preparation of a medicament for treating pneumonia caused by coronavirus. The traditional Chinese medicine composition comprises the following raw materials of active ingredients in parts by weight: 300-350 parts of prepared polygonum multiflorum, 400-440 parts of glossy privet fruit, 400-440 parts of mulberry, 400-440 parts of eclipta, 300-350 parts of white paeony root, 300-350 parts of astragalus mongholicus and 300-350 parts of rhizoma cibotii.

Further, the active ingredients of the traditional Chinese medicine preparation comprise the following raw materials in parts by weight: 320-350 parts of prepared fleece-flower root, 425-440 parts of glossy privet fruit, 425-440 parts of mulberry, 425-440 parts of yerbadetajo herb, 320-350 parts of white paeony root, 320-350 parts of astragalus root and 320-350 parts of rhizoma cibotii.

Further, the active ingredients of the traditional Chinese medicine preparation comprise the following raw materials in parts by weight: 344 parts of prepared fleece-flower root, 430.7 parts of glossy privet fruit, 430.7 parts of mulberry, 430.7 parts of yerbadetajo herb, 344 parts of white paeony root, 344 parts of astragalus root and 344 parts of east Asian tree fern rhizome.

Further, the Chinese medicinal preparation also comprises a pharmaceutically acceptable carrier and/or excipient.

Further, the Chinese medicinal preparation is granules, tablets, capsules, pills, oral solutions, oral suspensions or oral emulsions.

Further, the traditional Chinese medicine preparation is prepared by the following preparation method: 1) cutting radix polygoni multiflori preparata, glossy privet fruit, mulberry, yerbadetajo herb, white paeony root, astragalus and rhizoma cibotii, and soaking in water for 15-30 minutes; 2) boiling twice, adding 9-12 times of water for the first time, and decocting for 1.5-2.5 hours; 3) adding 7-9 times of water for the second time, decocting for 1.5-2.5 hours, and combining the decoctions; 4) filtering, and concentrating the filtrate under reduced pressure to 1000 m; 5) adding 0.5% of stevioside; 6) adding ethylparaben 0.15%, mixing, and packaging.

Further, the traditional Chinese medicine preparation is prepared by the following preparation method: 1) cutting radix Polygoni Multiflori Preparata, fructus Ligustri Lucidi, Mori fructus, Ecliptae herba, radix Paeoniae alba, radix astragali and rhizoma Cibotii, and soaking in water for 20 min; 2) decocting twice with boiling, adding 10 times of water for the first time, and decocting for 2 hr; 3) adding 8 times of water for the second time, decocting for 1.5 hr, and mixing decoctions; 4) filtering, and concentrating the filtrate under reduced pressure to 1000 m; 5) adding 0.5% of stevioside; 6) adding ethylparaben 0.15%, mixing, and packaging.

Further, the coronavirus is human coronavirus HCoV-229E.

The traditional Chinese medicine preparation can be used for treating pneumonia caused by coronavirus, and the treatment effect of the traditional Chinese medicine preparation is comprehensively evaluated by detecting indexes such as lung tissue lung index of mice, percentage change of peripheral blood immunocytes and the like through in-vivo experiments, and the results show that the traditional Chinese medicine preparation can obviously reduce the lung index of mice infected by coronavirus, and effectively improve the peripheral blood CD4 of the mice⁺T lymphocytes, CD8⁺The percentage of T lymphocytes and the percentage of total B lymphocytes show that the Chinese medicinal preparation has remarkable treatment effect.

Detailed Description

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 invention will be described in detail with reference to examples.

According to a typical embodiment of the invention, the application of a traditional Chinese medicine preparation in preparing a medicine for treating pneumonia caused by coronavirus is provided. The traditional Chinese medicine composition comprises the following raw materials of active ingredients in parts by weight: 300-350 parts of prepared polygonum multiflorum, 400-440 parts of glossy privet fruit, 400-440 parts of mulberry, 400-440 parts of eclipta, 300-350 parts of white paeony root, 300-350 parts of astragalus mongholicus and 300-350 parts of rhizoma cibotii.

Preferably, the active ingredients of the traditional Chinese medicine preparation comprise the following raw materials in parts by weight: 320-350 parts of prepared fleece-flower root, 425-440 parts of glossy privet fruit, 425-440 parts of mulberry, 425-440 parts of yerbadetajo herb, 320-350 parts of white paeony root, 320-350 parts of astragalus root and 320-350 parts of rhizoma cibotii. More preferably, the active ingredients of the Chinese medicinal preparation comprise the following raw materials in parts by weight: 344 parts of prepared fleece-flower root, 430.7 parts of glossy privet fruit, 430.7 parts of mulberry, 430.7 parts of yerbadetajo herb, 344 parts of white paeony root, 344 parts of astragalus root and 344 parts of east Asian tree fern rhizome. The traditional Chinese medicine preparation with the proportion has better effect of treating pneumonia caused by coronavirus.

The Chinese medicinal preparation can be solid, liquid or gel and the like. As used herein, the use of the term "pharmaceutically acceptable carrier and/or vehicle" means a non-toxic solid, an inert semi-solid liquid excipient, a diluent, any type of auxiliary formulation, or simply a sterile aqueous solution, e.g., saline. Some examples of materials that can be used as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose, starches such as corn starch and potato solution, cellulose and its derivatives such as carboxymethyl cellulose, ethyl cellulose and cellulose acetate, cyclodextrins; oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil, glycols such as propylene glycol, polyols such as glycerol, sorbitol, mannitol and polyvinyl esters such as ethyl laurate, ethyl oleate, agar, buffers such as aluminum hydroxide and magnesium hydroxide, alginic acid, pyrogen-free water, isotonic saline (isotonic saline), Ringer's solution, buffered solutions of ethanol and phosphate, and other non-toxic compatible substances for pharmaceutical formulation.

In one embodiment, the chinese medicinal formulation comprises one or more pharmaceutically acceptable carriers and/or one or more pharmaceutically acceptable diluents and/or one or more pharmaceutically acceptable excipients. The Chinese medicinal preparation may comprise one or more pharmaceutically acceptable carriers. The Chinese medicinal preparation may comprise one or more pharmaceutically acceptable diluents. The Chinese medicinal preparation may comprise one or more pharmaceutically acceptable excipients. The Chinese medicinal preparation of the invention can be granules, tablets, capsules, pills, oral solutions, oral suspensions, oral emulsions and the like, but is not limited to the dosage forms.

According to a typical embodiment of the invention, the traditional Chinese medicine preparation is prepared by the following preparation method: 1) cutting radix polygoni multiflori preparata, glossy privet fruit, mulberry, yerbadetajo herb, white paeony root, astragalus and rhizoma cibotii, and soaking in water for 15-30 minutes; 2) boiling twice, adding 9-12 times of water for the first time, and decocting for 1.5-2.5 hours; 3) adding 7-9 times of water for the second time, decocting for 1.5-2.5 hours, and combining the decoctions; 4) filtering, and concentrating the filtrate under reduced pressure to 1000 m; 5) adding 0.5% of stevioside; 6) adding ethylparaben 0.15%, mixing, and packaging. Preferably, the traditional Chinese medicine preparation is prepared by the following preparation method: 1) cutting radix Polygoni Multiflori Preparata, fructus Ligustri Lucidi, Mori fructus, Ecliptae herba, radix Paeoniae alba, radix astragali and rhizoma Cibotii, and soaking in water for 20 min; 2) decocting twice with boiling, adding 10 times of water for the first time, and decocting for 2 hr; 3) adding 8 times of water for the second time, decocting for 1.5 hr, and mixing decoctions; 4) filtering, and concentrating the filtrate under reduced pressure to 1000 m; 5) adding 0.5% of stevioside; 6) adding ethylparaben 0.15%, mixing, and packaging. The Chinese medicinal preparation prepared by the method can effectively treat pneumonia caused by coronavirus. Typically, the coronavirus is human coronavirus HCoV-229E. The following examples are provided to further illustrate the advantageous effects of the present invention.

Example 1

The raw materials of the traditional Chinese medicine preparation 1 and the preparation method are as follows:

1) cutting 344 parts of prepared polygonum multiflorum, 430.7 parts of glossy privet fruit, 430.7 parts of mulberry, 430.7 parts of yerbadetajo herb, 344 parts of white paeony root, 344 parts of astragalus root and 344 parts of rhizoma cibotii, and then adding water for soaking for 20 minutes;

2) decocting twice with boiling, adding 10 times of water for the first time, and decocting for 2 hr;

3) adding 8 times of water for the second time, decocting for 1.5 hr, and mixing decoctions;

4) filtering, and concentrating the filtrate under reduced pressure to 1000 m;

5) adding 0.5% of stevioside;

6) adding ethylparaben 0.15%, mixing, and packaging to obtain Chinese medicinal preparation 1.

Purpose of the experiment

The treatment effect of the tested medicament on the combined model of the syndrome of the coronavirus pneumonia cold-dampness epidemic toxin attacking the lung mice is evaluated from two aspects of lung inflammation and the percentage of immune cells in peripheral blood, and a basis is provided for clinical application.

Test site

ABSL-2 laboratory of institute of traditional Chinese medicine of Chinese academy of sciences and sciences

1 test Material

1.1 test drugs: the dosage of the traditional Chinese medicine preparation 1 prepared in the embodiment is as follows: orally administered 15ml each time, 3 times a day; storage conditions were as follows: sealing and storing in shade.

1.2 strains and cells

1.2.1 viral strains: human coronavirus (HCoV-229E), provided by institute of medical and biotechnology, of Chinese academy of medical sciences, was passaged in the laboratory and stored in a refrigerator at-80 ℃ for later use.

1.3 Experimental animals are shown in Table 1

TABLE 1

Species of

Rank of

Body weight

Number of

Sex

Certificate number

License number

Balb/c mice

SPF stage

13-15g

48

Male and female halves

1100112011004807

SCXK (Jing) 2016-

1.4 test reagents are shown in Table 2

TABLE 2

1.5 test instruments are shown in Table 3

TABLE 3

2 test drug dosage design

The clinical human dose of the traditional Chinese medicine preparation 1 is 45ml/60kg/d, and the dosage adopted in the experimental study is converted into the mouse dose of 16.5ml/kg/d and the mouse dose of 8.25ml/kg/d which is equivalent to 2 times and equal times of the clinical dosage for human. The mice were dosed at 0.2ml/10g/d by gavage during the experiment.

3 test methods and results

3.1 passage of Virus

Taking 25cm of A549 cells which have grown into a monolayer²Pouring out the culture solution, washing the cell surface with cell maintenance solution for 3 times, adding 200 μ l HCoV-229E virus solution, and placing at 37 deg.C with 5% CO₂Culturing in an incubator, observing cytopathic condition under an inverted microscope every day for 72-96h until 80% of cells have obvious pathological Changes (CPE), placing a cell culture bottle in a low-temperature refrigerator at minus 80 ℃ for cryopreservation, and repeatedly freezing and thawing virus liquid for 3 times for detecting virus virulence.

3.2 Virus titre assay

Collecting culture plate with single layer of A549 cells, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, diluting by 10 times, inoculating HCoV-229E virus solution with different titer, and 10 times diluting^-1-10^-8A total of 8 dilutions, 100. mu.l/well, 4 replicates per concentration, were included along with normal cell controls. Placing at 37 ℃ with 5% CO₂Culturing in an incubator, observing cytopathic condition under an inverted microscope every day, and recording the cytopathic condition of each well for 72-96 h. The 50% cytopathic concentration (TCID50) was calculated as Reed-Muench.

3.3 Effect of Chinese medicinal preparation 1 on BALB/c mouse Lung index of human coronavirus pneumonia and epidemic toxin attacking Lung

48 BALB/c mice are selected, the body mass (14 +/-1) g and the male and female parts are randomly divided into a normal group, an HCoV-229E infection group (hereinafter referred to as an infection group), a cold-dampness group, a cold-dampness + HCoV-229E infection group (hereinafter referred to as a cold-dampness infection group), a traditional Chinese medicine preparation 1 high-dose group (16.5ml/kg/d) and a traditional Chinese medicine preparation 1 low-dose group (8.25ml/kg/d) according to the body mass grade, and the female and male parts are 8 in each group. Except for the normal group and the infected group, the other 4 groups of mice are subjected to cold-dampness syndrome modeling, and the modeling method comprises the following steps: the mice were placed in a windless climatic chamber at 4 + -2 deg.C and 90% + -3% relative humidity for 4h daily for 7 days. On day 5 of the model building of cold-dampness syndrome, mice in each group were lightly anesthetized with ether except for the normal group and the cold-dampness group, and the mice were infected with 100TCID50HCoV-229E drops of nasal drops of 50. mu.L/mouse for 2 days continuously. On the infection day, the group with the large and small dosage of the Chinese medicinal preparation 1 begins to be administrated by intragastric administration, 0.02mL/g is continuously administrated for 3 days, and the other groups are administrated by intragastric administration with equal amount of distilled water. On day 8, the mice were weighed, orbital blood was collected in 2 parts, lungs were peeled off, weighed, and 3 parts, stored at 2-80 ℃, and one part was directly put into formalin for use.

Mouse lung index calculation formula:

lung index ═ lung wet weight (g) × 100/body weight (g)

The lung index inhibition rate calculation formula is as follows:

the therapeutic effect of the Chinese medicinal preparation 1 on the lung disease of human coronavirus pneumonia and epidemic toxin combined with mice is shown in Table 4.

TABLE 4

Note: comparison with Normal group^##p is less than 0.01; compared with the model group of the lung with cold-dampness stagnation^**p＜0.01

As shown in table 4, compared with the normal group, the lung index of the mice in the cold-dampness group has no obvious change, and the lung index of the mice in the infection group and the cold-dampness infection group is obviously increased and has obvious difference (P < 0.01); after the mice are given the Chinese medicinal preparation 1 dry prognosis, the lung indexes of the mice of 2 dose groups are obviously reduced, and the mice have significant difference (P <0.01) compared with a cold-dampness infected group. The lung index inhibition rates of mice in large and small dose groups of the traditional Chinese medicine preparation 1 are 70.18% and 54.40% respectively. The traditional Chinese medicine preparation 1 has obvious treatment effect on the combined model of the disease of the coronavirus pneumonia cold-dampness epidemic toxin attacking lung mice.

3.4 Effect of Chinese medicinal preparation 1 on the percentage of BALB/c mouse peripheral blood lymphocytes in the pattern of human coronavirus pneumonia and epidemic lung disease

When the mouse takes the eyeball and draws blood, 3 drops of blood (about 150 mu L) are directly dripped into a centrifuge tube filled with 10mL PBS, the mixture is evenly mixed by turning upside down, and the temperature is 1600 r.min^-1Centrifuging for 5min, carefully pouring out supernatant, adding 1mL of erythrocyte lysate into each tube, gently blowing and beating the heavy suspension cell sediment, performing room-temperature lysis for 5-10 min, adding 10mL of PBS to stop lysis when the liquid turns from turbid to clear, and performing centrifugation at 4 ℃ for 2000 r.min^-1Centrifuging for 5min, sucking the supernatant with a pipette tip, discarding, adding 10mL PBS per tube, and blowing to remove the cell precipitate, 4 deg.C，2000r·min^-1Centrifuging for 5min, discarding supernatant, adding 400 μ L blocking solution (PBS containing 5% FBS) into each tube, resuspending, transferring cell sap into 1.5mL EP tube, blocking at 4 deg.C for 30min, and then sealing at 4 deg.C 2000 r.min^-1Centrifuging for 5min, sucking off supernatant, discarding, adding 50 μ L of flow antibody (preparation method: PE-labeled anti-mouse CD19, PerCP-Cy5.5-labeled anti-mouse CD4, APC-labeled anti-mouse CD8a, adding 0.3 μ L of each antibody into 50 μ L of blocking solution), standing at 4 deg.C in dark for 30min, adding 1mL of PBS into each tube, 4 deg.C, 2000 r.min^-1Centrifuging for 5min, sucking out supernatant, discarding, resuspending cells with 200 μ L of 2% paraformaldehyde fixing solution, storing overnight at 4 deg.C in dark place, and detecting on the 2 nd day.

The therapeutic effect of the Chinese medicinal preparation 1 on the lung disease of human coronavirus pneumonia and epidemic toxin combined with mice is shown in Table 5.

TABLE 5

Note: comparison with Normal group^##p is less than 0.01; compared with the model group of the lung with cold-dampness stagnation^**p＜0.07

Note: # p <0.01 compared to normal; p <0.01 in comparison with the cold-damp-depressed lung model group

As shown in Table 5, the mice in the cold-dampness infected group had CD4 in their peripheral blood level compared with the normal group⁺T lymphocytes, CD8⁺The percentage of T lymphocytes and total B lymphocytes is obviously reduced (P)<0.05，P<0.01); group dry prognosis given 12 doses of the Chinese medicinal preparation, mouse peripheral blood CD4⁺T lymphocytes, CD8⁺The percentage of T lymphocytes and total B lymphocytes is obviously increased, and the percentage is obviously different from that of a cold-dampness infected group (P)<0.05，P<0.01)。

To summarize:

in the embodiment, the therapeutic effect of the traditional Chinese medicine preparation 1 is comprehensively evaluated by detecting indexes such as lung index of mouse lung tissue, percentage change of peripheral blood immune cells and the like through in vivo experiments. The experimental result shows that the traditional Chinese medicine preparation 1 can obviously reduce the lung index of a cold-dampness infected mouse, and the inhibition rates of the large dose and the small dose of the traditional Chinese medicine preparation 1 respectively reach 70.18% and 54.40%.The traditional Chinese medicine preparation 1 can effectively improve the mouse peripheral blood CD4⁺T lymphocytes, CD8⁺The percentage of T lymphocytes and total B lymphocytes is significantly different from that of the cold-dampness infected group (P)<0.05，P<0.01)。

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

Claims

1. The application of a traditional Chinese medicine preparation in preparing a medicine for treating pneumonia caused by coronavirus is characterized in that the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 300-350 parts of prepared polygonum multiflorum, 400-440 parts of glossy privet fruit, 400-440 parts of mulberry, 400-440 parts of eclipta, 300-350 parts of white paeony root, 300-350 parts of astragalus mongholicus and 300-350 parts of rhizoma cibotii.

2. The application of claim 1, wherein the effective components of the Chinese medicinal preparation comprise the following raw materials in parts by weight: 320-350 parts of prepared fleece-flower root, 425-440 parts of glossy privet fruit, 425-440 parts of mulberry, 425-440 parts of yerbadetajo herb, 320-350 parts of white paeony root, 320-350 parts of astragalus root and 320-350 parts of rhizoma cibotii.

3. The application of claim 2, wherein the effective components of the Chinese medicinal preparation comprise the following raw materials in parts by weight: 344 parts of prepared fleece-flower root, 430.7 parts of glossy privet fruit, 430.7 parts of mulberry, 430.7 parts of yerbadetajo herb, 344 parts of white paeony root, 344 parts of astragalus root and 344 parts of east Asian tree fern rhizome.

4. The use according to any one of claims 1 to 3, wherein the Chinese medicinal preparation further comprises a pharmaceutically acceptable carrier and/or excipient.

5. Use according to any one of claims 1 to 3, characterized in that the traditional Chinese medicine preparation is granules, tablets, capsules, pills, oral solutions, oral suspensions or oral emulsions.

6. The use according to any one of claims 1 to 3, wherein the Chinese medicinal preparation is prepared by the following preparation method:

1) cutting radix polygoni multiflori preparata, glossy privet fruit, mulberry, yerbadetajo herb, white paeony root, astragalus and rhizoma cibotii, and soaking in water for 15-30 minutes;

2) boiling twice, adding 9-12 times of water for the first time, and decocting for 1.5-2.5 hours;

3) adding 7-9 times of water for the second time, decocting for 1.5-2.5 hours, and combining the decoctions;

4) filtering, and concentrating the filtrate under reduced pressure to 1000 m;

5) adding 0.5% of stevioside;

6) adding ethylparaben 0.15%, mixing, and packaging.

7. The use of claim 6, wherein the Chinese medicinal preparation is prepared by the following preparation method:

1) cutting radix Polygoni Multiflori Preparata, fructus Ligustri Lucidi, Mori fructus, Ecliptae herba, radix Paeoniae alba, radix astragali and rhizoma Cibotii, and soaking in water for 20 min;

4) filtering, and concentrating the filtrate under reduced pressure to 1000 m;

5) adding 0.5% of stevioside;

6) adding ethylparaben 0.15%, mixing, and packaging.

8. Use according to any one of claims 1 to 3, wherein the coronavirus is human coronavirus HCoV-229E.