CN114344350A - Application of artemisia pigweed extract - Google Patents
Application of artemisia pigweed extract Download PDFInfo
- Publication number
- CN114344350A CN114344350A CN202111647965.6A CN202111647965A CN114344350A CN 114344350 A CN114344350 A CN 114344350A CN 202111647965 A CN202111647965 A CN 202111647965A CN 114344350 A CN114344350 A CN 114344350A
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- CN
- China
- Prior art keywords
- extract
- artemisia
- pigweed
- enterovirus
- ethyl acetate
- Prior art date
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Links
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- A—HUMAN NECESSITIES
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- A61K2236/30—Extraction of the material
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- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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Abstract
The invention provides application of artemisia pigweed extract in preparing a medicine for preventing and treating diseases caused by enterovirus infection. The invention finds a new application of the artemisia pigweed extract in preparing medicines for resisting enterovirus 71 and coxsackievirus A16 infection. The artemisia pigweed extract has the effect of inhibiting enterovirus infection at a cellular level, a molecular level and an animal experiment.
Description
Technical Field
The invention relates to the field of medicines, in particular to application of an artemisia pigweed extract.
Background
Enteroviruses are non-enveloped viruses containing single-stranded positive-strand RNA, are the causative agents of many human and animal diseases, and include enterovirus type 71 (enterovirus 71, EV 71), Coxsackievirus type A16 (Coxsackie virus A16, CVA 16), Coxsackievirus type A6 (Coxsackie virus A6, CVA 6), Coxsackievirus type B3 (Coxsackie virus B3, CVB 3), Poliovirus (PV), rhinovirus (rhinovirus) and the like, and cause various diseases such as hand-foot-and-mouth disease, herpetic angina, myocarditis, poliomyelitis, common cold, and the like, resulting in various diseases such as hand-foot-and-mouth herpes and inflammation, meningitis, acute myelogrittitis, cardiovascular diseases and the like, which may lead to death in severe cases. Enteroviruses are prevalent worldwide and have become a serious public health hazard.
At present, diseases caused by enterovirus infection are mainly controlled clinically by supporting treatment and symptomatic treatment methods, broad-spectrum conventional antiviral drugs such as ribavirin, acyclovir, ganciclovir and the like are used for inhibiting virus activity, and immune protection is exerted by immune regulators such as interferon, immunoglobulin and glucocorticoid. Since the use of broad-spectrum drugs leads to the emergence of drug-resistant viral strains, there is a current lack of novel antiviral drugs having different targets from those of conventional antiviral drugs. The natural medicine has the advantages that many chemical medicines do not have, such as low price, no pollution, low toxic and side effects, rich resources, obvious curative effect and the like, and has the potential to be used for treating enterovirus infection diseases. So far, a considerable part of Chinese herbal medicines and national medicines have been developed and applied to the field of antivirus, and the effect is remarkable. It is worth noting that the combined use of the active ingredients in herbs plays a crucial role in improving the antiviral efficacy. However, the Mongolian medicines with various types, abundant resources and wide distribution are not abundant in the research and development of new medicines, so that the value of many Mongolian medicines is not realized. Therefore, there is an urgent need to develop and develop a drug for preventing or treating diseases caused by enterovirus infection.
The herbal antiviral pathway can be divided into two, direct and indirect antiviral pathways, and some herbs can also have both pathways simultaneously. The former is the 'elimination of pathogens' in traditional Chinese medicine, and is mainly reflected in that the traditional Chinese medicine components inhibit the virus transmission process so as to play an antiviral role. The latter is also called 'strengthening body' in traditional Chinese medicine, and is mainly reflected in effectively regulating the immunity of the organism, activating the function of the organism and realizing virus resistance.
Herb of common Wormwood (Artemisia scopariaEt Kit.) is a herb of Artemisia of Compositae, seedling or tender stem and leaf, also called Bihai wormwood, northeast herba Artemisiae Scopariae, has bitter and pungent taste and cold nature, has effects of clearing lung-heat, relieving cough, expelling pus, etc., and is mainly used for lung heat, lung abscess, asthma, phlegm accumulation, throat sensation, lung prickle, common cold and cough, etc. The chemical components of the artemisia pigweed mainly comprise volatile oil, flavonoid, coumarins, acids, sterols, triterpenes, eneynes, ketones, esters, alcohols and aldehydes. In recent years, the artemisia pigweed has good effects of protecting the liver, promoting bile flow, resisting influenza, resisting inflammation, inhibiting bacteria, relieving fever, relieving cough, regulating immunity, killing insects, weeding, resisting anoxia, resisting lipolysis, treating primary senile dementia and the like, and particularly has wide application prospect in the aspect of anti-influenza treatment. Chlorogenic acid, p-hydroxyacetophenone and 6, 7-dimethoxy incense in the artemisia pigweed have the content of the coumarin which plays an important role in playing the drug effect, and the three components have good drug effect in the cholagogue aspect.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide an application of artemisia pigweed extract, which can prevent and treat diseases caused by enterovirus infection.
The invention provides application of artemisia pigweed extract in preparing a medicine for preventing and treating diseases caused by enterovirus infection.
Preferably, the enterovirus is enterovirus 71 and/or coxsackievirus a 16.
Preferably, the disease caused by an enterovirus infection comprises herpetic pharyngolaryngitis and/or hand-foot-and-mouth disease.
Preferably, the preventing or treating comprises inhibiting nucleic acid replication of enterovirus, inhibiting protein synthesis of enterovirus, reducing enterovirus titer, reducing infection of host by enterovirus or reducing mortality rate of host infected by enterovirus.
Preferably, the artemisia pigweed extract comprises one or more of an alcohol extract, an aqueous extract, volatile oil, petroleum ether extract, dichloromethane extract, ethyl acetate extract and n-butanol extract of artemisia pigweed.
Preferably, the preparation method of the artemisia pigweed alcohol extract comprises the following steps: mixing herba Artemisiae Annuae and 95% ethanol, soaking, heating under reflux, and filtering;
the preparation method of the artemisia pigweed aqueous extract comprises the following steps: mixing herba Artemisiae Annuae and water, soaking, heating under reflux, extracting, and filtering;
the preparation method of the artemisia pigweed volatile oil comprises the following steps: mixing herba Artemisiae Annuae and water, soaking, and extracting under reflux with volatile oil extractor.
Preferably, the preparation method of the artemisia pigweed petroleum ether extract comprises the following steps: mixing the artemisia pigweed alcohol extract with water to obtain a suspension, adding petroleum ether for extraction, combining upper layer petroleum ether extract, and drying to obtain an artemisia pigweed petroleum ether extract;
the preparation method of the artemisia pigweed methylene chloride extract comprises the following steps: mixing the artemisia pigweed alcohol extract with water to obtain a suspension, adding petroleum ether for extraction, taking the lower layer of water extract, adding dichloromethane, combining the lower layer of dichloromethane extract, and drying to obtain the artemisia pigweed dichloromethane extract.
Preferably, the preparation method of the artemisia pigweed ethyl acetate extract comprises the following steps: mixing the Artemisia scoparia ethanol extract with water to obtain suspension, adding petroleum ether for extraction, taking the lower layer water extract, adding dichloromethane, mixing the upper layer raffinate, adding ethyl acetate, layering, mixing the upper layer ethyl acetate extract, and drying to obtain the final product;
the preparation method of the artemisia pigweed n-butanol extract comprises the following steps: mixing the artemisia pigweed alcohol extract with water to obtain a suspension, adding petroleum ether for extraction, taking the lower layer water extract, adding dichloromethane, combining the upper layer raffinate, adding ethyl acetate, combining the lower layer raffinate, adding n-butyl alcohol, layering, combining the upper layer n-butyl alcohol extract, and drying to obtain the artemisia pigweed n-butyl alcohol extract.
The invention provides a medicament for preventing or treating diseases caused by enterovirus infection, which comprises artemisia pigweed and/or an artemisia pigweed extract and pharmaceutically acceptable auxiliary materials.
Compared with the prior art, the invention provides the application of the artemisia pigweed extract in preparing the medicine for preventing and treating the diseases caused by the enterovirus infection. The invention finds a new application of the artemisia pigweed extract in preparing medicines for resisting enterovirus 71 and coxsackievirus A16 infection. The artemisia pigweed extract has the effect of inhibiting enterovirus infection at a cellular level, a molecular level and an animal experiment.
Detailed Description
The invention provides application of artemisia pigweed extract, and the method can be realized by appropriately improving process parameters by referring to the content in the text by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides application of artemisia pigweed extract in preparing a medicine for preventing and treating diseases caused by enterovirus infection.
The enteroviruses comprise enterovirus 71 and/or coxsackievirus A16.
Diseases caused by enterovirus infection according to the present invention include, but are not limited to, herpetic pharyngolaryngitis and/or hand-foot-and-mouth disease.
The prevention and treatment of the invention comprises inhibition of nucleic acid replication of enterovirus, inhibition of protein synthesis of enterovirus, reduction of enterovirus titer, reduction of infection of enterovirus to a host or reduction of mortality of enterovirus infected host.
According to the invention, the artemisia pigweed extract preferably comprises one or more of artemisia pigweed alcohol extract, artemisia pigweed water extract, artemisia pigweed volatile oil, artemisia pigweed petroleum ether extract, artemisia pigweed dichloromethane extract, artemisia pigweed ethyl acetate extract and artemisia pigweed n-butyl alcohol extract; most preferably artemisia pigweed alcohol extract and artemisia pigweed ethyl acetate extract; particularly preferred is an ethyl acetate extract of artemisia pigweed.
In the invention, the preparation method of the artemisia pigweed alcohol extract comprises the following steps: mixing herba Artemisiae Annuae and 95% ethanol, soaking, heating under reflux, and filtering.
Wherein, the proportion of the artemisia pigweed and the 95 percent ethanol is preferably 1: (9-10); more preferably 1: 10; the heating reflux extraction time is preferably 2-6 h; more preferably 3 to 5 hours, most preferably 4 hours. The extraction times are 2-3 times. The filtration is preferably performed by gauze filtration, and more preferably by 4-layer gauze filtration. The filtrate is then freed of the solvent, preferably by distillation under reduced pressure.
In the invention, the preparation method of the artemisia pigweed aqueous extract comprises the following steps: mixing herba Artemisiae Annuae and water, soaking, heating under reflux, extracting, and filtering.
Wherein, the proportion of the artemisia pigweed and the water is preferably 1: (9-10); more preferably 1: 10; the heating reflux extraction time is preferably 4-6 h; most preferably 4 h. The extraction times are 2-3 times. The filtration is preferably performed by gauze filtration, and more preferably by 4-layer gauze filtration. The filtrate is then freed of the solvent, preferably by distillation under reduced pressure.
In the invention, the preparation method of the artemisia pigweed volatile oil comprises the following steps: mixing herba Artemisiae Annuae and water, soaking, and extracting under reflux with volatile oil extractor.
Wherein, the proportion of the artemisia pigweed and the water is preferably 1: (9-10); more preferably 1: 10; the heating reflux extraction time of the volatile oil extractor is preferably 6-8 h; most preferably 8 h.
In the invention, the preparation method of the artemisia pigweed petroleum ether extract comprises the following steps: mixing the artemisia pigweed alcohol extract with water to obtain a suspension, adding petroleum ether for extraction, combining upper layer petroleum ether extract, and drying to obtain the artemisia pigweed petroleum ether extract.
The mass ratio of the artemisia pigweed alcohol extract to the water is preferably 5 (3-5), and the mass volume ratio of the artemisia pigweed alcohol extract to the petroleum ether added each time is 5 (3-5) and g/mL. The number of times of extraction is preferably 25-30.
In the invention, the preparation method of the artemisia pigweed methylene chloride extract comprises the following steps: mixing the artemisia pigweed alcohol extract with water to obtain a suspension, adding petroleum ether for extraction, taking the lower layer of water extract, adding dichloromethane, combining the lower layer of dichloromethane extract, and drying to obtain the artemisia pigweed dichloromethane extract.
The mass-volume ratio of the artemisia pigweed alcohol extract to dichloromethane added each time is 5 (3-5), (g/mL).
Standing for more than 10min after each extraction. The number of times of extraction is preferably 25-30.
In the invention, the preparation method of the artemisia pigweed ethyl acetate extract comprises the following steps: mixing the Artemisia scoparia ethanol extract with water to obtain suspension, adding petroleum ether for extraction, taking the lower layer water extract, adding dichloromethane, mixing the upper layer raffinate, adding ethyl acetate, layering, mixing the upper layer ethyl acetate extract, and drying.
Standing for more than 10min after each extraction. The number of times of extraction is preferably 25-30.
The mass-volume ratio of the artemisia pigweed alcohol extract to the ethyl acetate added each time is 5 (3-5), (g/mL).
In the invention, the preparation method of the artemisia pigweed n-butyl alcohol extract comprises the following steps: mixing the artemisia pigweed alcohol extract with water to obtain a suspension, adding petroleum ether for extraction, taking the lower layer water extract, adding dichloromethane, combining the upper layer raffinate, adding ethyl acetate, combining the lower layer raffinate, adding n-butyl alcohol, layering, combining the upper layer n-butyl alcohol extract, and drying to obtain the artemisia pigweed n-butyl alcohol extract.
Standing for more than 10min after each extraction. The number of times of extraction is preferably 25-30.
The mass-volume ratio of the artemisia pigweed alcohol extract to n-butanol added each time is 5 (3-5), (g/mL).
The invention provides a medicament for preventing or treating diseases caused by enterovirus infection, which comprises artemisia pigweed and/or an artemisia pigweed extract and pharmaceutically acceptable auxiliary materials.
According to the invention, the artemisia pigweed extract preferably comprises one or more of artemisia pigweed alcohol extract, artemisia pigweed water extract, artemisia pigweed volatile oil, artemisia pigweed petroleum ether extract, artemisia pigweed dichloromethane extract, artemisia pigweed ethyl acetate extract and artemisia pigweed n-butyl alcohol extract; most preferably artemisia pigweed alcohol extract and artemisia pigweed ethyl acetate extract; particularly preferred is an ethyl acetate extract of artemisia pigweed.
The above extracts and the preparation method of the present invention have been described clearly, and are not described herein again.
The dosage form of the drug is not limited in the present invention, and may be powder, tablet, granule, injection, etc. known to those skilled in the art.
The pharmaceutically acceptable excipients are not limited in the present invention and are well known to those skilled in the art.
In an inhibition experiment of enterovirus infection, the inventor finds that the artemisia pigweed extract has an inhibition effect on cytopathic effect caused by enterovirus 71 and coxsackievirus A16, and has low cytotoxicity.
In the experiment for researching the enterovirus 71 type infection inhibition mechanism, the artemisia pigweed extract is found to inhibit virus infection by inhibiting RNA replication and protein synthesis of the enterovirus 71 type.
The inventor finds that the artemisia pigweed ethyl acetate extract has an obvious inhibiting effect on the content of virus protein in the enterovirus 71 infection process.
In addition, the artemisia scoparia ethyl acetate extract has a remarkable protection effect on a newborn suckling mouse infection process by enterovirus 71. Therefore, the artemisia scoparia ethyl acetate extract can be used as a candidate drug for resisting the enterovirus 71 infection, and further developed into a drug for preventing or treating diseases caused by the enterovirus infection.
The present invention provides a novel use of artemisia pigweed extract for the preparation of a medicament for preventing or treating a disease caused by an enterovirus infection in a subject.
The invention finds a new application of the artemisia pigweed extract in preparing medicines for resisting enterovirus 71 and coxsackievirus A16 infection. The artemisia pigweed extract has the effect of inhibiting enterovirus infection at a cellular level, a molecular level and an animal experiment.
The present invention provides a method of treating a disease caused by an enterovirus infection comprising administering the above drug and/or an extract of artemisia pigweed.
The specific mode of administration of the present invention may be oral or injectable.
In order to further illustrate the present invention, the following will describe in detail the application of an artemisia pigweed extract provided by the present invention with reference to the examples.
All the artemisia pigweed extracts tested by the invention are mixtures, unless otherwise indicated, all the instruments and reagents used in the examples are common commercial products, and the 1-day-old Balb/c suckling mouse described in example 5 is an offspring within 24 hours after the production of Balb/c pregnant mouse, and the pregnant mouse is purchased from a biological product of vinpoch.
Example 1: preparation method of various-level extracts of artemisia pigweed
(1) Artemisia scoparia alcohol extract
Soaking herba Artemisiae Annuae 500g in 10 times of 95% ethanol (5000 mL) for 4-6 hr, heating under reflux for 4 hr, extracting for 3 times, and mixing extractive solutions. Filtering with 4 layers of gauze, recovering solvent from the filtrate under reduced pressure with rotary evaporator, recovering 95% ethanol, and air drying to obtain 95% ethanol extract of herba Artemisiae Scopariae.
(2) Artemisia scoparia aqueous extract
Soaking herba Artemisiae Annuae 500g in 10 times of water (5000 mL) for 4-6 hr, heating under reflux for 4 hr, extracting for 3 times, and mixing extractive solutions. Filtering with 4 layers of gauze, recovering solvent from the filtrate under reduced pressure with rotary evaporator, and air drying to obtain water extract of herba Artemisiae Scopariae.
(3) Artemisia scoparia volatile oil
Soaking 500g of Artemisia scoparia L in 10 times of water (5000 mL) for 4-6 hr, and heating and refluxing for 8 hr with volatile oil extractor to obtain volatile oil extract of Artemisia scoparia L.
(4) Artemisia scoparia petroleum ether extract
Soaking herba Artemisiae Annuae 500g in 10 times of 95% ethanol (5000 mL) for 4-6 hr, heating and reflux extracting for 4 hr for 3 times, and mixing extractive solutions. Filtering with 4 layers of gauze, recovering solvent from the filtrate under reduced pressure with rotary evaporator, recovering 95% ethanol, and air drying to obtain 95% ethanol extract of herba Artemisiae Scopariae. Taking 50g of 95% ethanol extract of the artemisia pigweed, adding 500mL of 300-500mL of water to prepare a suspension, putting the suspension into a separating funnel, adding 500mL of petroleum ether with 300-500mL of petroleum ether each time, standing for more than 10 minutes, taking the upper petroleum ether layer extract after layering, repeatedly extracting for 25-30 times, combining all the petroleum ether extract, recovering, and airing to obtain the artemisia pigweed petroleum ether extract.
(5) Artemisia scoparia methylene dichloride extract
Soaking herba Artemisiae Annuae 500g in 10 times of 95% ethanol (5000 mL) for 4-6 hr, heating and reflux extracting for 4 hr for 3 times, and mixing extractive solutions. Filtering with 4 layers of gauze, recovering solvent from the filtrate under reduced pressure with rotary evaporator, recovering 95% ethanol, and air drying to obtain 95% ethanol extract of herba Artemisiae Scopariae. Taking 50g of 95% ethanol extract of the artemisia pigweed, adding 500mL of 300-500mL of water to prepare a suspension, putting the suspension into a separating funnel, adding 500mL of petroleum ether with 300-500mL of petroleum ether each time, standing for more than 10 minutes, taking petroleum ether layer extract after layering, repeatedly extracting for 25-30 times, combining all the petroleum ether extract, recovering, and drying in the air to obtain the artemisia pigweed petroleum ether extract. And continuously adding dichloromethane into the lower layer water suspension after the petroleum ether extraction, adding 500ml of dichloromethane each time, standing for more than 10 minutes, taking the lower layer dichloromethane layer extraction liquid after layering, repeatedly extracting for 25-30 times, combining all dichloromethane extraction liquids, recovering, and airing to obtain the artemisia pigweed dichloromethane extract.
(6) Artemisia scoparia ethyl acetate extract
Soaking herba Artemisiae Annuae 500g in 10 times of 95% ethanol (5000 mL) for 4-6 hr, heating and reflux extracting for 4 hr for 3 times, and mixing extractive solutions. Filtering with 4 layers of gauze, recovering solvent from the filtrate under reduced pressure with rotary evaporator, recovering 95% ethanol, and air drying to obtain 95% ethanol extract of herba Artemisiae Scopariae. Taking 50g of 95% ethanol extract of the artemisia pigweed, adding 500mL of 300-500mL of water to prepare a suspension, putting the suspension into a separating funnel, adding 500mL of petroleum ether with 300-500mL of petroleum ether each time, standing for more than 10 minutes, taking petroleum ether layer extract after layering, repeatedly extracting for 25-30 times, combining all the petroleum ether extract, recovering, and drying in the air to obtain the artemisia pigweed petroleum ether extract. And continuously adding dichloromethane into the lower layer water suspension after the petroleum ether extraction, adding 500ml of dichloromethane each time, standing for more than 10 minutes, taking the lower layer dichloromethane layer extraction liquid after layering, repeatedly extracting for 25-30 times, combining all dichloromethane extraction liquids, recovering, and airing to obtain the artemisia pigweed dichloromethane extract. And continuously adding ethyl acetate into the upper-layer water suspension after the extraction of the dichloromethane, adding 500ml of ethyl acetate into the upper-layer water suspension each time, standing for more than 10 minutes, taking the upper-layer ethyl acetate extract after layering, repeatedly extracting for 25-30 times, combining all the ethyl acetate extracts, recovering, and airing to obtain the artemisia pigweed ethyl acetate extract.
(7) N-butanol extract of artemisia pigweed
Soaking herba Artemisiae Annuae 500g in 10 times of 95% ethanol (5000 mL) for 4-6 hr, heating and reflux extracting for 4 hr for 3 times, and mixing extractive solutions. Filtering with 4 layers of gauze, recovering solvent from the filtrate under reduced pressure with rotary evaporator, recovering 95% ethanol, and air drying to obtain 95% ethanol extract of herba Artemisiae Scopariae. Taking 50g of 95% ethanol extract of the artemisia pigweed, adding 500mL of 300-500mL of water to prepare a suspension, putting the suspension into a separating funnel, adding 500mL of petroleum ether with 300-500mL of petroleum ether each time, standing for more than 10 minutes, taking petroleum ether layer extract after layering, repeatedly extracting for 25-30 times, combining all the petroleum ether extract, recovering, and drying in the air to obtain the artemisia pigweed petroleum ether extract. And continuously adding dichloromethane into the lower layer water suspension after the petroleum ether extraction, adding 500ml of dichloromethane each time, standing for more than 10 minutes, taking the lower layer dichloromethane layer extraction liquid after layering, repeatedly extracting for 25-30 times, combining all dichloromethane extraction liquids, recovering, and airing to obtain the artemisia pigweed dichloromethane extract. And continuously adding ethyl acetate into the upper-layer water suspension after the extraction of the dichloromethane, adding 500ml of ethyl acetate into the upper-layer water suspension each time, standing for more than 10 minutes, taking the upper-layer ethyl acetate extract after layering, repeatedly extracting for 25-30 times, combining all the ethyl acetate extracts, recovering, and airing to obtain the artemisia pigweed ethyl acetate extract. Adding n-butanol into the lower layer water suspension after ethyl acetate extraction, adding 500ml of n-butanol each time, standing for more than 10 minutes, taking the upper layer n-butanol extract after layering, repeatedly extracting for 25-30 times, combining all n-butanol extracts, recovering, and air drying to obtain the Artemisia scoparia n-butanol extract
Example 2: cytotoxicity and inhibition of cytopathic effects of Artemisia Sus Domestica extract caused by Enterovirus type 71
Taking RD cells in logarithmic growth phase, and taking the cells at 3X 104The density of each well was inoculated in a 96-well plate and cultured for 24 hours. RD cells at 37 ℃ and 5% CO2The culture medium used was DMEM cell culture medium supplemented with 10% fetal bovine serum (abbreviated as 10% FBS-DMEM, FBS (fetal bovine serum) and DMEM are purchased from Sigma, USA) containing a diabody (penicillin-streptomycin solution at a concentration of 100U/mL).
When detecting cytotoxicity of various extracts of Artemisia scoparia, aspirating off original cell culture solution, adding 100 μ L DMEM cell culture solution (2% FBS-DMEM) supplemented with 2% fetal bovine serum containing Artemisia scoparia extract with different concentrations into each well, marking the final concentration of the drug in the result, simultaneously setting cell control group added with 100 μ L DMEM-2% FBS, setting 3 parallel for each concentration, and placing into CO2Infection in a constant temperature incubator with 5% CO2Culturing the cells at 37 ℃; after 48 hours, adding 10 microliter of CCK-8 cell activity detection reagent (purchased from doctor, America) into each well, reacting for 1 hour at 37 ℃, and detecting the absorbance of the cells at 450 nm; based on the measured absorbance value of the cell, the magnitude of the absorbance value reflects the activity of the cell. Cytotoxic effect application CC50(cell viability 5)Drug concentration at 0%) for cell viability = drug group cell absorbance value/cell control group cell absorbance value × 100%; respectively calculating the drug concentration when the survival rate is 50% according to the cytotoxicity of the drugs with different concentrations to each hole, and using the half inhibitory concentration CC50Represents;
when the inhibition effect of various extracts of artemisia pigweed on the cytopathic effect caused by enterovirus 71 (EV 71) is detected, the original cell culture solution is aspirated off, and 50 mu l of 2% FBS-DMEM cell culture solution containing artemisia pigweed extracts with different concentrations is added into each well cell before 50 mu l of enterovirus 71 (genotype EV71-C4b, GengBank code KJ 508817) is added for infection, and the final concentration is indicated in the result; 50 μ L of live EV71 virus (multiplicity of infection 0.05) was added simultaneously, and a control group of cells not infected with live EV71 virus and a control group of virus not treated with the drug were set, 3 wells in parallel per concentration, in CO2 Constant temperature incubator, 5% CO2Culturing at 37 deg.C; after 48 hours, 10. mu.L of CCK-8 cell activity assay reagent (available from doctor, USA) was added to each well and reacted at room temperature for 1 hour, and absorbance at 450nm was measured as described above.
When the inhibition effect of various extracts of artemisia pigweed on cytopathic effect caused by coxsackie virus A16 (CVA 16) is detected, original cell culture solution is sucked away, and 50 mu l of 2% FBS-DMEM cell culture solution containing artemisia pigweed extracts with different concentrations is respectively added into each hole of cells before 50 mu l of coxsackie virus A16 (genotype CVA16-B1, GengBank accession number KF 055241.1) is infected, wherein the final concentration is indicated in the result; 50 μ L of live CVA16 virus (MOI = 0.05) was added simultaneously, while a control group of cells infected with live CVA16 virus and a control group of virus not treated with drug were placed, 3 parallel wells for each concentration, in CO2Constant temperature incubator, 5% CO2Culturing at 37 deg.C; after 48 hours, 10. mu.L of CCK-8 cell activity assay reagent (available from doctor, USA) was added to each well and reacted at room temperature for 1 hour, and absorbance at 450nm was measured as described above.
Inhibition rate of drug on virus-induced cytopathic effect =(drug group cell absorbance value-virus control group cell absorbance value)/(cell control group cell absorbance value-virus control group cell absorbance value) × 100%; respectively calculating the drug concentration with the inhibition rate of 50% according to the inhibition rate of different concentrations of drugs on cytopathic effect of each hole, and using half of the inhibition concentration EC50Represents;
independent experiments were repeated twice, and the results were averaged twice.
The experimental results are as follows:
TABLE 1 inhibition of RD cytopathic effect by EV71 by various Artemisia scoparia extracts
| Various extracts of artemisia pigweed | Concentration (μ g/ml) | Maximum inhibition ratio (%) |
| 95% ethanol | 500 | 73.56±2.81 |
| Ethyl acetate | 250 | 95.52±8.78 |
| Methylene dichloride | 500 | 38.47±3.81 |
| Petroleum ether | 2000 | 44.85±4.36 |
| N-butanol | 750 | 25.85±5.68 |
| Essential oils | 500 | 43.29±1.79 |
| Aqueous extracts | 750 | 44.17±3.05 |
TABLE 2 cytotoxicity of Artemisia scoparia Ether extract on RD cells
| Artemisia scoparia ethyl acetate extract (mu g/ml) | Cytotoxicity (%) |
| 15.63 | 0.00±0.00 |
| 31.25 | 4.51±2.24 |
| 62.5 | 9.06±3.21 |
| 125 | 12.25±2.43 |
| 250 | 13.08±0.52 |
| 500 | 14.73±1.21 |
| 1000 | 29.37±1.64 |
| 2000 | 57.43±2.79 |
TABLE 3 inhibition of RD cytopathic effect caused by EV71 by ethyl acetate extract of Artemisia scoparia
| Artemisia scoparia ethyl acetate extract (mu g/ml) | Inhibition ratio (%) |
| 15.63 | 0.00±0.00 |
| 31.25 | 16.67±4.07 |
| 62.5 | 42.58±9.64 |
| 125 | 72.25±4.98 |
| 250 | 95.52±8.78 |
| 500 | 63.24±3.65 |
| 1000 | 31.02±4.50 |
| 2000 | 18.31±3.74 |
TABLE 4 inhibition of RD cytopathic effect caused by CVA16 infection by Artemisia scoparia ethyl acetate extract
| Artemisia scoparia ethyl acetate extract (mu g/ml) | Inhibition ratio (%) |
| 15.63 | 16.54±1.74 |
| 31.25 | 32.68±6.11 |
| 62.5 | 41.16±4.93 |
| 125 | 62.75±7.53 |
| 250 | 84.35±3.74 |
| 500 | 39.03±4.65 |
| 1000 | 9.53±2.93 |
| 2000 | 0.00±0.00 |
From the above results, it can be concluded that the ethyl acetate extract of artemisia rupestris has less cytotoxicity at effective inhibitory concentrations against RD cytopathic effects caused by enterovirus type 71 infection. The artemisia scoparia ethyl acetate extract has an obvious inhibition effect on RD cytopathic effect caused by enterovirus 71, and the optimal inhibition rate is 95.52%; the compound has obvious inhibition effect on RD cytopathic effect caused by Coxsackie virus A16, and the optimal inhibition rate is 84.35%. Therefore, the artemisia scoparia ethyl acetate extract can be a candidate drug for resisting various enterovirus infections, and further developed into a drug for preventing or treating diseases caused by the enterovirus infections.
Example 3: inhibition effect of artemisia pigweed ethyl acetate extract on enterovirus 71 type nucleic acid replication
In an experiment for investigating the replication effect of the artemisia scoparia ethyl acetate extract on enterovirus 71-type nucleic acid, inoculating RD cells into a 24-pore plate for cell culture, and starting the experiment when the cell density reaches about 80%; after the cell culture supernatant was completely aspirated by a pipette, 500. mu.L of Artemisia scoparia ethyl acetate-2% FBS-DMEM solution was added to each well plate to treat the cells to a final concentration of: 250 mu g/ml; simultaneously adding 500 μ L of EV71 (multiplicity of infection MOI = 1) 2% FBS-DMEM solution, and using compound luteolin as a positive control group to make the concentration of the compound luteolin be 50 μ M; a control group of cells infected without addition of EV71 and a control group of viruses not treated with drug were set, 3 parallel wells for each concentration. Is placed in CO2In a constant temperature incubator at 5% CO2Culturing at 37 deg.C; after 16h, 250. mu.L Trazol lysis buffer was added to each wellCells (purchased from Beijing Kokai Kanji Co., Ltd.) were extracted and the EV71 RNA content in the cells and supernatant was quantitatively determined using the RNA extraction Kit for virus detection (purchased from Beijing Konji Co., Ltd.) and One Step SYBR PrimeScript RT-PCR Kit II (purchased from TaKaRa Japan) according to the instructions. Independent experiments were repeated twice, and the results were averaged twice.
The experimental results are as follows:
TABLE 5 inhibition of EV71 nucleic acid replication by Artemisia carvifolia ethyl acetate extract
| Group of | Viral control | Artemisia scoparia ethyl acetate extract | Luteolin | Cell controls |
| Viral nucleic acid content (%) | 100.00 ± 0.00 | 2.24 ± 0.64 | 26.63 ± 4.88 | 0.16 ± 0.11 |
The results can be used for concluding that the artemisia pigweed ethyl acetate extract has obvious inhibition effect on the content of virus nucleic acid in the enterovirus 71 infection process.
Example 4: inhibition effect of artemisia pigweed ethyl acetate extract on enterovirus 71 type protein synthesis
In an experiment for investigating the synthesis effect of the artemisia pigweed ethyl acetate extract on enterovirus 71 type protein, EV71-Luciferase report pseudovirus is used as a measurement basis for the expression level of the virus genome coding protein. The EV71-Luciferase report pseudovirus has a complete virus capsid, but the P1 gene region is deleted from the genome, and the Luciferase Luciferase gene is replaced by the Luciferase Luciferase gene, and the reading frame of the EV71 genome is consistent. The EV71-Luciferase report pseudovirus can normally infect host cells, can normally transcribe viral genomes and express viral proteins, can express Luciferase, and can reflect the expression level of the viral genome encoding proteins by quantitatively detecting the Luciferase expression level. Inoculating the RD cells into a 96-well plate for cell culture, and starting an experiment when the cell density reaches about 80%; the cell supernatant was completely aspirated away with a pipette, and 50. mu.L of Artemisia scoparia ethyl acetate-DMEM-2% FBS solution was added to the well plate to treat the cells to a final concentration: 250 mu g/ml; at the same time, 50. mu.L of prepackaged EV71-Luciferase pseudovirus (multiplicity of infection 0.5) DMEM-2% FBS solution was added, and the compound luteolin was used as a positive control group to a concentration of 50. mu.M, with 3 replicates per concentration. Finally discharging to CO2In a constant temperature incubator at 5% CO2Culturing at 37 deg.C; and after 16h, adding 100 mu L of Luciferase fluorescent substrate into each hole, transferring to a multi-hole plate, and measuring the Luciferase fluorescent value of the cells, wherein the size of the fluorescent value reflects the synthesis capacity of the EV71-Luciferase protein.
The control group of cells not infected with EV71-Luciferase and the control group of viruses not treated with the drug were prepared, and the same procedure was followed except for the addition of the different substances. Independent experiments were repeated twice, and the results were averaged twice.
The experimental results are as follows:
TABLE 6 Effect of Artemisia scoparia ethyl acetate extract on EV71 protein synthesis
| Group of | Viral control | Artemisia scoparia ethyl acetate extract | Luteolin | Cell controls |
| Expression level (%) | 100.00 ± 0.00 | 19.03 ± 8.59 | 41.35± 9.83 | 0.11 ± 0.2 |
The results can be used for concluding that the artemisia pigweed ethyl acetate extract has obvious inhibition effect on the content of virus protein in the enterovirus 71 infection process.
Example 5: artemisia scoparia ethyl acetate extract for reducing yield of enterovirus 71 type progeny virus
Taking RD cells in logarithmic growth phase, and culturing at 2.5X 105The density of each hole is inoculated on a 24-hole culture plate and cultured for 24 h. RD cells at 37 ℃ and 5% CO2The culture medium used was 10% FBS-DMEM containing the diabody (penicillin-streptomycin solution at a concentration of 100U/mL). Sucking off the original cell culture solution, and adding 500 μ l of 2% FBS-DMEM cell culture solution containing Artemisia scoparia ethyl acetate extract with different concentrations into each well cell to make the final concentration of the Artemisia scoparia ethyl acetate extract: 250 μ g/ml, triplicate wells per sample, and simultaneous addition of EV71 infection (MOI = 0.2), cells were cultured for 16h, and cell supernatants were collected. At the same time, virus control infected by only adding enterovirus 71 and virus-free control infected by no enterovirus 71 are setThe control group of virus-infected cells was operated as described above except that the substances were added.
Taking RD cells in logarithmic growth phase, and taking the cells at 3X 104The density of each hole is inoculated on a 96-hole culture plate and cultured for 24 h. RD cells at 37 ℃ and 5% CO2The culture medium used was 10% FBS-DMEM containing the diabody (penicillin-streptomycin solution at a concentration of 100U/mL). The stock culture was aspirated, 100. mu.l of 2% FBS-DMEM cell culture containing the cell supernatant at different dilutions were added to each well, 8 wells were each graded, and 100 ul of 2% FBS-DMEM was added to each well; the cells were cultured for 7 days, the number of cytopathic wells was observed, and the virus titer was determined by the Reed-Muench method. Independent experiments were repeated twice, and the results were averaged twice.
The experimental results are as follows:
TABLE 7 Effect of Artemisia scoparia ethyl acetate extract on EV71 progeny Virus production
| Group of | Viral control | Artemisia scoparia ethyl acetate extract | Cell controls |
| Viral titer (lgTCID)50） | 7.98 ± 0.67 | 4.54 ± 0.52 | 0.00 ± 0.00 |
From the above results, it can be concluded that the artemisia pigweed ethyl acetate extract has a significant effect of reducing the yield of the enterovirus 71 progeny virus, and can reduce the virus titer of the progeny by about 3 orders of magnitude (more than 1000 times).
Example 6: artemisia scoparia ethyl acetate extract for inhibiting enterovirus 71 infection of newborn suckling mice
Grouping 1-day-old Balb/c suckling mice: the placebo group (treated by placebo PBS after toxin attack), the artemisia pigweed ethyl acetate extract treated group (1.2 mg/g body weight) is administered after toxin attack by intragastric gavage, the artemisia pigweed ethyl acetate extract treated group (0.4 mg/g body weight) is administered after toxin attack by intragastric gavage, the non-toxin attack group (virus is replaced by PBS injection, and treatment is replaced by intragastric PBS), and 10-12 animals are selected in each group. Intracranial injection of 20ul lethal dose of enterovirus 71 type virus solution (1500000 TCID) into suckling mice with sterile insulin syringe50) (ii) a Subsequently, 20ul placebo or different doses of the ethyl acetate extract of artemisia pigweed were administered by direct gavage 6 consecutive days post-infection (days 0 to 6); placing a suckling mouse in a mouse cage, giving a mother mouse, water and food, and feeding the mother mouse with breast milk freely; the observation was continued for 16 days starting from day 0, and the number of deaths of suckling mice was recorded every day, and the mortality of suckling mice on day 16 was examined.
The experimental results are as follows:
TABLE 8 protection of Artemisia scoparia Ether extract treatment of neonatal rats infected with Enterovirus type 71
| Group of | Placebo | Gavage 1.2 mg/g | Gavage 0.4 mg/g | Without attacking toxin |
| Mortality (%) | 100 | 36.36 | 54.54 | 0 |
From the above results, it can be concluded that the artemisia pigweed ethyl acetate extract can effectively reduce the mortality rate caused by enterovirus type 71 infection of newborn suckling mice; the optimal dosage is 1.2 mg per g body weight of artemisia pigweed ethyl acetate extract, and the death rate of suckling mice can be reduced from 100% to 36.36%.
The artemisia scoparia ethyl acetate extract is proved to have obvious protective effect on the infection of the enterovirus 71 on newborn suckling mice; the animal model further proves that the artemisia pigweed ethyl acetate extract can effectively inhibit enterovirus 71 infection; therefore, the artemisia scoparia ethyl acetate extract can be used as a candidate drug for resisting the enterovirus 71 infection, and further developed into a drug for preventing or treating diseases caused by the enterovirus infection.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. Application of herba Artemisiae Annuae extract in preparing medicine for preventing and treating diseases caused by enterovirus infection is provided.
2. Use according to claim 1, wherein the enterovirus is an enterovirus type 71 and/or a coxsackievirus type a16 and/or a coxsackievirus type a6 and/or a coxsackievirus type B3 and/or a poliovirus and/or a rhinovirus.
3. Use according to claim 1, wherein the disease caused by an enterovirus infection comprises herpetic pharyngolaryngitis and/or hand-foot-and-mouth disease and/or viral myocarditis and/or poliomyelitis and/or the common cold.
4. The use of claim 1, wherein the control comprises inhibiting nucleic acid replication of an enterovirus, inhibiting protein synthesis of an enterovirus, reducing enterovirus titer, reducing infection of a host by an enterovirus, or reducing mortality of a host infected by an enterovirus.
5. The use of claim 1, wherein the artemisia pigweed extract comprises one or more of an alcohol extract, an aqueous extract, a volatile oil, a petroleum ether extract, a dichloromethane extract, an ethyl acetate extract and a n-butanol extract.
6. The use of claim 5, wherein the preparation method of the artemisia scoparia ethanol extract comprises the following steps: mixing herba Artemisiae Annuae and 95% ethanol, soaking, heating under reflux, and filtering;
the preparation method of the artemisia pigweed aqueous extract comprises the following steps: mixing herba Artemisiae Annuae and water, soaking, heating under reflux, extracting, and filtering;
the preparation method of the artemisia pigweed volatile oil comprises the following steps: mixing herba Artemisiae Annuae and water, soaking, and extracting under reflux with volatile oil extractor.
7. The use of claim 5, wherein the Artemisia rupestris petroleum ether extract is prepared by a method comprising: mixing the artemisia pigweed alcohol extract with water to obtain a suspension, adding petroleum ether for extraction, combining upper layer petroleum ether extract, and drying to obtain an artemisia pigweed petroleum ether extract;
the preparation method of the artemisia pigweed methylene chloride extract comprises the following steps: mixing the artemisia pigweed alcohol extract with water to obtain a suspension, adding petroleum ether for extraction, taking the lower layer of water extract, adding dichloromethane, combining the lower layer of dichloromethane extract, and drying to obtain the artemisia pigweed dichloromethane extract.
8. The use of claim 5, wherein the preparation method of the artemisia pigweed ethyl acetate extract comprises: mixing the Artemisia scoparia ethanol extract with water to obtain suspension, adding petroleum ether for extraction, taking the lower layer water extract, adding dichloromethane, mixing the upper layer raffinate, adding ethyl acetate, layering, mixing the upper layer ethyl acetate extract, and drying.
9. The use of claim 5, wherein the preparation method of the artemisia scoparia n-butanol extract comprises the following steps: mixing the artemisia pigweed alcohol extract with water to obtain a suspension, adding petroleum ether for extraction, taking the lower layer water extract, adding dichloromethane, combining the upper layer raffinate, adding ethyl acetate, combining the lower layer raffinate, adding n-butyl alcohol, layering, combining the upper layer n-butyl alcohol extract, and drying to obtain the artemisia pigweed n-butyl alcohol extract.
10. A medicine for preventing or treating diseases caused by enterovirus infection is characterized by comprising artemisia pigweed and/or an artemisia pigweed extract and pharmaceutically acceptable auxiliary materials.
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