CN113181229A - Application of cabbage type rape-isatis tinctoria G monomer addition system in inhibiting novel coronavirus SARS-CoV-2 - Google Patents
Application of cabbage type rape-isatis tinctoria G monomer addition system in inhibiting novel coronavirus SARS-CoV-2 Download PDFInfo
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- CN113181229A CN113181229A CN202110478468.1A CN202110478468A CN113181229A CN 113181229 A CN113181229 A CN 113181229A CN 202110478468 A CN202110478468 A CN 202110478468A CN 113181229 A CN113181229 A CN 113181229A
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Abstract
The invention belongs to the field of biotechnology, and discloses application of a cabbage type rape-isatis tinctoria G monomer addition system in inhibition of novel coronavirus SARS-CoV-2. The invention discovers for the first time that the cabbage type rape-isatis tinctoria G monomer addition line extract has obvious effect of resisting SARS-CoV-2 virus on a cell infection model and has no toxic or side effect on cells. The cabbage type rape-isatis tinctoria additional line has the advantage of large-scale planting, can provide sufficient raw materials for production and extraction of an anti-SARS-CoV-2 preparation, has low cost and high yield, can be used as antiviral medicinal and edible vegetables, feeds and the like for development and utilization, can be used as health-care food for human and animals, and can ensure the health of the animals and the human.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to application of a cabbage type rape-isatis tinctoria G monomer additional system in inhibiting novel coronavirus SARS-CoV-2
Background
The novel coronavirus pneumonia (CoVID-19) is caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). However, there is no known effective new method for treating coronary pneumonia or anti-SARS-CoV-2 virus, and therefore, it is important to research and develop a novel anti-SARS-CoV-2 virus preparation or drug.
The cabbage type rape-isatis tinctoria additional line (isatis tinctoria or oil-coated isatis tinctoria for short) is an additional line formed after the cabbage type rape and isatis tinctoria cells are hybridized and fused. The rape has multiple functions and purposes, the leaves and the flower shoots can be used for vegetables, the seeds can be used for oil extraction, the rapeseed dregs and the green feed can be eaten by animals, and the rape flowers also have ornamental value. The rape is widely planted in the world, the source is wide, and the yield is high. While isatis tinctoria is an important medicinal plant in China, the root of isatis tinctoria is isatis root, the leaf of isatis tinctoria is isatis leaf, and isatis tinctoria has antibacterial, endotoxin-resisting, anticancer, immunoregulation, blood circulation-promoting, blood stasis-removing and antiviral effects, in "diagnosis and treatment scheme for pneumonia infected by novel coronavirus" (trial first to seventh edition), abbreviated as "diagnosis and treatment scheme" and 13 provinces recommend a traditional Chinese medicine treatment scheme group, isatis tinctoria is one of the medicines, but the use frequency of 18 used traditional Chinese medicines is the second to last, and is only 2 times (old and natural, etc., the characteristics and application of immunoregulation antiviral traditional Chinese medicines [ J ]. Chinese herbal medicines, volume 51, 6, 3-month 2020); in related research and news reports, the compound isatis root instead of the isatis root has the effect of resisting the new coronavirus at the in vitro cell level. Isatis tinctoria is also much lower than oilseed rape in terms of source and yield. The cabbage type rape-Isatis tinctoria additional line is obtained by hybridizing somatic cells of cabbage type rape and Isatis tinctoria, backcrossing the formed somatic cell hybrid with cabbage type rape continuously for multiple generations, and adding 7 chromosomes of Isatis tinctoria to rape respectively to obtain 7 additional lines (A, B, C, D, E, F, G). At present, the addition lines are mainly used for rape genetic breeding and analysis of isatis indigotica fort genome structure and medicinal components. Although some related research reports show that some addition systems in the brassica napus-isatis tinctoria addition system have certain antiviral effect on influenza viruses, for example, the 5 monomer addition systems (B monomer, C monomer, D monomer, E monomer and F monomer) and the disomic addition system (D doublet) are extracted by using water as a solvent in the Huang-Luciling laboratory of Chinese academy of sciences, and the D doublet is found to have certain inhibition effect on the H1N1 influenza viruses. However, there has been no report on the effect of different addition lines of Brassica napus-Isatis indigotica on the resistance to SARS-CoV-2 virus. Influenza viruses belong to the orthomyxoviridae family and SARS-CoV-2 viruses belong to the coronaviridae family. According to phylogenetic trees, coronaviruses can be divided into four genera: alpha, beta, gamma and delta, wherein the beta coronavirus can be divided into A, B, C and D four independent subgroups, MERS-CoV which is known to cause serious diseases belongs to the beta coronavirus C subgroup, SARS-CoV belongs to the beta coronavirus B subgroup, and the novel coronavirus and SARS-CoV genome have higher homology and are named as SARS-CoV-2. Lihui et al (2019 novel coronavirus antiviral treatment available drug available period [ J ]. China tuberculosis and journal of respiration, 2020, Vol 43, No 3) report that clinically common antiviral drugs including oseltamivir, peramivir, zanamivir and other drugs having a good effect on influenza viruses are ineffective against SARS-CoV-2. Although many studies on SARS-CoV-2 have been published, the current understanding of this novel coronavirus is only in Bingshan mountain corner, the pathogenesis of SARS-CoV-2 infection and the molecular mechanisms of virus-host interaction remain unclear, and there is no specific drug for treating this virus.
The drug effect of the traditional Chinese medicine is closely related to the content and the composition of active substances, and the leaching and the composition of the active substances of the traditional Chinese medicine are influenced by different extraction methods. In the invention, methanol is used as a solvent, ultrasonic treatment is carried out at 55 ℃, effective components of different addition systems are extracted by a method of rotary evaporation at 50 ℃ on a rotary evaporator, and the extracted product is dried at 50 ℃ and then used for subsequent tests. Research reports exist (Rouyun, etc., the influence of different drying processes on effective components in the isatis root water extract [ J ]. Chinese herbal medicines, No. 42, No. 8 of 2011 in 8), and different drying modes have great influence on the contents of formic acid, salicylic acid, adenosine and polysaccharide in the isatis root water extract. Li shang bin and the like research the influence of 3 extraction methods on the content of flavonoid active ingredients in the tetrastigma hemsleyanum ethanol extract and the proliferation activity of anti-lung cancer cells a549, and as a result, it is found that different extraction processes have certain influence on the leaching of the tetrastigma hemsleyanum flavonoid substances, and further influence the leaching of active substances in the tetrastigma hemsleyanum which exert a main-effect tumor inhibition effect (li shang bin and the like, influence of different extraction methods on the content of the tetrastigma hemsleyanum ethanol extract and the proliferation inhibition of lung cancer cells a549 [ J ]. zhejiang journal of traditional Chinese medicine, vol.54, No. 5, 2019).
In the invention, the extracts of different addition lines of cabbage type rape-isatis tinctoria are screened to resist SARS-CoV-2 virus, and the result shows that the extract of one addition line has obvious inhibition effect on SARS-CoV-2 virus, has obvious effect of resisting new coronavirus and relieving the damage of new coronavirus to mouse lung tissue in a mouse infection model with cell level and new coronavirus lethality, reduces weight loss caused by SARS-CoV-2 infection, and improves the survival rate of mice. With the outbreak of new coronary pneumonia, the scientific attempt of anti-viral infection and treatment to health and medical care is heavy, and the challenge of large-scale production is quickly faced when searching for anti-viral drugs, the cabbage type rape-isatis indigotica fort additional line has the advantage of large-scale planting, sufficient raw materials can be provided for production and extraction of anti-SARS-CoV-2 preparations, and meanwhile, the cabbage type rape-isatis indigotica additional line can also be developed and utilized as medicinal and edible vegetables, feeds and the like, and can be used as health food for human and animals to guarantee the health of the animals and the human.
Disclosure of Invention
The invention aims to make up the defects of the prior art and solve the problem that severe acute respiratory syndrome coronavirus (SARS-CoV-2) is difficult to prevent and treat, and provides an application of a cabbage type rape-isatis tinctoria G monomer additional system in preparing a preparation for treating or preventing novel coronavirus SARS-CoV-2 infection.
In order to achieve the purpose, the invention adopts the following technical measures:
the application of cabbage type rape-isatis tinctoria G monomer additive in preparing medicine for preventing and treating SARS-CoV-2 infection,
the application comprises the following steps:
the application of cabbage type rape-isatis tinctoria G monomer addition system in the preparation of SARS-CoV-2 inhibiting medicine;
the application of cabbage type rape-isatis tinctoria G monomer additional system in preparing medicine for preventing and treating SARS-CoV-2 infection;
the application of cabbage type rape-isatis tinctoria G monomer addition system in the preparation of medicines for relieving the weight reduction after SARS-CoV-2 infection;
the application of cabbage type rape-isatis tinctoria G monomer addition system in preparing medicine for raising the survival rate of SARS-CoV-2 infected body;
the application of cabbage type rape-isatis tinctoria G monomer additive in preparing medicine for lowering virus content in body's lung after SARS-CoV-2 infection.
The application of cabbage type rape-isatis tinctoria G monomer additional system in preparing medicine for preventing and treating mouse SARS-CoV-2 infection;
the application of cabbage type rape-isatis tinctoria G monomer additional system in preparing medicine for raising the survival rate of mouse SARS-CoV-2 infection;
the application of the cabbage type rape-isatis tinctoria G monomer additional system in preparing a medicine for relieving the weight reduction of mice after SARS-CoV-2 infection;
or the cabbage type rape-isatis tinctoria G monomer additional system is applied to the preparation of the medicine for reducing the virus content in the lung of the body after SARS-CoV-2 infection of mice.
In the above applications, preferably, SARS-CoV-2 in mouse SARS-CoV-2 infection is a novel coronavirus SARS-CoV-2/WBP-1, with the collection number being CCTCC NO: v202031.
In the above applications, preferably, the main active ingredient of the drug is an extract of Brassica napus-Isatis indigotica fort monomer addition system, and the extraction method comprises:
mixing cabbage type rape-Isatis tinctoria G monomer addition system powder with methanol, performing ultrasonic treatment at 50-60 deg.C, collecting supernatant, evaporating off methanol to obtain methanol crude extract; dissolving the crude extract with single distilled water, evaporating to remove methanol, extracting with ethyl acetate to obtain lower layer containing effective components, and evaporating water to dry.
The protection scope of the invention also includes: a method for constructing a drug screening model of a mouse infected with a novel coronavirus comprises the following steps: infected with a preservation number of CCTCC NO: v202031 mice, using Brassica napus-Isatis tinctoria G monomer addition line extract as therapeutic drug standard, screening other therapeutic infections with a preservation number of CCTCC NO: v202031.
Compared with the prior art, the invention has the following advantages:
1) the cabbage type rape-isatis tinctoria G monomer addition line extract has obvious SARS-CoV-2 virus resisting effect in cell infection model and no toxic side effect on cell.
2) The cabbage type rape-isatis tinctoria additional line has the advantage of large-scale planting, can provide sufficient raw materials for production and extraction of an anti-SARS-CoV-2 preparation, and has low cost and large yield.
3) The cabbage type rape-isatis tinctoria additional line has the advantages of being natural and healthy, free of toxic and side effects, free of drug resistance and the like.
4) The cabbage type rape-isatis tinctoria additional line can be developed and utilized as antiviral medicinal and edible vegetables, feeds and the like, and can be used as health-care food for human and animals to guarantee the health of the animals and the human.
Drawings
FIG. 1 Effect of different addition lines of Brassica napus-Isatis tinctoria on the proliferation of SARS-CoV-2 virus at the cellular level in vitro.
Wherein A, B, C, D-1, E-1, F-1 and G are rape-isatin monomer addition systems; d-2 and F-2 are binary addition systems.
FIG. 2 is a schematic representation of the cytotoxicity of Brassica napus-Isatis tinctoria G monomer addition line extract on Caco-2 cells.
FIG. 3 the effect of different amounts of Brassica napus-Isatis tinctoria G monomer addition line extract on the proliferation of SARS-CoV-2 virus.
FIG. 4 is a graph showing the effect of Brassica napus-Isatis tinctoria G monomer addition line extract on the body weight of SARS-CoV-2 virus infected mice. FIG. 5 is a schematic representation of the effect of Brassica napus-Isatis tinctoria G monomer addition line extracts on the survival of mice infected with SARS-CoV-2 virus.
FIG. 6 is a schematic diagram showing the effect of Brassica napus-Isatis tinctoria G monomer addition line extract on the lung morphology of SARS-CoV-2 virus infected mice.
FIG. 7 Effect of Brassica napus-Isatis tinctoria G monomer addition line extract on lung histopathological changes of SARS-CoV-2 virus infected mice.
The specific implementation mode is as follows:
for a better understanding of the present disclosure, the following examples are provided to illustrate the present disclosure, but the present disclosure is not limited to the following examples. Unless otherwise specified, the test methods and conditions in the examples of the present invention are conventional methods. The technical scheme of the invention is a conventional scheme in the field if no special description exists; the reagents or materials are commercially available, unless otherwise specified. All tests related to the live SARS-CoV-2 virus were performed in the biosafety third-level laboratory (ABSL 3).
Currently, antiviral drug evaluation models are largely classified into an in vitro model (in vitro model) and an in vivo model (in vivo model).
The in vitro model mainly uses various cell lines to evaluate the medicine, and has the advantages of providing a large number of cells with the same genetic character as research objects, being convenient to operate, eliminating the influence of other external factors, detecting the toxicity, effective concentration and the like of the medicine, and providing more bases for later mechanism research. In vivo models various models of animal infection are generally used, and the overall effect of the drug in living animals is measured by various phenotypic indicators after drug treatment. The method has the advantage of being capable of carrying out real and systematic evaluation on the effect of the candidate drug in the living body. In the invention, the human colon cancer cell line Caco-2 is adopted to measure the in vitro anti-SARS-CoV-2 effect of the cabbage type rape-isatis indigotica fort different addition line extracts; the in vivo anti-SARS-CoV-2 effect of the Brassica napus-Isatis tinctoria addition line extract was evaluated using a mouse adapted strain WBP-1 infection model.
Experimental materials:
(1) cell line, experimental animal and virus required by experiment
Cell line: caco-2 cells were stored in the university of Huazhong agriculture laboratory;
experimental animals: SPF grade 4 to 6 week old Balb/c mice, purchased from the Experimental animals center of the university of Sanxia;
the novel coronavirus SARS-CoV-2/WBP-1 has the preservation number of CCTCC NO: v202031, which has been disclosed in CN111961653A, and in the present invention, the virus is referred to simply as novel coronavirus WBP-1 or mouse adapted strain WBP-1;
(2) cabbage type rape-woad additional line required by experiment and parent thereof
The Brassica napus-Isatis tinctoria addition line and its parent used in the experiment are provided by the team of Li-Byun teachers at plant science and technology institute of Huazhong university
The cabbage type rape-woad addition line is formed by hybridizing somatic cells of cabbage type rape and woad, a formed somatic Cell hybrid and the cabbage type rape are backcrossed continuously for multiple generations, 7 chromosomes of woad are respectively added to the rape, and then 7 monomer addition lines (A, B, C, D, E, F, G) are obtained, wherein a specific preparation process of the cabbage type rape-woad monomer addition line is described in 'Development of a complex of monomeric addition lines between Brassica napus and Isatis indica' (Kang Lei et al,2014, Plant Cell); reference is made to the creation and cytological analysis of the Brassica napus-Isatis indigotica fort binary addition line (Yang Han et al, proceedings of oil crops, 2016, 38(3): 281) 286).
(3) Reagents required for the experiment:
DMEM medium, Fetal Bovine Serum (FBS) were purchased from GIBCO;
cell activity detection kit: TransDetect Cell Counting Kit (CCK) was purchased from Beijing Quanji gold organisms.
Example 1:
preparation of brassica napus-isatis indigotica additional line extract:
harvesting the whole wild cabbage type rape-woad different addition lines planted in the field, naturally drying in the shade or drying at low temperature, crushing, sieving by a 80-mesh sieve, and collecting powder for preparing coarse extraction, wherein the specific extraction steps are as follows:
1) 100g of different attachment system powders and 1000mL of methanol were added to a 2000mL Erlenmeyer flask. Ultrasonic treating at 55 deg.C for 40-60min, filtering, and collecting supernatant and residue respectively;
2) adding 1000mL of methanol into the filter residue in the step 1, continuing ultrasonic treatment, circulating for 5-6 times according to the step 1, and combining all obtained supernatants;
3) evaporating methanol from the supernatant in the step 2 at 50 ℃ in a rotary evaporator;
4) dissolving the crude extract with single distilled water to evaporate methanol, extracting with equal volume of ethyl acetate, collecting the lower layer containing effective components, flowing out from the lower end of separating funnel, collecting the upper layer containing ethyl acetate, recovering the upper layer, and repeatedly extracting the lower layer for 4-5 times until the upper layer becomes clear light color;
5) evaporating water from the extracted lower water-soluble substance at 50 ℃ by using a rotary evaporator, taking down the rotary bottle, placing the rotary bottle in a 50 ℃ oven, drying and weighing;
6) dissolving the dried extract with single distilled water, transferring into a sterilization bottle, and sealing with ethyl acetate solution for later use.
The extraction method of Brassica napus and Isatis tinctoria parents is the same as above.
Example 2:
effect of Brassica napus-Isatis tinctoria different addition line extracts on the proliferation of SARS-CoV-2 Virus at the in vitro cellular level
1. Cell culture
After 2 passages, the frozen and recovered Caco-2 cells are subjected to expanded culture by using a DMEM medium containing 10% fetal calf serum and double antibodies (penicillin 100U/ml and streptomycin 100 ug/ml).
2. Brassica napus-isatis additional line extract for treating cells and infecting with new coronavirus WBP-1
1) Digesting and passaging Caco-2 cells with good growth state, and adjusting cell density to 1 × 10 with cell culture medium5Per ml, 1ml per well was inoculated in 12-well plates.
2) When the cell growth monolayer density reaches 80%, extracts of different addition lines of the cabbage type rape-isatis indigotica fort are added into Caco-2 cells in a 12-well plate, the concentration of the extracts is 200mg/mL, and the working concentration is 400 mu g/mL.
3) Treating different addition line extracts for 24h, infecting with new coronavirus (MOI of 1), incubating at 37 deg.C for 1h, discarding virus incubation solution, adding culture medium containing corresponding addition line extract with the same concentration, and further culturing for 24 h.
4) And collecting virus cell culture solution after 24h, and performing immunoblotting detection.
3. Western blot experiment (Western Blotting)
After Caco-2 cells are infected by the new coronavirus (WBP-1), the influence of the brassica napus-isatis tinctoria additional line extract on the proliferation of the new coronavirus is verified by a Western Blotting experiment, and the specific steps are as follows:
1) and (3) adding 5x protein loading into the cell culture virus solution collected in the step (2), boiling in boiling water for 10min, and centrifuging for later use.
2) SDS-PAGE electrophoresis: preparing 10% polyamide gel as separation gel, performing sample application electrophoresis, keeping constant pressure of concentrated gel at 80V for 30min, and keeping constant pressure of separation gel at 120V for 1-1.5 h.
3) Film transfer: and pre-cooling the membrane transferring liquid in advance during membrane transferring, putting the PAGE gel subjected to electrophoresis into a membrane transferring groove, and transferring the membrane for 1h at constant pressure of 100V.
4) And (3) sealing: and taking out the NC membrane after the membrane is transferred, and sealing the NC membrane for 1h by using sealing liquid.
5) Primary antibody and secondary antibody incubation: the blocked NC membrane was incubated for 2h or overnight at 4 deg.C, and after removal of the primary antibody, washed 6 times with 1 × TBST, 5min each time. The primary antibody was incubated for membrane washing followed by incubation with HRP-labeled secondary antibody for 1h, 6 washes with 1x TBST, 5min each.
6) Color development: color development was performed using Thermo's ECL color development kit.
As shown in FIG. 1, of the 9 addition lines of Brassica napus-Isatis tinctoria, both the F-1 and G-monomer addition lines had some effect against the novel coronavirus, while the other 7 addition lines had no significant difference from the control, i.e., no inhibitory effect on the novel coronavirus WBP-1. The G monomer has obvious effect of resisting new coronavirus, the monomer has stronger inhibition effect on the new coronavirus WBP-1 than isatis tinctoria, the multiplication of the new coronavirus on cells can be obviously inhibited, and after the virus is infected, the virus content in the cell fluid of the G monomer treatment group is obviously lower than that of an untreated infected control group.
Example 3:
inhibition of SARS-CoV-2 virus proliferation by Brassica napus-Isatis tinctoria G monomer addition lines at the in vitro cell level:
according to the results of example 2, the G monomer addition line extract has a significant inhibitory effect on the new coronavirus at the working concentration of 4mg/mL, and example 3 further confirms the anti-new coronavirus effect of the G monomer addition line extract and the inhibitory effect of different using concentrations thereof on the proliferation of the new coronavirus.
1. Toxic effects of monomeric G extract on cells
1) Digesting and passaging Caco-2 cells which grow well, and adjusting the cell density to be 2 multiplied by 10 by using cell growth liquid (DMEM culture medium + 10% fetal calf serum + double antibody)6Inoculating 96-well plates with each well being 100 mul;
2) mu.l of G monomer extract prepared from culture medium (DMEM medium + 10% serum + double antibody) and different concentrations was added to each well and mixed well. Setting 7 concentration gradients, wherein the concentration of the added G monomers is 0 mug/mL respectively; 50 mu g/mL; 100 mu g/mL; 200 mu g/mL; 400 mug/mL; 800 mug/mL; 1600 ug/mL.
3) Setting cell control at the same time, placing at 37 deg.C and 5% CO2Culturing in an incubator.
4) Culturing for 48h, measuring cell activity with TransDetect cell counting box, adding 10 μ l of CCK reagent into 96-well plate cell after culturing, incubating at 37 deg.C in dark for 2h, detecting OD450nm reading with microplate reader, and calculating
Cell survival rate:
cell survival (%). percent vs. drug treated/untreated control 100%
As shown in FIG. 2, the cell viability was measured in response to the toxic effect of the G monomer extract on Caco-2 cells, and it can be seen from FIG. 2 that the cell activity was above 80% at a working concentration of G monomer of 400. mu.g/mL.
2. Inhibitory Effect of monomer extract of G at various concentrations on SARS-CoV-2 Virus proliferation
1) Digesting and passaging Caco-2 cells with good growth state, and adjusting cell density to 1 × 10 with cell culture medium5Per ml, 1ml per well was inoculated in 12-well plates.
2) When the cell growth monolayer density reached 80%, the G monomer extract was added to the 12-well plate at a concentration of 200mg/mL, and the concentrations of the G monomer extract were 0. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, and 400. mu.g/mL, respectively.
3) Treating the G monomer extracts with different concentrations for 24h, infecting new coronavirus (MOI is 1), incubating in an incubator at 37 ℃ for 1h, discarding virus incubation liquid, adding culture medium containing G monomer extract with corresponding concentration, and continuing to culture cells for 24 h.
4) After 24h, the virus cell culture fluid was collected and subjected to immunoblotting (Western Blotting) detection.
As shown in FIG. 3, the WB assay showed that the intensity of the SARS-CoV-2-NP protein decreased with the increase of the concentration of the G monomer, indicating that the extraction of the G monomer had an inhibitory effect on the proliferation of SARS-CoV-2 and was dose-dependent, and that the inhibitory effect on SARS-CoV-2 increased with the increase of the amount of the G monomer extract.
Example 4:
evaluation of antiviral Effect of G monomer extract in mouse lethal infection model
The in vivo effect evaluation of the G monomer against the new coronavirus is carried out in a lethal infection model of the new coronavirus mouse, and the mouse animal test comprises the following specific steps:
1) randomly dividing female BALB/c mice with the age of 6 weeks into 2 major groups, wherein the first major group is a group without virus infection, and is respectively a PBS control group, a Brassica napus (Brassica napus) parent group, an Isatis indigotica (Isatis indigotica) parent group and a G monomer addition line group, and each group comprises 8 mice; the second major group is a group infected with SARS-CoV-2 virus, which is PBS + SARS-CoV-2 control group, Brassica napus (Brassica napus) parent + SARS-CoV-2 group, Isatis indigotica (Isatis indigotica) parent + SARS-CoV-2 group, and G monomer addition line group + SARS-CoV-2, each group contains 10 mice.
2) Gavage was performed on mice with Brassica napus, Isatis tinctoria, and G monomer 3 plant extract and PBS for one week, once a day, and 100. mu.L (200mg/mL) per mouse.
3) One week after the plant extract is infused, new coronavirus WBP-1 challenge infection is carried out, and each mouse is infected with 2 × 10 drops of nose5PFU novel coronavirus mouse adapted strain SARS-CoV-2/WBP-1, infection dose 50. mu.l, mice body weight and survival were observed and recorded daily for 10 consecutive days after virus infection.
4) And drawing a weight change curve and a survival rate curve according to the statistical result.
5) 40 female BALB/c mice of 6 weeks old were randomly divided into 4 groups, which were PBS + SARS-CoV-2 control group, Brassica napus (Brassica napus) parent + SARS-CoV-2 group, Isatis indigotica (Isatis indigotica) parent + SARS-CoV-2 group, and G monomer addition line group + SARS-CoV-2, 10 mice per group, and the administration and infection were performed as above, and the mice were dissected on day 5 after SARS-CoV-2 infection, and 3 mice per group were dissected randomly, and lung tissue samples were taken, and the collected lung tissue samples were fixed with tissue fixative (4% PFA) for histopathological analysis.
The results of anti-SARS-CoV-2 in mice show that the G monomer also has better anti-new coronavirus effect in animals.
The weight change results of the mice are shown in fig. 4 and table 1, the mice which are not infected with the new coronavirus and are drenched with the PBS and the 3 plant extracts have no weight reduction during the monitoring period, and the weight gradually increases along with the increase of the daily age, which indicates that the G monomer and the parent extracts thereof have no influence on the growth and the health of the mice, and the use is healthy and safe; in all 4 groups infected with the new coronavirus, the body weight of mice began to decrease after challenge, the body weight of the PBS group and the isatis indigotica parent group gradually decreased after infection and all died on day 7 after infection, the body weight of the rape group also continuously decreased after infection and all died on day 8 after infection, but the body weight of the G monomer group began to recover on day 9 after decrease.
TABLE 1 Effect of Brassica napus-Isatis tinctoria G monomer addition line extracts on the body weight of mice infected with SARS-CoV-2 Virus
The results of the survival rate of mice are shown in fig. 5 and table 2, and the mice were all alive in 4 groups not infected with the new coronavirus (in fig. 5, the lines of the PBS group, the Brassica napus group, the Radix lsatidis group, and the G monosome group coincide); survival of mice in G monomer treated group was 50% in 4 groups infected with the corresponding new coronavirus; the survival rate of mice in the rape parent group, the isatis indigotica parent group and the PBS group is 0.
TABLE 2 Effect of Brassica napus-Isatis tinctoria G monomer addition line extracts on the survival of mice infected with SARS-CoV-2 Virus
The appearance morphology of lung tissues shows that the G monomer extract can relieve the change of the morphology of lung tissues caused by the infection of new coronavirus (figure 6); the lung histopathological section detection result is shown in fig. 7: severe inflammatory cell infiltration, pulmonary congestion, was observed in the lungs of the control mice, whereas the damage of the lung tissue by the virus in the G monomer addition line group was significantly reduced.
The above results indicate that the G monomer addition line has significant anti-new coronavirus effects both at the cellular level and in mice. In view of the above results, the G monomer can be used for the preparation of a preparation for preventing or treating SARS-CoV-2 infection.
Claims (5)
1. Application of Brassica napus-Isatis tinctoria G monomer addition system in preparation of preparation for treating or preventing novel coronavirus SARS-CoV-2 infection is provided.
2. The application of cabbage type rape-isatis tinctoria G monomer addition system in the preparation of SARS-CoV-2 inhibiting medicine;
the application of cabbage type rape-isatis tinctoria G monomer additional system in preparing medicine for preventing and treating SARS-CoV-2 infection;
the application of cabbage type rape-isatis tinctoria G monomer addition system in the preparation of medicines for relieving the weight reduction after SARS-CoV-2 infection;
the application of cabbage type rape-isatis tinctoria G monomer addition system in preparing medicine for raising the survival rate of SARS-CoV-2 infected body;
the application of cabbage type rape-isatis tinctoria G monomer additive in preparing medicine for lowering virus content in body's lung after SARS-CoV-2 infection.
3. The use according to claim 3 or 4 or 5 or 6, wherein the infected subject is a mouse, and the collection number of the infected SARS-CoV-2 virus is CCTCC NO: v202031.
4. The use according to claim 1 or 2 or 3 or 4 or 5 or 6, wherein the main effective component of the medicament is Brassica napus-Isatis indigotica fort monomer addition line extract, and the extraction method comprises:
mixing cabbage type rape-Isatis tinctoria G monomer addition system powder with methanol, performing ultrasonic treatment at 50-60 deg.C, collecting supernatant, evaporating off methanol to obtain methanol crude extract; dissolving the crude extract with single distilled water, evaporating to remove methanol, extracting with ethyl acetate to obtain lower layer containing effective components, and evaporating water to dry.
5. A method for constructing a drug screening model of a mouse infected with a novel coronavirus comprises the following steps: infected with a preservation number of CCTCC NO: v202031 mice, using Brassica napus-Isatis tinctoria G monomer addition line extract as therapeutic drug standard, screening other therapeutic infections with a preservation number of CCTCC NO: v202031.
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