CN112220898A

CN112220898A - Anti-coronavirus composition, preparation method and application thereof

Info

Publication number: CN112220898A
Application number: CN202011247622.6A
Authority: CN
Inventors: 李守黔; 杨细飞; 房师松; 许本洪; 谢永美; 李守跃; 李光陆; 周友洲; 周誉; 李光胜
Original assignee: Guizhou Jinqianguo Biotechnology Co ltd
Current assignee: Guizhou Jinqianguo Biotechnology Co ltd
Priority date: 2020-11-10
Filing date: 2020-11-10
Publication date: 2021-01-15
Also published as: CN114010737A

Abstract

The invention relates to an anti-coronavirus composition, a preparation method and application thereof, belonging to the technical field of medicines. The anti-coronavirus composition comprises the following components in parts by weight: 5-100 parts of honey, 5-100 parts of roxburgh rose, 1-50 parts of glabrous greenbrier rhizome, 1-30 parts of green lemon, 1-30 parts of plantain herb, 1-30 parts of walnut, 1-30 parts of indigowoad root, 1-30 parts of lucid ganoderma and 1-30 parts of revival cinquefoil herb. The anti-coronavirus composition of the present invention has excellent activity against novel coronavirus SARS-CoV-2.

Description

Anti-coronavirus composition, preparation method and application thereof

Technical Field

The invention relates to an anti-coronavirus composition, a preparation method and application thereof, belonging to the technical field of medicines.

Background

The new coronary pneumonia (COVID-19) caused by the novel coronavirus (SARS-CoV-2) is a serious public health event, and the outbreak has caused huge casualties and economic losses so far, but at present, no specific medicine aiming at the SARS-CoV-2 with definite curative effect exists clinically. In addition, most patients are treated by antibiotics, interferons, oxygen supply, glucocorticoids, traditional Chinese medicines and other medicines, but the medicines are not novel specific medicines for coronavirus. At present, part of potential antiviral drugs pass through a rapid review channel, but clinical tests are not finished yet and the effectiveness is to be determined.

In 2020, 10 and 22 days, the antiviral drug Reidesvir (trade name: Veklury) approved by the US FDA is sold on the market, and is suitable for treating patients with new crown pneumonia which is 12 years old or more, has the weight of at least 40 kg and needs hospitalization. In 5 months of 2020, the FDA grants redciclovir emergency use authorization, allowing medical personnel to use hospitalized patients with new coronary pneumonia. However, the efficacy of Reidesciclovir is controversial. The world health organization publishes the clinical test results of the medicines such as the Reidesciclovir and the hydroxychloroquine in 10 months and 15 days, and the medicines are said to be 'almost ineffective' for reducing the fatality rate of the new coronary pneumonia. Therefore, the development of novel antiviral drugs having proprietary intellectual property rights is urgently required.

Disclosure of Invention

The first object of the present invention is to provide an anti-coronavirus composition.

To achieve the first object of the present invention, the anti-coronavirus composition comprises by weight: 5-100 parts of honey, 5-100 parts of roxburgh rose, 1-50 parts of glabrous greenbrier rhizome, 1-30 parts of green lemon, 1-30 parts of plantain herb, 1-30 parts of walnut, 1-30 parts of indigowoad root, 1-30 parts of lucid ganoderma and 1-30 parts of revival cinquefoil herb.

In one embodiment, the composition comprises: 10-50 parts of honey, 10-50 parts of roxburgh rose, 5-30 parts of glabrous greenbrier rhizome, 5-30 parts of green lemon, 2-10 parts of plantain herb, 2-10 parts of walnut, 2-10 parts of indigowoad root, 2-10 parts of lucid ganoderma and 2-10 parts of revival grass.

In one embodiment, the composition comprises: 20-40 parts of honey, 20-40 parts of roxburgh rose, 10-30 parts of glabrous greenbrier rhizome, 5-15 parts of green lemon, 3-10 parts of plantain herb, 2-5 parts of walnut, 2-5 parts of indigowoad root, 2-5 parts of lucid ganoderma and 2-5 parts of revival grass.

In one embodiment, the composition is prepared by a method comprising: pulverizing or slicing Mel, fructus Rosae Normalis, rhizoma Smilacis Glabrae, fructus Citri Limoniae, herba plantaginis, semen Juglandis, herba Elephantopi scaberis, Ganoderma and revival grass, extracting with ethanol, percolating, collecting extractive solution or percolate, adding Mel, mixing, and making into oral preparation; or fermenting or soaking the honey, the roxburgh rose, the glabrous greenbrier rhizome, the green lemon, the plantain herb, the walnut, the elephantopus scaber, the lucid ganoderma and the revival grass to prepare an oral preparation.

In a specific embodiment, the oral preparation is a wine, an oral liquid or a tincture.

The second purpose of the invention is to provide a preparation method of the anti-coronavirus composition.

To achieve the second object of the present invention, the method comprises: pulverizing or slicing Mel, fructus Rosae Normalis, rhizoma Smilacis Glabrae, fructus Citri Limoniae, herba plantaginis, semen Juglandis, herba Elephantopi scaberis, Ganoderma and revival grass, extracting with ethanol, percolating, collecting extractive solution or percolate, adding Mel, mixing, and making into oral preparation; or fermenting or soaking the honey, the roxburgh rose, the glabrous greenbrier rhizome, the green lemon, the plantain herb, the walnut, the elephantopus scaber, the lucid ganoderma and the revival grass to prepare an oral preparation.

The third purpose of the invention is to provide the application of the anti-coronavirus composition in the preparation of medicines for preventing and/or treating diseases caused by coronavirus.

In a specific embodiment, the coronavirus comprises a novel coronavirus SARS-CoV-2, Severe acute respiratory syndrome SARS, middle east respiratory syndrome MERS, or human enterocoronavirus.

In a specific embodiment, the coronavirus is a novel coronavirus SARS-CoV-2.

Has the advantages that:

1) the composition is prepared from common medicinal materials and food materials through scientific formulation and is used for resisting coronavirus.

2) The materials used by the composition provided by the invention are all derived from pure natural Chinese herbal medicines or conventional foods, and the composition has the characteristics of wide sources, low price, greenness, health, safety, no toxic or side effect and the like, and is suitable for large-scale popularization and use.

3) The anti-coronavirus composition of the present invention has excellent activity against novel coronavirus SARS-CoV-2.

Detailed Description

In a specific embodiment, the coronavirus is a novel coronavirus SARS-CoV-2.

The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.

In the present invention, all parts and percentages are by weight unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified; in the invention, the roxburgh rose, the glabrous greenbrier rhizome, the green lemon, the plantain herb, the walnut, the elephantopus scaber, the lucid ganoderma and the revival grass can be fresh products or dried products.

Example 1

Taking 50g of honey, 50g of roxburgh rose, 30g of glabrous greenbrier rhizome, 20g of green lemon, 8g of plantain herb, 5g of walnut, 5g of elephantopus scaber, 5g of lucid ganoderma and 5g of revival grass, adding water which is 4 times of the total weight of the components, fermenting for 30 days at 15-40 ℃ in the dark under a sealed condition, filtering, and collecting liquid medicine to obtain the traditional Chinese medicine.

Example 2

Taking 60g of honey, 40g of roxburgh rose, 25g of glabrous greenbrier rhizome, 20g of green lemon, 15g of plantain herb, 10g of walnut, 10g of scabrous elephantfoot herb, 10g of lucid ganoderma and 10g of revival grass, adding water which is 4 times of the total weight of the components, fermenting for 30 days at 15-40 ℃ in the dark under a sealed condition, filtering, and collecting liquid medicine to obtain the traditional Chinese medicine.

Example 3

Taking 20g of honey, 30g of roxburgh rose, 20g of glabrous greenbrier rhizome, 10g of green lemon, 5g of plantain herb, 5g of walnut, 5g of elephantopus scaber, 5g of lucid ganoderma and 5g of revival grass, adding water which is 4 times of the total weight of the components, fermenting for 30 days at 15-40 ℃ in the dark under a sealed condition, filtering, and collecting liquid medicine to obtain the traditional Chinese medicine.

Comparative example 1

Taking 50g of honey, 50g of roxburgh rose, 30g of glabrous greenbrier rhizome, 20g of green lemon, 8g of plantain herb, 5g of walnut, 5g of elephantopus scaber, 5g of lucid ganoderma and 5g of common resurrection grass, crushing into coarse powder, soaking in 50-degree white spirit, performing percolation extraction at the flow rate of 1ml/min, collecting percolate with the weight 2 times of the formula weight, adding 5g of honey, and uniformly mixing to obtain the traditional Chinese medicine.

Comparative example 2

Taking 50g of honey, 50g of roxburgh rose, 30g of glabrous greenbrier rhizome, 20g of green lemon, 8g of plantain herb, 5g of walnut, 5g of elephantopus scaber, 5g of lucid ganoderma and 5g of common resurrection grass, crushing into coarse powder, soaking in 50-degree white spirit, performing percolation extraction at the flow rate of 3ml/min, collecting 3 times of percolation liquid according to the weight of the prescription, adding 20g of honey, and uniformly mixing to obtain the traditional Chinese medicine.

Comparative example 3

Taking 60g of honey, 60g of roxburgh rose, 40g of glabrous greenbrier rhizome, 30g of green lemon, 10g of plantain herb, 10g of walnut, 10g of elephantopus scaber, 10g of lucid ganoderma and 10g of revival grass, adding water with the weight 6 times of the total weight of the medicinal materials, soaking for half an hour, extracting for 2 times, 2 hours each time, filtering, combining extracting solutions, adding 10g of honey, and uniformly mixing to obtain the traditional Chinese medicine.

Comparative example 4

Adding water of which the weight is 4 times of the total weight of the components into 35g of honey, 30g of roxburgh rose, 20g of glabrous greenbrier rhizome, 10g of green lemon, 7g of plantain herb, 4g of walnut, 4g of elephantopus scaber, 4g of lucid ganoderma and 4g of revival grass, fermenting for 30 days in a shade, filtering and collecting fermentation liquor to obtain the traditional Chinese medicine.

Comparative example 5

Adding water of which the weight is 4 times of the total weight of the components into 35g of honey, 100g of roxburgh rose, 20g of glabrous greenbrier rhizome, 10g of green lemon, 7g of plantain herb, 4g of walnut, 4g of elephantopus scaber, 4g of lucid ganoderma and 4g of revival grass, fermenting for 30 days in a shade, filtering and collecting fermentation liquor to obtain the traditional Chinese medicine.

Comparative example 6

Adding water of which the weight is 4 times of the total weight of the components into 35g of honey, 30g of roxburgh rose, 80g of glabrous greenbrier rhizome, 10g of green lemon, 7g of plantain herb, 4g of walnut, 4g of elephantopus scaber, 4g of lucid ganoderma and 4g of revival grass, fermenting for 30 days in a shade, filtering and collecting fermentation liquor to obtain the traditional Chinese medicine.

Comparative example 7

Adding water of which the weight is 4 times of the total weight of the components into 35g of honey, 30g of roxburgh rose, 20g of glabrous greenbrier rhizome, 50g of green lemon, 7g of plantain herb, 4g of walnut, 4g of elephantopus scaber, 4g of lucid ganoderma and 4g of revival grass, fermenting for 30 days in a shade, filtering and collecting fermentation liquor to obtain the traditional Chinese medicine.

Comparative example 8

Adding water of which the weight is 4 times of the total weight of honey 35g, glabrous greenbrier rhizome 20g, green lemon 10g, plantain herb 7g, walnut 4g, elephantopus scaber 4g, lucid ganoderma 4g and resurrection grass 4g into the components, fermenting the components in a shade place for 30 days, filtering the fermented liquid and collecting the fermented liquid to obtain the traditional Chinese medicine composition.

Comparative example 9

Adding water 4 times of the total weight of honey 35g, fructus Rosae Normalis 30g, green lemon 10g, herba plantaginis 7g, semen Juglandis 4g, herba Elephantopi scaberis 4g, Ganoderma 4g, and herba Polygoni revitalis 4g, fermenting in shade for 30 days, filtering, and collecting the fermentation broth.

Comparative example 10

Adding water 4 times of the total weight of honey 35g, fructus Rosae Normalis 30g, rhizoma Smilacis Glabrae 20g, green lemon 10g, semen Juglandis 4g, herba Elephantopi scaberis 4g, Ganoderma 4g, and herba Inulae Cappae 4g, fermenting in shade for 30 days, filtering, and collecting the fermentation liquid.

Comparative example 11

Adding water of which the weight is 4 times of the total weight of 35g of honey, 30g of roxburgh rose, 20g of glabrous greenbrier rhizome, 10g of green lemon, 7g of plantain herb, 4g of walnut, 4g of elephantopus scaber and 4g of revival grass into the mixture, fermenting the mixture in a shade for 30 days, filtering the mixture, and collecting fermentation liquor to obtain the traditional Chinese medicine.

Comparative example 12

Adding water 4 times of the total weight of honey 35g, fructus Rosae Normalis 30g, rhizoma Smilacis Glabrae 20g, herba plantaginis 7g, semen Juglandis 4g, herba Elephantopi scaberis 4g, Ganoderma 4g, and herba Inulae Cappae 4g, fermenting in shade for 30 days, filtering, and collecting the fermentation liquid.

Comparative example 13

Adding water 4 times the weight of Ganoderma 100g, fermenting in shade for 30 days, filtering, and collecting the fermentation broth.

Comparative example 14

Taking 100g of plantain, adding 4 times of water by weight, fermenting in shade for 30 days, filtering, and collecting fermentation liquor.

Test example 1

Anti-novel coronavirus study

(1) Cytotoxicity test

Taking out Vero E6 cells frozen in liquid nitrogen tank, and performing standard proceduresAnd (5) performing resuscitation. After the cells are passed through two generations, the logarithmically grown cells are taken, 2000 cells are inoculated into a 96-well plate per well, the side holes are filled with normal saline, 100 microliters of culture medium is added into each hole, after the cells are attached to the wall, the fermentation liquor in each embodiment is diluted to a certain concentration by the culture medium, added into the cells and cultured for 72 hours. Adding 20 microliters of MTT (5mg/ml) into each hole, absorbing the liquid by using a discharging gun after the action for 4 hours, adding 150 microliters of DMSO into each hole, oscillating the mixture in a shaking table for about 10 minutes, and measuring the absorbance value of 570 by using a microplate reader; calculating the inhibition rate according to the absorbance of the data, and then calculating the half cytotoxic concentration CC₅₀。

(2) Plaque Reduction Neutralization Test (PRNT)

1) Vero E6 cells, 20 ten thousand cells per well in 24-well plates, were cultured overnight to allow attachment.

2) Each serum sample was diluted stepwise in 2-fold gradient with DMEM medium containing 2% inactivated FBS, starting at 5-fold dilution, for a total of 6 concentration gradients, i.e., 5, 10, 20, 40, 80, 160. The volume of each diluted sample was 125. mu.L. The specific method comprises the following steps: firstly, 36 serum samples (6 immunization groups multiplied by 6 animals/group) are centrifuged, and the liquid flows to the bottom of an EP tube; adding 200 mu L of DMEM medium containing 2% inactivated FBS into a centrifuge tube containing 50 mu L of serum, and uniformly mixing to obtain a sample diluted by 5 times; taking out 125 mu L of the solution, and adding the solution into 125 mu L of DMEM medium containing 2% inactivated FBS to obtain a sample diluted by 10 times; and by analogy, gradually diluting to obtain samples diluted by 20 times, 40 times, 80 times and 160 times respectively.

3) SARS-Cov-2 virus was diluted to 400PFU/mL with DMEM medium containing 2% inactivated FBS.

4) Mixing the diluted serum sample with SARS-Cov-2 virus in equal volume, and incubating for 1 hour at 37 ℃; meanwhile, DMEM medium containing 2% inactivated FBS is used as a blank control group, mixed with SARS-Cov-2 virus in equal volume, subjected to parallel test, and incubated at 37 ℃ for 1 hour.

5) The Vero cell medium was discarded, and the heat-incubated virus-serum mixture was added to the cells at 200. mu.L per well.

6) The incubator was incubated for 1 hour, during which the plates were shaken every 15 minutes.

7) After 1 hour the virus-containing medium was discarded and washed once with PBS.

8) The low melting point agarose 5% in concentration was melted by heating at 65-100 degrees and diluted to 1% concentration with 37 degrees pre-warmed DMEM medium containing 2% inactivated FBS when it was cooled to be slightly non-scalding (about 40-60 degrees). Then 250. mu.L per well (24 well plate in step 7) was added.

9) The incubator was incubated for 48 hours.

10) The medium was discarded, fixed with 4% paraformaldehyde for 15 minutes, and then stained with 0.2% crystal violet.

11) Counting the number of plaques

Calculating the half effective concentration EC according to the number of the plaques₅₀. Selection index SI ═ CC₅₀/EC₅₀. The results of the in vitro experiments are shown in table 1.

TABLE 1 study of the activity of fermentation broths against neocoronaviruses

Note: the values of the median cytotoxic concentration and median effective concentration in the table are the percentage of the medium diluted with broth; the plaque reduction neutralization experiment is carried out under the condition that the fermentation liquor has no cytotoxicity; selection index SI ═ CC₅₀/EC₅₀。

The results of the cytotoxicity test and plaque reduction neutralization test are shown in Table 1. As shown in the table, the selectivity index of the example 1-3 is 6.40-50.00, which is higher than that of the comparative example 2.53-5.70, wherein the half inhibitory concentration CC of the fermentation liquid of the example 1 on Vero E6 cells₅₀6.00%, half the effective concentration EC for SARS-CoV-2₅₀The index was chosen to be 50 at 0.12%, while in other examples the index was chosen to be much lower than in example 1. It was shown that the fermentation broth of example 1 had excellent anti-SARS-CoV-2 activity.

Claims

1. An anti-coronavirus composition, wherein the composition comprises, by weight: 5-100 parts of honey, 5-100 parts of roxburgh rose, 1-50 parts of glabrous greenbrier rhizome, 1-30 parts of green lemon, 1-30 parts of plantain herb, 1-30 parts of walnut, 1-30 parts of indigowoad root, 1-30 parts of lucid ganoderma and 1-30 parts of revival cinquefoil herb.

2. The anti-coronavirus composition of claim 1, wherein the composition comprises: 10-50 parts of honey, 10-50 parts of roxburgh rose, 5-30 parts of glabrous greenbrier rhizome, 5-30 parts of green lemon, 2-10 parts of plantain herb, 2-10 parts of walnut, 2-10 parts of indigowoad root, 2-10 parts of lucid ganoderma and 2-10 parts of revival grass.

3. The anti-coronavirus composition according to claim 1 or 2, wherein the composition comprises: 20-40 parts of honey, 20-40 parts of roxburgh rose, 10-30 parts of glabrous greenbrier rhizome, 5-15 parts of green lemon, 3-10 parts of plantain herb, 2-5 parts of walnut, 2-5 parts of indigowoad root, 2-5 parts of lucid ganoderma and 2-5 parts of revival grass.

4. The anti-coronavirus composition according to any one of claims 1-3, wherein the composition is prepared by a method comprising: pulverizing or slicing Mel, fructus Rosae Normalis, rhizoma Smilacis Glabrae, fructus Citri Limoniae, herba plantaginis, semen Juglandis, herba Elephantopi scaberis, Ganoderma and revival grass, extracting with ethanol, percolating, collecting extractive solution or percolate, adding Mel, mixing, and making into oral preparation; or fermenting or soaking the honey, the roxburgh rose, the glabrous greenbrier rhizome, the green lemon, the plantain herb, the walnut, the elephantopus scaber, the lucid ganoderma and the revival grass to prepare an oral preparation.

5. The anti-coronavirus composition of claim 4, wherein the oral formulation is a wine, an oral liquid, or a tincture.

6. A process for the preparation of an anti-coronavirus composition according to any one of claims 1 to 5, said process comprising: pulverizing or slicing Mel, fructus Rosae Normalis, rhizoma Smilacis Glabrae, fructus Citri Limoniae, herba plantaginis, semen Juglandis, herba Elephantopi scaberis, Ganoderma and revival grass, extracting with ethanol, percolating, collecting extractive solution or percolate, adding Mel, mixing, and making into oral preparation; or fermenting or soaking the honey, the roxburgh rose, the glabrous greenbrier rhizome, the green lemon, the plantain herb, the walnut, the elephantopus scaber, the lucid ganoderma and the revival grass to prepare an oral preparation.

7. The method for preparing the composition against neocoronaviruses according to claim 6, wherein the oral preparation is a medicated wine, an oral liquid or a tincture.

8. Use of the anti-coronavirus composition according to any one of claims 1-5 or the anti-coronavirus composition prepared by the method of claim 6 or 7 in the preparation of a medicament for preventing and/or treating diseases caused by coronavirus.

9. Use according to claim 8, wherein the coronavirus comprises a novel coronavirus, severe acute respiratory syndrome, middle east respiratory syndrome or human enterocoronavirus.

10. Use according to claim 9, characterized in that the coronavirus is the novel coronavirus SARS-CoV-2.