CN114452310A - Plant extract for preventing novel coronavirus infection and preparation method and application thereof - Google Patents
Plant extract for preventing novel coronavirus infection and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a plant extract for preventing novel coronavirus infection and a preparation method and application thereof, belonging to the technical field of medicines. The study finds that the celosia seeds extract adopted by the invention can obviously reduce the expression level of ACE2 protein and block the combination of S protein on the surface of coronavirus and ACE2, and shows that the celosia seeds extract prepared by the invention has the effect of preventing and/or treating coronavirus infection diseases.
Description
Technical Field
The invention relates to a plant extract for preventing novel coronavirus infection and a preparation method and application thereof, belonging to the technical field of medicines.
Background
Coronaviruses are RNA viruses, a large group of viruses that are widely found in nature. 2019-nCoV is the 7 th coronavirus which is known to infect human, the remaining 6 are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, respectively. Among them, 2019-nCoV infection can cause novel coronavirus pneumonia COVID-19, SARS-CoV infection can cause severe acute respiratory syndrome, and MERS-CoV infection can cause middle east respiratory syndrome. It is seen that coronavirus infection poses a great threat to human health.
Angiotensin converting enzyme 2 (ACE 2) is widely present in human organs such as heart, lung and kidney, and coronavirus is replicated by binding its surface S protein to specific ACE2 on infected cell membrane and entering into cell via endocytosis of cell under the mediation of ACE 2. Also, ACE2 in lung tissue is not only an invasive receptor for coronaviruses, but it may also be involved in the development of lung injury. Therefore, the blocking agent of the binding of the S protein on the surface of the coronavirus and the ACE2 can effectively inhibit virus infection.
Currently, the results of research on drugs that block binding of the S protein on the surface of coronaviruses to ACE2 are not ideal. For example, drugs that simply block viral entry, such as P4, P5, etc., may have an effect on the physiology (blood pressure regulation) of the ACE2 receptor itself; while inhibiting classical renin-angiotensin (RAS) can reduce inflammatory response, adverse reactions such as hyperkalemia, hypotension, cough and the like caused by the RAS restrict the use of the drugs.
Clinically, the coronavirus infection is also treated by using Chinese herbal medicines, and the advantages of treating the coronavirus infection by using the Chinese herbal medicines are represented by early intervention, blocking the disease course, relieving symptoms, shortening the treatment time, relieving complications, avoiding toxic and side effects caused by large doses of hormones and the like. During SARS, after healthy volunteers continuously take the mulberry-chrysanthemum-jade-screen powder prepared by the Chinese medicine research institute of hong Kong university, the large green leaves and the baical skullcap root, almost no flu-like symptoms exist, and the Chinese herbal medicine preparation is prompted to have good prevention effect on virus infection; the COVID-19 epidemic prevention and control proves the unique functions of the traditional Chinese medicine in the aspects of preventing new crowns, controlling the progress of diseases and the like again. The medicine which can block the combination of the S protein on the surface of the coronavirus and the ACE2 and further prevent the coronavirus infection is also allowed to be obtained by being developed from the traditional Chinese medicine.
The semen Celosiae (Celosiae seed) is dried mature seed of semen Celosiae (Ce losia argentea L.) of Celosia of Amaranthaceae (Amaranthaceae), and is a pharmacopoeia collection variety. The semen Celosiae has effects of reducing blood sugar, resisting oxidation, protecting liver, treating cataract, resisting tumor, and mitosis. The feather cockscomb is widely distributed and has low price.
At present, no report exists about the application of the feather cockscomb seed extract in the preparation of medicines for preventing and/or treating coronavirus infection diseases.
Disclosure of Invention
Therefore, the invention aims to provide a new application of the feather cockscomb seed extract, namely an application of the feather cockscomb seed extract in preparing medicines for preventing and/or treating coronavirus infection diseases.
In order to solve the problems, the invention provides the application of the plant extract in preparing the medicine, wherein the plant extract is the feather cockscomb seed extract; the medicament has at least one of the following uses:
(a) preventing and/or treating coronavirus infection diseases; and/or the presence of a gas in the atmosphere,
(b) inhibiting angiotensin converting enzyme 2 or reducing angiotensin converting enzyme 2 expression level.
In one embodiment of the present invention, the method for preparing the feather cockscomb seed extract comprises the steps of extracting feather cockscomb seeds as a raw material with an organic solvent and/or water as an extraction reagent; the extraction solvent is 30-95 vt% alcohol water solution.
In one embodiment of the invention, the organic solvent is 60 to 95 vt% alcohol aqueous solution.
In one embodiment of the present invention, the method for preparing the feather cockscomb seed extract comprises the following steps: the feather cockscomb seed extract is prepared by crushing feather cockscomb seeds to obtain powder, adding 10-15 times of alcohol-water solution with volume concentration of 60-90% by weight into the powder, performing reflux extraction at 55-70 ℃ for 1-3 times for 1-3 hours each time, combining extracting solutions, concentrating to obtain feather cockscomb seed extract, and drying to obtain the feather cockscomb seed extract.
In an embodiment of the invention, the preparation method of the feather cockscomb seed extract further comprises the steps of adding water to dissolve the feather cockscomb seed extract, centrifuging, taking supernatant, and then subjecting the supernatant to macroporous resin adsorption and/or polyamide chromatography column and/or extraction;
the macroporous resin adsorption step and the polyamide chromatographic column step both comprise the steps of taking supernate, washing impurities by adopting 0-20 vt% of alcohol-water solution, then analyzing by adopting 70-95 vt% of alcohol-water solution, and collecting an analysis solution;
the extraction step comprises the steps of extracting supernate by using petroleum ether, ethyl acetate and n-butanol as extracting agents or extracting by using petroleum ether and ethyl acetate as extracting agents or extracting by using petroleum ether and n-butanol as extracting agents, and collecting an ethyl acetate extraction organic phase or an n-butanol extraction organic phase.
In an embodiment of the present invention, the preparation method of the feather cockscomb seed extract further comprises the steps of taking the supernatant, performing the extraction step, and performing the macroporous resin adsorption step to obtain the feather cockscomb seed extract; or taking the supernatant, carrying out the polyamide chromatographic column step, and then carrying out the extraction step to obtain the feather cockscomb seed extract.
In one embodiment of the invention, in the macroporous resin adsorption step and/or the polyamide chromatographic column step, the elution flow rate is controlled to be 2-4 BV/h, and the elution volume of each elution reagent is 2-4 BV;
in the extraction step, the amount of petroleum ether, ethyl acetate or n-butanol is 2-3 times of the weight of the supernatant, and each extracting agent is used for 2-3 times of extraction.
In one embodiment of the present invention, the macroporous resin column used in the macroporous resin adsorption step is selected from a D101 macroporous resin column or an AB-8 macroporous resin column.
In one embodiment of the present invention, the alcohol aqueous solution is a mixed solution of ethanol and water or a mixed solution of methanol and water.
In one embodiment of the invention, the coronavirus comprises an alphacoronavirus and/or a betacoronavirus.
In one embodiment of the invention, the coronavirus comprises HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and/or 2019-nCoV.
In one embodiment of the invention, the coronavirus infection diseases include respiratory, digestive and nervous system diseases.
In one embodiment of the invention, the coronavirus infectious disease comprises a cold, fever, frontal sinusitis, otitis media, pharyngitis, chronic bronchitis, pneumonia, pleural effusion, various respiratory syndromes, acute gastroenteritis, cardiopulmonary disease, immune hypofunction, repeated infection, lung injury and/or organ failure.
In one embodiment of the invention, the angiotensin converting enzyme 2 is pulmonary angiotensin converting enzyme 2.
In one embodiment of the present invention, the angiotensin converting enzyme 2 is angiotensin converting enzyme 2 of alveolar epithelial cells and pulmonary bronchi.
In one embodiment of the invention, the medicine is the feather cockscomb seed extract, and conventional auxiliary materials are added according to a conventional process to prepare clinically acceptable gels, creams, tablets, capsules, powders, mixtures, pills, granules, solutions, syrups, decocted ointments, suppositories, aerosols, emplastrums, ointments, injections, sprays, liniments, tinctures, wet dressings, pastes, lotions or sustained and controlled release preparations.
The invention also provides a preparation method of the plant extract, which comprises the step of extracting the feather cockscomb seeds serving as a raw material by using an organic solvent and/or water as an extraction reagent; the extraction solvent is 30-95 vt% alcohol water solution.
In one embodiment of the invention, the organic solvent is 60 to 95 vt% alcohol aqueous solution.
In one embodiment of the present invention, the preparation method of the plant extract comprises the steps of: the feather cockscomb seed extract is prepared by crushing feather cockscomb seeds to obtain powder, adding 10-15 times of alcohol-water solution with volume concentration of 60-90% by weight into the powder, performing reflux extraction at 55-70 ℃ for 1-3 times for 1-3 hours each time, combining extracting solutions, concentrating to obtain feather cockscomb seed extract, and drying to obtain the feather cockscomb seed extract.
In an embodiment of the invention, the preparation method of the plant extract further comprises the steps of adding water to dissolve the feather cockscomb seed extract, centrifuging, taking supernatant, and then subjecting the supernatant to macroporous resin adsorption and/or polyamide chromatography column and/or extraction;
the macroporous resin adsorption step and the polyamide chromatographic column step both comprise the steps of taking supernate, washing impurities by adopting 0-20 vt% of alcohol-water solution, then analyzing by adopting 70-95 vt% of alcohol-water solution, and collecting an analysis solution;
the extraction step comprises the steps of extracting supernate by using petroleum ether, ethyl acetate and n-butanol as extracting agents or extracting by using petroleum ether and ethyl acetate as extracting agents or extracting by using petroleum ether and n-butanol as extracting agents, and collecting an ethyl acetate extraction organic phase or an n-butanol extraction organic phase.
In an embodiment of the present invention, the preparation method of the plant extract further comprises taking the supernatant, performing the extraction step, and performing the macroporous resin adsorption step to obtain the feather cockscomb seed extract; or taking the supernatant, carrying out the polyamide chromatographic column step, and then carrying out the extraction step to obtain the feather cockscomb seed extract.
In one embodiment of the invention, in the macroporous resin adsorption step and/or the polyamide chromatographic column step, the elution flow rate is controlled to be 2-4 BV/h, and the elution volume of each elution reagent is 2-4 BV;
in the extraction step, the amount of petroleum ether, ethyl acetate or n-butanol is 2-3 times of the weight of the supernatant, and each extracting agent is used for 2-3 times of extraction.
In one embodiment of the present invention, the macroporous resin column used in the macroporous resin adsorption step is selected from a D101 macroporous resin column or an AB-8 macroporous resin column.
In one embodiment of the present invention, the alcohol aqueous solution is a mixed solution of ethanol and water or a mixed solution of methanol and water.
The invention also provides the plant extract prepared by the preparation method of the plant extract.
The invention also provides a medicament for preventing and/or treating coronavirus infection, which contains the plant extract.
In one embodiment of the invention, the coronavirus comprises an alphacoronavirus and/or a betacoronavirus.
In one embodiment of the invention, the coronavirus comprises HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and/or 2019-nCoV.
In one embodiment of the invention, the coronavirus infectious diseases include respiratory, digestive and nervous system diseases.
In one embodiment of the invention, the coronavirus infectious disease comprises a cold, fever, frontal sinusitis, otitis media, pharyngitis, chronic bronchitis, pneumonia, pleural effusion, various respiratory syndromes, acute gastroenteritis, cardiopulmonary disease, immune hypofunction, repeated infection, lung injury and/or organ failure.
In one embodiment of the invention, the medicament is the plant extract, and is prepared into clinically acceptable gels, creams, tablets, capsules, powders, mixtures, pills, granules, solutions, syrups, decocted ointments, suppositories, aerosols, emplastrums, ointments, injections, sprays, liniments, tinctures, wet dressings, pastes, lotions or sustained and controlled release preparations by adding conventional auxiliary materials according to a conventional process.
The invention also provides a medicine for inhibiting angiotensin converting enzyme 2, which contains the plant extract.
In one embodiment of the invention, the angiotensin converting enzyme 2 is pulmonary angiotensin converting enzyme 2.
In one embodiment of the present invention, the angiotensin converting enzyme 2 is angiotensin converting enzyme 2 of alveolar epithelial cells and pulmonary bronchi.
The invention also provides a medicine for reducing the expression level of angiotensin converting enzyme 2, and the medicine contains the plant extract.
In one embodiment of the invention, the angiotensin converting enzyme 2 is pulmonary angiotensin converting enzyme 2.
In one embodiment of the present invention, the angiotensin converting enzyme 2 is angiotensin converting enzyme 2 of alveolar epithelial cells and pulmonary bronchi.
Has the advantages that:
1. the study finds that the celosia seeds extract adopted by the invention can obviously reduce the expression level of ACE2 protein and block the combination of S protein on the surface of coronavirus and ACE2, and shows that the celosia seeds extract prepared by the invention has the effect of preventing and/or treating coronavirus infection diseases.
2. The application of the feather cockscomb seed extract in preparing the medicine for preventing and/or treating coronavirus infection provided by the invention is that the ACE2 protein expression can be obviously reduced by using the feather cockscomb seed extract obtained by extracting with 30-95 vt% alcohol-water solution as an extraction solvent. Dissolving the feather cockscomb seed extract in water, centrifuging, taking supernatant, and then carrying out macroporous resin adsorption and/or polyamide chromatographic column and/or extraction on the supernatant; the step of jointly controlling the macroporous resin adsorption and the step of polyamide chromatographic column both comprise the steps of taking supernate, washing impurities by adopting 0-20 vt% of alcohol-water solution, then analyzing by adopting 70-95 vt% of alcohol-water solution, and collecting an analysis solution; effective components can be effectively enriched by macroporous resin adsorption or polyamide chromatographic column, and the obtained feather cockscomb seed extract has the capability of better reducing the expression of ACE2 protein; in the same way, the extraction step comprises the steps of extracting the supernate by using petroleum ether, ethyl acetate and n-butyl alcohol as an extracting agent or extracting by using petroleum ether and ethyl acetate as an extracting agent or extracting by using petroleum ether and n-butyl alcohol as an extracting agent, collecting an ethyl acetate extraction organic phase or an n-butyl alcohol extraction organic phase, enriching effective components in the ethyl acetate extraction organic phase or the n-butyl alcohol extraction organic phase, and obtaining the celosia seeds extract with the capability of better reducing the expression of the ACE2 protein.
3. The application of the feather cockscomb seed extract in preparing the medicine for preventing and/or treating coronavirus infection provided by the invention is that the feather cockscomb seed extract is obtained by extracting supernatant liquid and then carrying out macroporous resin adsorption; or taking the supernatant, carrying out polyamide chromatography column, and extracting to obtain the feather cockscomb seed extract; the obtained feather cockscomb seed extract has the capability of better reducing the expression of ACE2 protein.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the expression level of ACE2 protein in mouse lung tissue of test groups 1-6;
FIG. 2 shows the expression level of ACE2 protein in mouse lung tissue of test groups 7-9;
FIG. 3 is a Western blot electrophoresis result of ACE2 protein and beta-actin protein in mouse lung tissues of test drug groups 1-6;
FIG. 4 shows Western blot electrophoresis results of ACE2 protein and beta-actin protein in mouse lung tissues of test groups 7-9.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The feather cockscomb seed medicinal material related in the following examples is purchased from the Bozhou medicinal material market in Anhui; SPF-grade male ICR mice (3-5 weeks old, 11-13 g in weight) referred to in the examples below were purchased from Tokyo Wintolite.
Example 1: preparation method of semen Celosiae extract
The method comprises the following specific steps:
pulverizing semen Celosiae to obtain semen Celosiae powder; adding 10 times of 60% ethanol water solution into semen Celosiae powder, heating and reflux-extracting at 55 deg.C for 3 times, 1 for 2 hr, and 2 and 3 for 1 hr respectively, and mixing extractive solutions; concentrating the extractive solution at 50 deg.C under reduced pressure until no alcohol smell exists to obtain semen Celosiae extract; and concentrating the feather cockscomb seed extract at 50 ℃ under reduced pressure, and drying to obtain the feather cockscomb seed extract 1A.
Example 2: preparation method of semen Celosiae extract
The method comprises the following specific steps:
pulverizing semen Celosiae to obtain semen Celosiae powder; adding 10 times of 60% ethanol water solution into semen Celosiae powder, heating and reflux-extracting at 55 deg.C for 3 times, 1 for 2 hr, and 2 and 3 for 1 hr respectively, and mixing extractive solutions; concentrating the extractive solution under reduced pressure until no alcohol smell exists to obtain semen Celosiae extract; adding pure water into the feather cockscomb seed extract, and adding 1L of pure water into the feather cockscomb seed extract extracted according to 1kg of raw material (feather cockscomb seed powder) for suspension to obtain suspension; centrifuging the suspension at 3500rpm for 10min, and collecting supernatant; purifying the supernatant by D101 macroporous resin column chromatography (diameter of macroporous resin column is 8cm, height of macroporous resin column is 70cm, column volume is 3.5L), sequentially eluting with pure water, 20% (v/v) and 95% (v/v) ethanol-water gradient (flow rate of gradient elution is 3BV/h, each gradient has 3 column volumes), respectively collecting eluates, concentrating under reduced pressure at 50 deg.C, and drying to powder to obtain semen Celosiae extract 1B, semen Celosiae extract 1C, and semen Celosiae extract 1D.
Example 3: preparation method of semen Celosiae extract
The method comprises the following specific steps:
pulverizing semen Celosiae to obtain semen Celosiae powder; adding 15 times of ethanol water solution with volume concentration of 90% into semen Celosiae powder, and heating and reflux-extracting at 65 deg.C for 3 hr to obtain extractive solution; concentrating the extractive solution at 50 deg.C under reduced pressure until no alcohol smell exists to obtain semen Celosiae extract; and (3) continuing to perform decompression concentration on the feather cockscomb seed extract at 50 ℃, and drying to obtain the feather cockscomb seed extract 2A.
Example 4: preparation method of semen Celosiae extract
The method comprises the following specific steps:
pulverizing semen Celosiae to obtain semen Celosiae powder; adding 15 times of ethanol water solution with volume concentration of 90% into semen Celosiae powder, and heating and reflux-extracting at 65 deg.C for 3 hr to obtain extractive solution; concentrating the extractive solution at 50 deg.C under reduced pressure until no alcohol smell exists to obtain semen Celosiae extract; adding pure water into the feather cockscomb seed extract, and adding 1L of pure water into the feather cockscomb seed extract obtained by extracting 1kg of raw material (feather cockscomb seed powder) for suspension to obtain suspension; centrifuging the suspension at 3500rpm for 10min, and collecting supernatant; sequentially extracting the supernatant with 2 times of petroleum ether, ethyl acetate and n-butanol as extracting agents, extracting each extracting agent for 2 times, respectively collecting organic phases of petroleum ether, ethyl acetate and n-butanol extraction liquid, concentrating under reduced pressure at 50 ℃, and drying to obtain semen Celosiae extract 2B, semen Celosiae extract 2C and semen Celosiae extract 2D.
Example 5: preparation method of semen Celosiae extract
The method comprises the following specific steps:
pulverizing semen Celosiae to obtain semen Celosiae powder; adding 15 times of ethanol water solution with volume concentration of 90% into semen Celosiae powder, and heating and reflux-extracting at 65 deg.C for 3 hr to obtain extractive solution; concentrating the extractive solution at 50 deg.C under reduced pressure until no alcohol smell exists to obtain semen Celosiae extract; adding pure water into the feather cockscomb seed extract, and adding 1L of pure water into the feather cockscomb seed extract extracted according to 1kg of raw material (feather cockscomb seed powder) for suspension to obtain suspension; centrifuging the suspension at 3500rpm for 10min, and collecting supernatant; sequentially extracting the supernatant with 2 times of petroleum ether, ethyl acetate and n-butanol as extractant, each extractant extracting for 3 times, collecting organic phase of n-butanol extractive solution, concentrating under reduced pressure at 50 deg.C, and drying to obtain organic phase powder; adding 10% (v/v) ethanol into the organic phase powder, and adding 100mL 10% (v/v) ethanol into the organic phase powder extracted from 1kg of the original medicinal material (feather cockscomb seed powder) for suspension to obtain a mixed solution; centrifuging the mixed solution at 3500rpm for 10min, and collecting supernatant; purifying the supernatant by AB-8 macroporous resin column (diameter of macroporous resin column is 8cm, height of macroporous resin column is 70cm, column volume is 3.5L), sequentially eluting with 20% (v/v), 75% (v/v) and 95% (v/v) ethanol-water gradient (flow rate of gradient elution is 3BV/h, each gradient has 3 column volumes), respectively collecting eluates, concentrating under reduced pressure at 50 deg.C, and drying to powder to obtain semen Celosiae extract 2E, semen Celosiae extract 2F, and semen Celosiae extract 2G.
Example 6: preparation method of semen Celosiae extract
The method comprises the following specific steps:
pulverizing semen Celosiae to obtain semen Celosiae powder; adding ethanol water solution with volume concentration of 80% 12 times of the weight of the powder of feather cockscomb seed, heating and reflux-extracting at 70 deg.C for 2 times, each for 2 hr, and mixing extractive solutions; concentrating the extractive solution at 50 deg.C under reduced pressure until no alcohol smell exists to obtain semen Celosiae extract; and (3) continuing to perform decompression concentration on the feather cockscomb seed extract at 50 ℃, and drying to obtain the feather cockscomb seed extract 3A.
Example 7: preparation method of semen Celosiae extract
The method comprises the following specific steps:
pulverizing semen Celosiae to obtain semen Celosiae powder; adding ethanol water solution with volume concentration of 80% 12 times of the weight of the powder of feather cockscomb seed, heating and reflux-extracting at 70 deg.C for 2 times, each for 2 hr, and mixing extractive solutions; concentrating the extractive solution at 50 deg.C under reduced pressure until no alcohol smell exists to obtain semen Celosiae extract; adding pure water into the feather cockscomb seed extract, and adding 1L of pure water into the feather cockscomb seed extract obtained by extracting 1kg of raw material (feather cockscomb seed powder) for suspension to obtain suspension; centrifuging the suspension at 3500rpm for 10min, and collecting supernatant; purifying the supernatant by polyamide chromatography column (diameter of column is 8cm, height is 70cm, column volume is 3.5L), sequentially performing gradient elution with pure water and 95% (v/v) ethanol (flow rate of gradient elution is 3BV/h, each gradient is 3 column volumes), collecting 95% (v/v) ethanol eluate, concentrating under reduced pressure at 50 deg.C, and drying to obtain semen Celosiae extract 3B.
Example 8: preparation method of semen Celosiae extract
The method comprises the following specific steps:
pulverizing semen Celosiae to obtain semen Celosiae powder; adding ethanol water solution with volume concentration of 80% 12 times of the weight of the powder of feather cockscomb seed, heating and reflux-extracting at 70 deg.C for 2 times, each for 2 hr, and mixing extractive solutions; concentrating the extractive solution at 50 deg.C under reduced pressure until no alcohol smell exists to obtain semen Celosiae extract; adding pure water into the feather cockscomb seed extract, and adding 1L of pure water into the feather cockscomb seed extract obtained by extracting 1kg of raw material (feather cockscomb seed powder) for suspension to obtain suspension; centrifuging the suspension at 3500rpm for 10min, and collecting supernatant; purifying the supernatant by polyamide chromatography column (diameter of column is 8cm, height is 70cm, column volume is 3.5L), sequentially gradient-eluting with pure water and 95% (v/v) ethanol (flow rate of gradient elution is 3BV/h, each gradient is 3 column volumes), collecting 95% (v/v) ethanol eluate, and concentrating under reduced pressure at 50 deg.C to obtain soft extract; adding pure water into the thick extract, and adding 100mL of pure water into the thick extract extracted according to 1kg of the raw medicinal material (feather cockscomb seed powder) for suspension to obtain a mixed solution; sequentially extracting the mixed solution with 3 times of petroleum ether and ethyl acetate as extracting agents, extracting each extracting agent for 3 times, respectively collecting organic phases of petroleum ether and ethyl acetate extraction solutions, concentrating under reduced pressure at 50 ℃, drying to powder to obtain a feather cockscomb seed extract 3C and a feather cockscomb seed extract 3D, collecting a water phase extracted by ethyl acetate, concentrating under reduced pressure at 50 ℃, drying to powder to obtain a feather cockscomb seed extract 3E.
Experimental example 1: effect of feather cockscomb seed extract on coronavirus-infected mice
The method comprises the following specific steps:
1. preparation of reagent and test solution
1.1, reagents
RIPA lysate (pecan day, cat # P0013B), PMSF (pecan day, cat # ST506), BCA protein quantification kit (pecan day, cat # P0010), 5 × Loading Buffer (Loading Buffer, pecan day, cat # P0015L), tris (i), glycine (BBI), sds (BBI), methanol (husband test, AR), tween-20 (husband test, AR), KCl (husband test), NaCl (husband test), Marker (Thermo, cat # 26619), transfer filter paper (pecan day), PVDF membrane (pecan day, 0.45 μm specification), Western solution set (including rapid wash blocking solution, primary and secondary antibody diluents, pecan day, cat # P0023), ACE2 rabbit primary antibody (pecan AB, cat # 252), β -actin primary antibody (goat anti-rabbit primary antibody), rabbit anti-rabbit monoclonal antibody (HRP), goat anti-tumor Marker (ECL), pecan anti-tumor Marker (bbl), bbl # 5008, cat # E411-03), SDS-PAGE gel rapid formulation kit (bi yun tian, cat # P0012 AC).
The preparation method of the TBST buffer solution comprises the following steps: 50mL of TrisHCL (1M, pH7.5), 8g of NaCl, 0.2g of KCL and 200.5mL of Tween, and adding distilled water to a constant volume of 1L.
Preparing an electrophoresis buffer solution: 3.03g Tris base (Tris base), 14.4g glycine, deionized water to 1L, at 4 degrees C storage.
Transfer Buffer (Transfer Buffer) preparation: 3.03g Tris base, 14.4g glycine, 200mL methanol (added before use, volatile), adding deionized water to 1L, and storing at 4 ℃.
1.2 preparation of test substance solution
0.5% of CMC-Na aqueous solution by mass fraction: weighing CMC-Na, and adding distilled water to prepare a CMC-Na aqueous solution with the mass fraction of 0.5%.
4mg/mL test solution: weighing a test object, adding a CMC-Na aqueous solution with the mass fraction of 0.5 percent to prepare a test object solution with the concentration of 4 mg/mL.
2. Test method
30 mice were taken and randomly divided into 10 groups of 3 mice each, 10 groups were: blank control group (CO), test article groups 1-9 to which test articles 1-9 were administered, wherein test articles 1-9 were 1A, 1C, 1D, 2A, 2D, 2F, 3B, 3D, and 3E prepared in examples 1-8, respectively.
After the experiment is started, the blank group of mice is gavaged with 0.5% CMC-Na aqueous solution in a dosage of 0.2g/kg in the morning, the test group of 1-9 mice is gavaged with 1-9 of 4mg/mL test solution in a dosage of 0.2g/kg in the morning, and the administration is continuously carried out for 2 weeks.
After administration for 1h for the last 1 day, the mice were euthanized, peripheral blood of the mice was collected, and serum was separated and frozen at-80 ℃ for future use; dissecting a mouse, taking lung tissues of the mouse, weighing, and freezing and storing at-80 ℃ for later use; western blot is adopted to detect the expression level of ACE2 protein in mouse lung tissues, and the detection results are shown in Table 1 and figures 1-4 (one plate of electrophoresis can only run 6 tested substances, two plates are used for running, and blank control is added respectively); the Excel is adopted to carry out input and statistical analysis on the data, the measurement data in the experimental result is represented by mean plus minus plus minus standard deviation (mean plus minus SD), the software carries out single-factor variance analysis on the comparison between groups by adopting SPASS13.0, and the comparison difference between the groups has statistical significance when P is less than 0.05;
the method for detecting the expression level of ACE2 protein in mouse lung tissues by using Western blot comprises the following steps:
(1) extraction of total lung protein:
preparing a protein lysate: referring to RIPA lysate specification, PMSF was added to RIPA to inhibit protein degradation according to PMSF: RIPA lysate 1: 100(v/v) were prepared as protein lysates. Cutting lung tissue into pieces, and adding 200 μ L of lysis solution into each 20mg of lung tissue; grinding with glass homogenizer on ice, and cracking on ice for 30 min;
precooling the centrifuge in advance, and setting the speed to 12000rpm, 4 ℃ and 10 min;
thirdly, 3) taking the supernatant, and determining the total protein concentration in order to keep the sample loading amount consistent among different processed samples and facilitate the analysis result; preparing a 96-well plate, adding 18 mu L of PBS buffer solution and 2 mu L of protein supernatant obtained in the step (2 mu L) and 200 mu L of BCA reagent (the volume ratio of the solution A to the solution B in the BCA protein quantification kit is 50: 1) according to the description of the BCA protein quantification kit, wherein each protein sample corresponds to 2-3 multiple wells;
fourthly, after incubation in a incubator at 37 ℃ for 30min, carefully blowing bubbles by a blower; measuring OD value by using an enzyme-labeling instrument at the wavelength of 562nm, and calculating the total protein concentration;
carefully sucking the residual supernatant in the centrifuge tube to a new Eppendorf tube, mixing according to the proportion of adding 1 mu L of loading buffer into every 4 mu L of protein sample, firstly whirling, and then centrifuging for a short time; placing the mixture in a boiling water bath for 5-10 min, and placing the mixture into a refrigerator at the temperature of-70 ℃ for storage after the protein is boiled;
(2) gel preparation and electrophoresis:
firstly, preparing separation gel according to the required concentration, adding the separation gel into a glass plate to the middle of a green frame, and adding water to seal the liquid level; standing at room temperature for about 40min for gelation, and at this time, an obvious interface between the separation gel and water can be seen;
preparing stacking gel, taking out the water phase, adding the stacking gel without bubbles, immediately inserting the comb, placing the comb into an electrophoresis tank after gelation, adding electrophoresis buffer solution, and pulling out the comb;
centrifuging the sample for a short time, and spin-drying water drops outside the tube wall and spin-drying the protein sample in the tube wall;
adding a protein sample (60 mu g of total protein is added into each hole) and a Marker into the sample adding hole, and adding a sample loading buffer solution into a lane without adding the sample;
fifthly, using 80V for the sample in the concentrated gel, using 120V for constant voltage electrophoresis after the front edge of the bromophenol blue enters the separation gel, and carrying out electrophoresis for 2-4 h;
seventhly, stopping electrophoresis when the front edge of the bromophenol blue just runs out of the gel;
tilting the glass plate under the environment of water wetting, and cutting a corner at one end of the glue to make a mark;
(3) transfer film (wet process):
precooling a Transfer Buffer at 4 ℃ to remove gas;
taking out the glue, removing the redundant glue, cutting corners, marking, measuring the size, and immersing in a Transfer buffer for 30 min;
cutting 6 pieces of small filter paper smaller than the glue, and immersing the filter paper in a Transfer buffer for 10 min;
cutting off a PVDF membrane smaller than the size of the glue, marking a cutting angle corresponding to the glue, soaking in methanol for 5min, soaking and washing with distilled water to make the membrane hydrophilic and transparent, and soaking in a Transfer buffer for 15 min;
soaking the fiber mat;
sixthly, opening the clamp, and covering the black surface with a fiber pad, filter paper multiplied by 3-glue, a membrane, filter paper multiplied by 3-fiber pad in sequence, wherein air bubbles are not required to be generated, and pressing;
seventhly, closing the clamp, placing the black surface of the film rotating groove into the film rotating groove, and placing the film rotating groove into a washbasin containing a large number of ice blocks;
ninthly, constant current 200mA transfer, beta-actin internal reference for 60min and ACE2 protein for 120 min;
(4) immune labeling and developing:
putting the membrane into a sealing box, adding a quick sealing liquid, and sealing for 10min at room temperature;
taking out the membrane, washing the membrane for 3 times by using TBST buffer solution, and washing for 10min each time;
③ diluting the primary antibody with an antibody diluent (antibody dilution ratio, refer to the antibody specification, dilution ratio 1: 1000 in this example), adding the diluted primary antibody into a primary antibody incubation box, and standing overnight at 4 ℃;
taking out the membrane, washing the membrane for 3 times by using TBST buffer solution, and washing for 10min each time;
diluting the secondary antibody marked by HRP with an antibody diluent (the dilution ratio of the antibody is 1: 1000 according to the specification of the antibody), adding the diluted secondary antibody into a secondary antibody incubation box, and incubating for 2 hours at room temperature;
sixthly, taking out the membrane, washing the membrane for 3 times by TBST, and washing for 10min each time;
seventhly, mixing the solution A and the solution B of the ECL kit in equal volume, adding the mixture on the membrane on the surface with the protein, and developing the membrane by a gel imager.
3. Test results
As can be seen from table 1 and fig. 1 to 4, compared with the blank control group, the expression level of ACE2 protein in the lung tissues of the test groups 6, 7, and 8 mice was significantly reduced (P <0.01), the expression level of ACE2 protein in the lung tissues of the test groups 1, 3, 4, and 5 mice was significantly reduced (P <0.05), and the expression levels of ACE2 protein in the lung tissues of the test groups 2 and 9 mice were statistically different but not statistically significant. Therefore, the celosia seeds extracts 1A, 1D, 2A, 2D, 2F, 3B and 3D can prevent the surface S protein of the coronavirus from being combined with ACE2 by reducing the expression level of ACE2 protein, so as to achieve the purpose of preventing and/or treating coronavirus infection.
Moreover, as can be seen from fig. 1-2, from the inhibitory activity of the test substances 1-3, the test substance 3 > the test substance 1 > the test substance 2, which indicates that the feather cockscomb seed extract obtained by ethanol extraction of feather cockscomb seed has a certain activity, and the purification by a chromatographic column can further enrich the effective components in the feather cockscomb seed extract and remove the ineffective components; from the aspect of the inhibitory activity of the test substances 4-6, the test substance 6 is more than the test substance 5 is more than the test substance 4, which shows that the effect of performing primary extraction on the feather cockscomb seed extract obtained by ethanol extraction of feather cockscomb seeds is equivalent to that of primary chromatographic column purification, and the inhibitory activity of the feather cockscomb seed extract can be further improved by performing chromatographic column purification on the extracted active ingredients; from the inhibitory activity of the test substances 7-9, the test substance 8 is more than the test substance 7 is more than the test substance 9, which shows that when the celosia seeds extract obtained by ethanol extraction of the celosia seeds is subjected to chromatographic purification, the polyamide chromatographic column can be used for better enriching the effective components in the celosia seeds extract by using a larger-pore resin column, and the inhibitory activity of the active components purified by the chromatographic column can be further improved by extracting the active components.
TABLE 1 expression levels of ACE2 protein in lung tissue of different groups of mice
| Group of | ACE2/β-actin | Group of | ACE2/β-actin |
| Blank control group (CO) | 1.46±0.09 | Blank control group (CO) | 1.45±0.08 |
| Test group 1 | 1.20±0.12* | Test group 7 | 0.95±0.1** |
| Test object group 2 | 1.41±0.08 | Test object group 8 | 0.52±0.18** |
| Test group 3 | 1.00±0.16* | Test object group 9 | 1.3±0.13 |
| Test group 4 | 1.25±0.08* | ||
| Test group 5 | 1.13±0.09* | ||
| Test group 6 | 0.48±0.14** |
Note: denotes P <0.05 compared to the blank control group (t-test); denotes P <0.01 compared to the blank control group (t-test).
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The application of the plant extract in preparing the medicine is characterized in that the plant extract is a feather cockscomb seed extract; the medicament has at least one of the following uses:
(a) preventing and/or treating coronavirus infection diseases; and/or the presence of a gas in the gas,
(b) inhibiting angiotensin converting enzyme 2 or reducing angiotensin converting enzyme 2 expression level.
2. The use as claimed in claim 1, wherein the preparation method of the feather cockscomb seed extract comprises the steps of extracting feather cockscomb seed with organic solvent and/or water as extraction reagent; the extraction solvent is 30-95 vt% alcohol water solution.
3. The use according to claim 1 or 2, wherein the celosia seeds extract is prepared by a method comprising the steps of: the feather cockscomb seed extract is prepared by crushing feather cockscomb seeds to obtain powder, adding 10-15 times of alcohol-water solution with volume concentration of 60-90% by weight into the powder, performing reflux extraction at 55-70 ℃ for 1-3 times for 1-3 hours each time, combining extracting solutions, concentrating to obtain feather cockscomb seed extract, and drying to obtain the feather cockscomb seed extract.
4. The use as claimed in claim 3, wherein the preparation method of the feather cockscomb seed extract further comprises the steps of dissolving the feather cockscomb seed extract in water, centrifuging, collecting the supernatant, and then subjecting the supernatant to macroporous resin adsorption and/or polyamide chromatography column and/or extraction;
the macroporous resin adsorption step and the polyamide chromatographic column step both comprise the steps of taking supernate, washing impurities by adopting 0-20 vt% of alcohol-water solution, then analyzing by adopting 70-95 vt% of alcohol-water solution, and collecting analysis liquid;
the extraction step comprises the steps of extracting supernate by using petroleum ether, ethyl acetate and n-butanol as extracting agents or extracting by using petroleum ether and ethyl acetate as extracting agents or extracting by using petroleum ether and n-butanol as extracting agents, and collecting an ethyl acetate extraction organic phase or an n-butanol extraction organic phase.
5. The use of claim 4, wherein the preparation method of the feather cockscomb seed extract further comprises the steps of taking the supernatant, performing the extraction step, and performing the macroporous resin adsorption step to obtain the feather cockscomb seed extract; or taking the supernatant, carrying out the polyamide chromatographic column step, and then carrying out the extraction step to obtain the feather cockscomb seed extract.
6. A preparation method of a plant extract is characterized in that the preparation method of the plant extract comprises the step of extracting feather cockscomb seeds serving as a raw material by taking an organic solvent and/or water as an extraction reagent; the extraction solvent is 30-95 vt% alcohol water solution.
7. A plant extract obtained by the method for producing a plant extract according to claim 6.
8. A medicament for preventing and/or treating coronavirus infection, comprising the plant extract of claim 7.
9. A medicament for inhibiting angiotensin converting enzyme 2, which comprises the plant extract according to claim 7.
10. A pharmaceutical agent for reducing the expression level of angiotensin converting enzyme 2, which comprises the plant extract according to claim 7.
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