CN112957388B - Application of brassica napus-isatis tinctoria E monomer addition system in inhibiting influenza virus - Google Patents
Application of brassica napus-isatis tinctoria E monomer addition system in inhibiting influenza virus Download PDFInfo
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- CN112957388B CN112957388B CN202110478900.7A CN202110478900A CN112957388B CN 112957388 B CN112957388 B CN 112957388B CN 202110478900 A CN202110478900 A CN 202110478900A CN 112957388 B CN112957388 B CN 112957388B
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- isatis
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Abstract
The invention belongs to the technical field of biology, and particularly relates to application of a brassica napus-isatis indigotica fort E monomer additional system in inhibiting influenza viruses. The invention provides a method for extracting active ingredients of a cabbage type rape-isatis tinctoria E monomer addition system, and an anti-influenza virus experiment with the extract prepared by the method shows that only the E monomer addition system has obvious anti-influenza virus effect on cell and animal levels in A, D, E, F4 monomer addition systems and A, B, C3 double-body addition systems, and has obvious anti-influenza virus effect on human influenza PR8, swine influenza G4 and avian influenza H5N 6. Can be developed and utilized as antiviral medicinal and edible vegetables, feeds and the like, can be used as health-care food for human and animals, and can ensure the health of the animals and human beings.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a brassica napus-isatis indigotica fort E monomer additional system in inhibiting influenza viruses.
Background
Influenza virus is a major pathogen of humans and animals, belongs to the genus influenza virus of the family orthomyxoviridae, and is a highly contagious disease on a global scale. Influenza viruses are classified into types A, B, C and D, wherein the antigenicity of the influenza A virus is easy to change, and the influenza A virus causes a worldwide pandemic for many times. In the influenza pandemic of 1918-1919, at least 2000-4000 million people die of influenza all over the world; the influenza B virus is also relatively strong in pathogenicity to human, but the influenza B virus is not found to cause a worldwide pandemic; influenza c virus causes only non-obvious or mild upper respiratory infections in humans, rarely causing epidemics; influenza type d virus is currently reported only in pigs, cattle and goats. Influenza a was successfully isolated in 1933, influenza b was obtained in 1940, and influenza c was not successfully isolated until 1949. Influenza a viruses can be further divided into 17H (H1-H17) subtypes and 10N (N1-N10) subtypes, depending on the surface glycoproteins Hemagglutinin (HA) and Neuraminidase (NA) of the virion. Human influenza viruses are mainly H1, H2 and H3 subtypes, while most of the highly pathogenic avian influenza which is seriously harmed at present are H5, H7 and H9 subtypes, since 1997, more and more people are detected to sporadically infect a plurality of subtypes of avian influenza A viruses, and a few animal influenza viruses are adapted to human and can cause human influenza pandemics.
Currently, the prevention of influenza virus infection is mainly vaccination, and the treatment drugs mainly use some neuraminidase inhibitors and ion channel M2 protein inhibitors, however, the effectiveness of the drugs is limited, and drug resistance can be generated, and the wide antiviral drug resistance limits the clinical application of the inhibitors in preventing and treating influenza infection. Seasonal influenza viruses infect 5% -15% of the population each year, causing about 50 million deaths worldwide. Despite decades of monitoring and pharmaceutical and non-pharmaceutical intervention, influenza viruses continue to become prevalent around the world each year. Annual recurrence of influenza is attributed to the continuous evolution of influenza viruses, enabling them to escape previous infection or vaccination-induced immunity. The frequency of the drug-resistant strains of the influenza viruses is faster and faster, the amantadine drug-resistant strains appear in 2003, the oseltamivir drug-resistant strains appear in 2007, and the peramivir drug-resistant strains appear in 2013. How to guarantee human health is a highly concerned problem in all countries of the world, and the development of new effective drugs for inhibiting and preventing influenza virus is imperative. Clinical and experimental evidence also suggests that prevention and treatment of influenza infection requires not only blocking viral transmission, but also reducing the inflammatory response of the viral infection to the host and damage to the lung epithelium, protecting epithelial cells from unwanted cell death. Therefore, in the course of preventing influenza, in addition to the development of drugs such as booster vaccines and inhibitors, other biological agents having a high anti-influenza effect should be actively developed.
The cabbage type rape-Isatis tinctoria additional line (abbreviated as Isatis tinctoria or oil-plated rape) is an additional line formed by crossing and fusing cabbage type rape (Brassica napus) and Isatis indigotica (Isatis indigotica) somatic cells. 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 the isatis tinctoria is an important medicinal plant in China, the root of the isatis tinctoria is isatis root, the leaf of the isatis tinctoria is dyers woad leaf, and the isatis tinctoria has the functions of resisting bacteria, resisting endotoxin, resisting cancer, regulating immunity, promoting blood circulation to remove blood stasis and resisting virus, but the source and the yield of the isatis tinctoria areBut much lower than rape. Cabbage type rape-isatis is produced through the somatic hybridization between cabbage type rape and isatis, the formed somatic cell hybrid is backcrossed with cabbage type rape continuously for several generations to obtain serial cabbage type rape plants with isatis chromosome attached. Through distant hybridization, new types and new properties of plants can be established, the genetic basis of the plants is widened, and the genetic diversity of the plants is enriched, so that the plants serving as new varieties which can meet the requirements of production and life and have application value are obtained. Different genetic varieties have different degrees of differences in functions and properties. Zhaoyanpeng (germplasm identification of anti-tumor Chinese herbal medicine cat ginseng and activity comparison research of anti-tumor Chinese herbal medicine cat ginseng and kindred species [ doctor thesis ]]2006) the in vitro antitumor activity of 9 kinds of kiwi fruits was studied, and IC of Actinidia valvata for human hepatoma cell line SMMC-7221 was found50The activity of the compound reaches 6.53 mu g/ml, the activity of the compound is 8 times of that of the kiwi fruit with big seeds, and the IC of the kiwi fruit with big seeds on human liver cancer cell strains5054.22 mug/ml, 217.85 mug/ml of kiwi fruit, which is a common mixed product of large-seed kiwi fruit, black-pistil kiwi fruit and Chinese kiwi fruit, has a much better inhibiting effect than small-leaf kiwi fruit, and the IC of large-seed kiwi fruit50The inhibition activity to liver cancer cells is lower when the concentration is more than 243 mug/ml; in vitro inhibition tests of human gastric cancer cell strain SGC-7901 and human lung cancer cell strain A549, only the kiwi fruit with big seed and the kiwi fruit with Hubei province in 9 test materials have strong inhibition effect.
At present, the brassica napus-isatis tinctoria additional line is mainly used for rape genetic breeding and analysis of isatis tinctoria genome structure and medicinal components, and no particularly report is provided on antiviral activity research of the additional line. The extraction of 5 monomer addition lines (B monomer, C monomer, D monomer, E monomer and F monomer) and a disomic addition line (D doublet) is carried out in a Huangqi laboratory of Chinese academy of sciences of traditional Chinese medicine by using water as a solvent, and the finding shows that only the D doublet has a certain inhibition effect on H1N1 influenza virus, but the D doublet addition band has male sterile cytoplasm and has antiviral activity only by adding a pair of isatis tinctoria chromosomes, so that the Huangqi laboratory can be preserved and propagated only by using a tissue culture method and cannot be applied on a large scale.
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. 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).
Five times of pandemics are caused in the global scope since the influenza virus is discovered in the beginning of 20 th century, one outbreak is caused in about ten years, huge loss is caused in the global scope, the health and medical care are heavily burdened by the anti-viral infection and treatment, the challenge of large-scale production is quickly faced by scientific attempt for searching the anti-viral drug, the cabbage type rape-isatis seed addition system has the advantage of large-scale planting, sufficient raw materials can be provided for the production and extraction of anti-influenza preparations, and meanwhile, the isatis root rape can also be developed and utilized as medicinal and edible vegetables, feeds and the like to be used as health food for human and animals, so that the health of the animals and the human is ensured, and the influenza virus can be better controlled.
Disclosure of Invention
The invention aims to make up the defects of the prior art and provides a method for extracting the effective components of the cabbage type rape-isatis indigotica E monomer addition system, which is simple.
The invention also aims to provide application of a product prepared by the extraction method of the active ingredients of the brassica napus-isatis indigotica E monomer addition system in preparing a preparation for treating or preventing influenza virus infection, and provides a green, safe and effective medicine source for preventing and controlling influenza virus.
In order to achieve the purpose, the invention adopts the following technical measures:
a method for extracting active ingredients of cabbage type rape-isatis tinctoria E monomer addition system comprises the following steps:
mixing cabbage type rape-isatis tinctoria E monomer addition system powder with methanol, performing ultrasonic treatment at 50-60 ℃, and taking supernatant to evaporate methanol to obtain a 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, evaporating water, and oven drying to obtain Brassica napus-Isatis indigotica fort E monomer addition system extract.
The protection scope of the invention also includes: the extract of rape-isatis indigotica fort E monomer addition system extracted by the method is applied to the preparation of the medicine for treating or preventing influenza virus infection.
The extract of rape-isatis indigotica fort E monomer addition system extracted by the method is applied to the preparation of the medicine for inhibiting the replication of influenza virus.
The extract of rape-isatis indigotica fort E monomer addition line extracted by the method is applied to the preparation of drugs for inhibiting influenza virus replication and cell apoptosis.
The extract of rape-isatis indigotica fort E monomer addition system extracted by the method is applied to the preparation of the medicine for promoting cell growth.
In the above applications, the influenza viruses include, but are not limited to: human influenza virus PR8 (subtype H1N 1), swine influenza virus G4 (subtype H1N 1) and avian influenza virus H5N 6.
Compared with the prior art, the invention has the following advantages:
1. the invention provides a method for extracting an effective component of a cabbage type rape-isatis tinctoria E monomer addition system. According to the invention, effective components of different addition systems are extracted by a method of rotary evaporation at 50 ℃ on a rotary evaporator after methanol is used as a solvent and ultrasonic treatment at 55 ℃, and the extracted product is dried at 50 ℃ and then used for subsequent tests, and the results show that only the E monomer addition system has obvious anti-influenza virus effects at the cell and animal levels in A, D, E, F4 monomer addition systems and A, B, C3 double-body addition systems, has obvious anti-influenza virus effects on human influenza PR8, swine influenza G4 and avian influenza H5N6, and has no toxic effect on cells.
2. The invention discovers for the first time that the effective components of the cabbage type rape-isatis E monomer addition line extracted by the method have no toxicity, can promote cell growth, improve cell activity and can obviously inhibit apoptosis. After the E monomer addition line extract is added into the A549 cells growing into a monolayer for 48 hours, the apoptosis condition is detected by a flow cytometer, and the result shows that the E monomer addition line extract obviously inhibits the apoptosis.
2. The brassica napus-isatis tinctoria E monomer addition line extract can improve weight loss caused by influenza virus infection, obviously inhibit the proliferation of the influenza virus in a mouse body, improve the survival rate of the mouse, improve lung tissue lesion caused by the influenza virus infection, and has an obvious effect of resisting the influenza virus infection.
3. The cabbage type rape-isatis E monomer addition system is natural and healthy, has no toxic or side effect, can be developed and utilized as antiviral medicinal and edible vegetables, feeds and the like, is used as health-care food for people and animals, and guarantees the health of the animals and the human beings.
Description of the drawings:
FIG. 1 Effect of cell growth status of Brassica napus-Isatis indigotica E monomer addition line extracts.
FIG. 2 is a schematic diagram showing cytotoxicity of Brassica napus-Isatis tinctoria E monomer addition line extract on A549 and MDCK cells.
FIG. 3 is a schematic diagram showing the effect of Brassica napus-Isatis tinctoria E monomer addition line extract on A549 cell activity.
FIG. 4 Effect (fluorescence intensity) of Brassica napus-Isatis tinctoria E monomer extract on the proliferation of avian influenza virus H5N6-GFP fluorescent strain.
FIG. 5 shows the inhibitory effect of Brassica napus-Isatis Indigotica E monomer extract on the proliferation of human influenza virus PR8 and swine influenza virus G4 (TCID)50Method to determine viral titer).
FIG. 6 is a graph showing the effect of Brassica napus-Isatis tinctoria E monomer extract on body weight in influenza virus infected mice.
FIG. 7 is a schematic diagram showing the effect of Brassica napus-Isatis tinctoria E monomer extract on serum inflammatory cytokines in mice infected with influenza virus.
FIG. 8 is a schematic diagram showing the effect of Brassica napus-Isatis tinctoria E monomer extract on lung viral load of influenza virus infected mice.
FIG. 9 Effect of Brassica napus-Isatis tinctoria E monomer extract on lung histopathological morphology of influenza 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.
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 in-vitro anti-influenza effect of the extracts of different addition lines of the cabbage type rape-isatis tinctoria is measured by adopting a human alveolar macrophage system A549; the in vivo anti-influenza effect of the brassica napus-isatis indigotica parents and the extracts of different monomer addition lines is evaluated by adopting an H5N6 influenza virus mouse infection model.
Experimental materials:
(1) cell line, experimental animal and virus required by experiment
Cell line: a549 (human alveolar macrophage line) and MDCK (canine kidney epithelial cell line) cells are stored in laboratories of university of agriculture in Huazhong;
experimental animals: SPF grade 4 to 6 week old Balb/c mice, purchased from the Experimental animals center of the university of Sanxia;
experimental strains: human influenza A virus PR8 (subtype H1N 1), swine influenza virus G4 (subtype H1N 1) and avian influenza virus H5N 6.
(2) Cabbage type rape-woad additional line required by experiment and parent thereof
The brassica napus-isatis indica addition line and its parents used in the experiments were provided by the team of the plum recons teachers at the institute of plant science and technology at the university of Huazhong agriculture.
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 obtained 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 obtained in step 2) in a rotary evaporator at 50 ℃.
4) Dissolving with single distilled water to evaporate crude extract of 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 to obtain clear light color for 4-5 times.
5) And (3) evaporating the water of the extracted lower-layer 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 influenza Virus proliferation at in vitro cellular level
1. Cell culture
After frozen and recovered A549 cells are subjected to 2 passages, DMEM medium containing 10% fetal calf serum and double antibody (penicillin 100U/ml and streptomycin 100ug/ml) is used for expanding culture.
2. Brassica napus-isatis additional line extract for treating cells and infecting influenza virus
1) Digesting and passaging A549 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 Brassica napus-isatis tinctoria are added into A549 cells in a 12-well plate, and the final acting concentrations of the extracts are all 200 mug/mL.
3) Treating different addition line extracts for 24H, then infecting an influenza virus H5N6-GFP green fluorescent strain (MOI of 0.01), incubating in an incubator at 37 ℃ for 1H, discarding virus incubation liquid, adding a culture medium containing the corresponding addition line extract with the same concentration, and continuing to culture the cells for 24H.
4) And after 24H, observing the fluorescence condition under an inverted microscope, measuring the fluorescence intensity of different samples by using a multifunctional microplate reader, and adding green fluorescent protein at the end of an influenza virus NS1 gene to construct an H5N6-GFP green fluorescent strain, wherein the green fluorescent protein is expressed along with the replication of H5N6, and further the replication level of the H5N6 influenza virus can be represented by the expression intensity of the green fluorescence, and the fluorescence is stronger when the virus is more proliferated.
The results are shown in table 1, in the brassica napus-isatis indigotica parent and 7 different addition lines, the E monomer and the isatis indigotica have obvious anti-influenza virus effect, but the E monomer has more obvious effect and lowest fluorescence intensity, and the result shows that the E monomer addition line can obviously inhibit the proliferation of influenza virus H5N 6.
TABLE 1 Effect of Brassica napus-Isatis indigotica parent and different episomal extracts on influenza Virus proliferation
| Sample numbering | OD485/530 (fluorescence intensity) |
| Monomer A | 3396 |
| D monomer | 2880 |
| E monomer | 1316 |
| F monomer | 2140 |
| A double body | 3479 |
| B double body | 3402 |
| C double body | 2559 |
| Brassica napus | 3320 |
| Isatis tinctoria | 1871 |
| Positive control (infection) | 4105 |
| Negative control (no infection) | 0 |
Example 3:
inhibition of influenza virus proliferation at the cellular level in vitro by E monomer addition line extracts
From the results of example 2, it is known that the brassica napus-isatis indigotica E monomer extract has a significant inhibitory effect on avian influenza virus H5N6, and example 3 further verifies the anti-influenza virus effect and the cytotoxic effect on cells of the brassica napus-isatis indigotica E monomer extract.
1. Toxic effects of E monomer extract on cells
1) Taking well-grown A549 and MDCK cells for digestion and passage, and using cell growth liquid (DMEM medium + 10% fetal bovine serum + double cells)Anti) to adjust the cell density to 2X 106Inoculating 96-well plates with each well being 100 mul;
2) mu.l of E monomer extract prepared from culture medium (DMEM medium + 10% serum + double antibody) and different concentrations was added to each well and mixed well. 6 concentration gradients were set, each gradient having 3 replicate wells with final concentrations of 0. mu.g/ml, 12.5. mu.g/ml, 25. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml, respectively.
3) Simultaneously setting cell blank control, placing at 37 deg.C and 5% CO2Culturing in an incubator.
4) After 24 and 48 hours of culture, measuring the cell activity by using a TransDetect cell counting box, adding 10 mu l of CCK reagent into the cells of a 96-well plate after the culture is processed, incubating the cells for 2 hours in a dark place at 37 ℃, detecting OD450nm reading by using an enzyme-linked immunosorbent assay, and calculating the cell survival rate:
cell survival (%). percent vs. drug treated/untreated control 100%
The cell survival rate is measured to reflect the toxic effect of the E monomer extract on cells, the test results are shown in figure 1 and figure 2, the Brassica napus-isatis indigotica E monomer extract has no toxicity on the cells, the activities of A549 cells and MDCK cells are both kept above 90%, and in addition, in the A549 cells, the E monomer extract has no toxicity, can promote the cell growth and improve the cell activity.
Further experiments show that after the E monomer addition line extract is added into the A549 cells growing into a monolayer for 48 hours, the apoptosis condition is detected by a flow cytometer, and the result is shown in figure 3, wherein the E monomer addition line extract obviously inhibits the apoptosis.
2. Immunofluorescence method for detecting inhibition effect of E monomer extract on influenza virus proliferation at cell level
1) Digesting and passaging A549 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%, adding a DMEM medium containing the E monomer extract with the final concentration of 200 mu g/ml into a 12-well plate; meanwhile, an anti-influenza medicament oseltamivir positive control group is arranged, and the final concentration of oseltamivir is 200 mug/ml; the negative control group was not treated.
3) Treating the E monomer extract and the medicine for 24H respectively, infecting H5N6-GFP influenza virus fluorescent virus (MOI of 0.01), incubating in an incubator at 37 deg.C for 1H, discarding virus incubation solution, adding culture medium containing plant extract and medicine with the same concentration, and continuously culturing cells
4) After the virus infection 24, the virus growth was judged by microscopic observation based on the fluorescence intensity, and the fluorescence increased as the virus growth increased.
The results are shown in fig. 4, compared with the untreated negative control group, the E monomer group can significantly reduce the proliferation of influenza virus, and the effect can be comparable to that of oseltamivir, which is an anti-influenza drug clinically used at present.
3. E monomer extract has effect of inhibiting proliferation of human influenza virus PR8 and swine influenza virus G4
1) Digesting and passaging A549 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%, adding DMEM medium containing E monomer extract (with a final concentration of 200 mug/ml) into a 12-well plate; a blank control group was also set.
3) Treating the E monomer extract for 24h, respectively infecting human influenza virus PR8 and swine influenza virus G4(MOI of 0.01), incubating in an incubator at 37 ℃ for 1h, discarding virus incubation liquid, adding culture medium containing plant extract with the same concentration, and continuing to culture cells.
4) Collecting virus cell culture solution after 24h, and adopting TCID50The method determines the virus titer.
As a result, as shown in fig. 5, the E monomer extract can significantly inhibit the proliferation of human influenza virus PR8 and swine influenza virus G4.
In addition, in the research of anti-influenza viruses of different addition lines of Brassica napus-Isatis tinctoria, the C binary addition line has a certain inhibiting effect on avian influenza virus H5N6, but has no anti-virus activity on H1N1 influenza virus.
Example 4:
evaluation and screening of antiviral Effect of Brassica napus-Isatis tinctoria parent and various addition line extracts in mouse influenza Virus infection model
The evaluation of the in vivo effect of the isatis root parent and 7 different addition lines on anti-influenza virus is carried out in an influenza virus mouse infection model, and the concrete steps of the mouse animal test are as follows
1) Female BALB/C mice of 8 weeks old were randomly divided into 10 groups, which were PBS control group, Brassica napus parent group, Isatis tinctoria parent group, A monomer addition line group, D monomer addition line group, E monomer addition line group, F monomer addition line group, A doublet addition line group, B doublet addition line group, C doublet addition line group, and 5 mice per group.
2) The cabbage type rape, the isatis tinctoria and the additive lines are subjected to intragastric administration on mice by 9 plant extracts and PBS, the intragastric administration is carried out once a day, the intragastric administration is carried out 9 times, and 20mg of the extract is administered to each mouse every time.
3) After the plant extract is perfused into stomach, 0.5LD is adopted50Influenza virus H5N6 was subjected to a nasal drop challenge infection, and the body weight and survival of mice were observed and recorded daily for 10 consecutive days after influenza infection.
4) Blood collection and dissection of mice and lung tissue sample collection, blood sample treatment: standing the collected non-anticoagulant sample at 4 ℃ overnight, after separating out serum, centrifuging at 4 ℃ and 1000rpm for 5min, taking supernatant, and freezing at-80 ℃ for detecting the content of the cell factor. And (3) lung tissue treatment: adding 1ml sterile PBS (1ml PBS/lung) into the collected lung tissue sample, homogenizing and breaking, centrifuging to obtain supernatant, freezing at-80 deg.C, and adopting EID50The method can be used for detecting the content of influenza virus.
The anti-influenza result in mice shows that the E monomer addition system also has a better anti-influenza virus effect in animals, and the anti-influenza virus effect is consistent with the cell level anti-virus effect.
The results of the weight change of the mice show that the mice are healthy after being challenged by the group of cabbage type rape-isatis indigotica fort parent isatis and the E monomer, and the weight reduction of the mice caused by the infection of influenza virus can be obviously inhibited (figure 6). The brassica napus-isatis indigotica parent brassica napus and the rest of the episomes do not show anti-influenza virus effect, and have no obvious difference with a PBS control group.
The detection of serum inflammatory cytokines shows that the E monomer and the IL-1 beta and TNF-alpha of the indigenous isatis indigenous group are obviously lower than those of a PBS control group and other plant extracts (figure 7), which indicates that the cabbage type rape-isatis indigenous monomer additional line extract has an anti-inflammatory effect in the infection of influenza viruses and reduces the inflammation level of organisms.
The lung viral load assay results of mice infected with influenza virus showed that the viral load of influenza virus of the E monomer addition line group in the lung was significantly lower than that of the other addition line groups (fig. 8).
Example 5:
evaluation of antiviral Effect of E monomer addition line extract in mouse influenza Virus infection model
Based on the animal experiment results in example 4, the E monomer was further subjected to animal experiments, and the specific steps of the mouse animal experiments were as follows
1) Female BALB/c mice, 8 weeks old, were randomly divided into 2 groups of 8 mice each, PBS control group and E monomer addition line group.
2) And (3) performing gavage on the E monomer addition system extract and PBS on the mice, wherein the gavage is performed once every other day for 5 times, and 20mg of the extract is administered to each mouse.
3) After gavage, use 10LD50Influenza virus H5N6 was used for the treatment of the mice by nasal administration, the survival of the mice was observed and recorded daily after infection with influenza virus, the monitoring and recording were continued for 14 days, and the lung tissue was subjected to histopathological section observation.
The results show that in 10 LDs50All PBS mice died under challenge dose infection, while E monomer group mice still had a 12.5% survival rate. Pathological sections of the mouse lung show that after the mouse is infected with influenza virus, a large amount of inflammatory cells seep out from the mouse lung of the PBS group, and the mouse has no complete alveolar morphology; the E monomer extract was able to alleviate lung damage from influenza infection (figure 9).
The above results indicate that the E monomer has significant anti-influenza virus effects both at the cellular level and in mice. In view of the above results, the E monomer can be used for the preparation of an agent for preventing or treating influenza virus infection.
Claims (4)
1. The application of rape-isatis tinctoria E monomer addition line extract in preparing a medicament for treating or preventing influenza virus infection is as follows: human influenza virus PR8, swine influenza virus G4, or avian influenza virus H5N 6;
the extraction method of the rape-isatis tinctoria E monomer addition line extract comprises the following steps:
mixing cabbage type rape-isatis tinctoria E monomer addition system powder with methanol, performing ultrasonic treatment at 50-60 ℃, and taking supernatant to evaporate methanol to obtain a 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, evaporating water, and oven drying to obtain Brassica napus-Isatis indigotica fort E monomer addition system extract.
2. The application of rape-isatis tinctoria E monomer addition system extract in preparing a medicament for inhibiting the replication of influenza viruses, wherein the influenza viruses are as follows: human influenza virus PR8, swine influenza virus G4, or avian influenza virus H5N 6;
the extraction method of the rape-isatis tinctoria E monomer addition line extract comprises the following steps:
mixing cabbage type rape-isatis tinctoria E monomer addition system powder with methanol, performing ultrasonic treatment at 50-60 ℃, and taking supernatant to evaporate methanol to obtain a 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, evaporating water, and oven drying to obtain Brassica napus-Isatis indigotica fort E monomer addition system extract.
3. The application of rape-isatis tinctoria E monomer addition line extract in preparing a medicament for inhibiting the replication of influenza viruses and inhibiting apoptosis simultaneously, wherein the influenza viruses are as follows: human influenza virus PR8, swine influenza virus G4, avian influenza virus H5N 6;
the extraction method of the rape-isatis tinctoria E monomer addition line extract comprises the following steps:
mixing cabbage type rape-isatis tinctoria E monomer addition system powder with methanol, performing ultrasonic treatment at 50-60 ℃, and taking supernatant to evaporate methanol to obtain a 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, evaporating water, and oven drying to obtain Brassica napus-Isatis indigotica fort E monomer addition system extract.
4. The application of rape-isatis indigotica fort E monomer addition line extract in preparing a medicament for inhibiting the replication of influenza virus, inhibiting apoptosis and promoting cell growth, wherein the influenza virus comprises: human influenza virus PR8, swine influenza virus G4, avian influenza virus H5N 6;
the extraction method of the rape-isatis tinctoria E monomer addition line extract comprises the following steps:
mixing cabbage type rape-isatis tinctoria E monomer addition system powder with methanol, performing ultrasonic treatment at 50-60 ℃, and taking supernatant to evaporate methanol to obtain a 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, evaporating water, and oven drying to obtain Brassica napus-Isatis indigotica fort E monomer addition system extract.
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