CN111773258B - Mongolian medicine for resisting influenza virus, pharmaceutical composition, preparation method and application thereof - Google Patents
Mongolian medicine for resisting influenza virus, pharmaceutical composition, preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- 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, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Abstract
The invention relates to a Mongolian medicine for resisting influenza virus, a pharmaceutical composition, a preparation method and an application thereof, and provides an application of belvedere fruit and/or a belvedere fruit water extract and/or a belvedere fruit alcohol extract in preparation of the pharmaceutical composition or the pharmaceutical preparation for resisting influenza virus, and provides a pharmaceutical composition prepared from the following raw materials in parts by weight: 45-55 parts of sargentgloryvine root, 25-35 parts of inula flower and 15-25 parts of fructus kochiae. The pharmaceutical composition disclosed by the invention has excellent anti-H1N 1 and/or anti-H3N 2 influenza virus effect and no toxic or side effect.
Description
Technical Field
The disclosure relates to a Mongolian medicine for resisting influenza virus, a pharmaceutical composition, a preparation method and application thereof.
Background
The information in this background section is only for enhancement of some understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Influenza viruses are called influenza viruses for short and are divided into three types, namely A (A), B (B) and C (C). The influenza virus can cause infection and morbidity of various animals such as human, poultry, pigs, horses, bats and the like, and is the pathogen of epidemic diseases of human and animals such as human influenza, avian influenza, swine influenza, horse influenza and the like. Human influenza is mainly caused by influenza a and influenza b viruses. Influenza a viruses often have antigenic variation and can be further divided into subtypes H1N1, H3N2, H5N1, H7N9 and the like (wherein H and N represent two surface glycoproteins of influenza viruses, respectively).
For influenza patients, western medicines such as interferon, amantadine and oseltamivir are mostly used for treatment, but the western medicines have great side effects and are easy to generate drug resistance. Aiming at the problem, the inula flower has obvious curative effect on influenza and does not generate drug resistance through previous research of the inventor team.
Disclosure of Invention
In view of the above background art, the present inventors have conducted intensive studies on inula flowers, and found that inula flowers alone have an excellent effect against influenza viruses such as H1N1, but have weak resistance against H3N 2. In view of the problem, the present disclosure provides an anti-influenza virus Mongolian medicine, a pharmaceutical composition and a preparation method and application thereof, wherein the anti-influenza virus Mongolian medicine is compatible with Salicornia bigelovii and Inulae flos, and the obtained pharmaceutical composition has excellent anti-H1N 1 effect and excellent anti-H3N 2 effect.
Specifically, the following technical scheme is adopted in the disclosure:
in a first aspect of the present disclosure there is provided the use of broom cypress fruit and/or a water extract of broom cypress fruit and/or an alcohol extract of broom cypress fruit for the manufacture of a pharmaceutical composition or formulation for combating influenza viruses.
In a second aspect of the present disclosure, there is provided the use of broom cypress fruit and/or a water extract of broom cypress fruit and/or a alcohol extract of broom cypress fruit for the preparation of a pharmaceutical composition or pharmaceutical formulation capable of inhibiting the cytopathic effects caused by influenza virus infection and the replication of viral genomic RNA in influenza virus infected cells.
In the present disclosure, the Kochia scoparia (L.) schrad, a plant of chenopodiaceae, and the aerial part of the Kochia scoparia and the fruit of the Kochia scoparia have an anti-influenza virus effect in the present disclosure.
The invention relates to a preparation method of a traditional Chinese medicine for treating skin itch, and the traditional Chinese medicine is mainly prepared from fructus kochiae, and the inventor surprisingly finds that the fructus kochiae and/or the water extract and/or the alcohol extract of the fructus kochiae have excellent anti-influenza virus effects, especially can effectively inhibit cytopathic effect caused by influenza virus infection and can effectively inhibit the replication of virus genome RNA in cells infected by the influenza virus.
In one or some embodiments of the disclosure, the influenza virus is an influenza a virus that is an influenza a H1N1 virus and/or an influenza a H3N2 virus.
In one or some embodiments of the present disclosure, the aqueous extract of kochia scoparia is prepared by the following method:
decocting aerial parts of Kochiae fructus in water, and filtering; decocting the residue with water, and mixing filtrates to obtain fructus Kochiae water extract.
In one or some embodiments of the present disclosure, the alcohol extract of kochia scoparia is an alcohol extract of kochia scoparia.
Further, the fructus kochiae ethanol extract is prepared by the following method:
decocting aerial parts of Kochiae fructus with ethanol solution, and filtering; decocting the residue with ethanol solution, and mixing the filtrates to obtain Kochiae fructus ethanol extract.
Furthermore, the volume fraction of the ethanol solution is 90-95%, and the active ingredients for resisting the influenza virus can be effectively extracted.
In a third aspect of the present disclosure, there is provided a pharmaceutical composition against influenza virus, comprising kochia scoparia or an active ingredient of kochia scoparia.
In one or some embodiments of the disclosure, the effective component of the broom cypress fruit is prepared by water extraction or alcohol extraction.
In one or some embodiments of the disclosure, the pharmaceutical composition is prepared from the following raw materials in parts by weight:
45-55 parts of sargentgloryvine root, 25-35 parts of inula flower and 15-25 parts of fructus kochiae.
Further, the pharmaceutical composition is prepared from the following raw materials in parts by weight:
50 parts of sargentgloryvine root, 30 parts of inula flower and 20 parts of fructus kochiae.
Furthermore, the overground part and seeds of the agriophyllum squarrosum, the flowers of the inula flower and the fruits of the broom cypress fruit are used as medicines, and tests prove that the effect of resisting influenza viruses is more excellent when the overground part and the seeds of the agriophyllum squarrosum, the flowers of the inula flower and the fruits of the broom cypress fruit are used as medicines.
In the present disclosure, the Agriophyllum squarrosum is a mongolian medicine named chu dawn (the classic on mongolian medicine), and is known as Agriophyllum squarrosum (L.) moq. The related records indicate that: treating pestilence, headache, red eye, jaundice, kidney heat, causalgia of urethra, thirst, aphtha, stomach-ache, and toxic heat.
In the present disclosure, the Inula flower is a mongolian drug named alatan-dasule-the division thereof, known as Inula britannica l.var.japonica (Thubn.) franch.et sav, belonging to Inula plants of the family compositae. The indications are as follows according to related records: it can be used for treating "sticky" pricking pain, "complication", fracture, and incised wound.
The pharmaceutical composition disclosed by the invention is constructed according to the traditional Mongolian medicine theory and the traditional Chinese medicine theory, comprises one traditional Chinese medicine and two Mongolian medicines, is an organic whole, is free from one component and can not be used, and the components have synergistic interaction, so that the technical problem of single H1N1 resistance can be effectively solved. Through test verification, the inventor finds that the overall effect of the pharmaceutical composition is remarkably reduced by reducing any pharmaceutical raw material or replacing the raw material components of the pharmaceutical composition with the bulk drugs with similar drug properties; other raw material components are added, so that the technical effect of the pharmaceutical composition is not obviously improved, and the overall technical effect is reduced.
In a fourth aspect of the present disclosure, there is provided a preparation method of the pharmaceutical composition against influenza virus, the preparation method comprising the steps of:
mixing the raw materials in a certain weight ratio, and extracting with ethanol solution or water for several times to obtain extractive solution, i.e. the pharmaceutical composition.
In one or some embodiments of the disclosure, the ethanol solution has a volume fraction of 90 to 95%, and can effectively extract the active ingredients against influenza viruses.
In a fifth aspect of the disclosure, a pharmaceutical preparation for resisting influenza virus is provided, which is prepared from the pharmaceutical composition and pharmaceutically acceptable excipients.
In one or some embodiments of the present disclosure, the pharmaceutical preparation may be prepared into various pharmaceutical dosage forms, such as decoction, pill, capsule, tablet, powder, granule, oral liquid, or the like.
In one or some embodiments of the present disclosure, pharmaceutically acceptable excipients in the pharmaceutical formulation include, but are not limited to: diluents include, but are not limited to, sorbitol, mannitol, starch, lactose, powdered or microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, sugars, and the like; filling agent: sodium carboxymethylcellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatinized starch, etc.; disintegrating agent: dry starch, crospovidone, croscarmellose sodium, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and the like; lubricant: magnesium stearate, sodium lauryl sulfate or aerosil; other adjuvants include one or more of binding agent, flowing agent, composite forming agent, plasticizer, colorant, sweetener, viscosity enhancer, preservative or antioxidant.
Compared with the related technologies known by the inventor, one technical scheme of the disclosure has the following beneficial effects:
the pharmaceutical composition disclosed by the invention has excellent anti-H1N 1 and/or anti-H3N 2 influenza virus effect and no toxic or side effect, and has more excellent anti-H3N 2 influenza virus effect compared with a single medicinal material. Further, the subsequent verification of an influenza animal model shows that the pharmaceutical composition disclosed by the invention has an obvious effect of relieving general symptoms of influenza, and the main symptoms comprise sweating, defervescence, pain relieving and the like.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the features, steps, operations and/or combinations thereof.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.
Example 1 preparation of extracts of fruit and aerial parts of Kochia scoparia
(1) Aqueous extracts
Taking 50g of dried mature fruits of belvedere fruit, adding 15 times of water, decocting for 40 minutes, filtering with 4 layers of gauze, adding 10 times of water to residues, decocting for 20 minutes, combining filtrates, and placing the combined filtrates in a water bath kettle to be pasty to obtain 12g of pasty extract of the belvedere fruit.
Taking 50g of the overground part (without fruits) of the dried fructus kochiae, adding 16 times of water, decocting for 40 minutes, filtering 4 layers of gauze, adding 10 times of water into residues, decocting for 20 minutes, combining filtrates, and putting the mixed filtrates on a water bath pot to form paste to obtain 11g of the overground part water extract of the paste fructus kochiae.
(2) Ethanol extract
Taking 50g of dried and mature fruits of broom cypress fruit, adding 15 times of 95 v/v% ethanol solution, decocting for 30 minutes, filtering with 4 layers of gauze, adding 10 times of 95 v/v% ethanol solution on residues, decocting for 15 minutes, combining the filtrates, putting the mixture on a water bath pot to form paste, and 3.9g of ethanol extract of overground part of paste broom cypress fruit.
Taking 50g of the overground part of the dry fructus kochiae, adding 14 times of 95 v/v% ethanol solution, decocting for 30 minutes, filtering by 4 layers of gauze, adding 10 times of 95 v/v% ethanol solution on residues, decocting for 15 minutes, combining the filtrate, putting the mixture on a water bath pot until the mixture is pasty, and 3.7g of the ethanol extract of the overground part of the pasty fructus kochiae.
EXAMPLE 2 preparation of Compound extract
Mixing dried flos Inulae, dried fruit of Kochiae fructus, aerial part of herba Salicorniae Glabrae and seed at a mass ratio of 2:3:5, collecting 50g of mixed medicinal materials, pulverizing, adding 14 times of water, decocting for 30 min, filtering with 4 layers of gauze, adding 7 times of water to the residue, decocting for 15 min, and mixing filtrates to obtain paste, i.e. compound preparation water extract.
Mixing dried flos Inulae, dried fruit of Kochiae fructus, aerial part and seed of herba Salicorniae Bicoloris at a mass ratio of 2:3:5, pulverizing, performing primary reflux with 15 times of 95% ethanol solution by mass volume for 4 hr, performing secondary reflux with 12 times of 95% ethanol solution by mass volume for 4 hr, performing tertiary reflux with 10 times of 95% ethanol solution by mass volume for 4 hr, concentrating the extractive solution under reduced pressure, suspending in water, sequentially extracting with petroleum ether and dichloromethane to obtain petroleum ether extract, dichloromethane extract and ethanol extract, i.e. petroleum ether extract, dichloromethane extract and ethanol extract of compound preparation.
EXAMPLE 3 anti-influenza Virus of drugs
Preparation of extracts of flowers and aerial parts of Inula japonica
(1) Water extract
Taking 50g of dried inula flower, adding 12 times of water by mass, decocting for 30 minutes, filtering with 4 layers of gauze, adding 8 times of water to residues, decocting for 15 minutes, and combining filtrates to form a paste. The paste was formed on a water bath, first in a large evaporation dish and then in a small evaporation dish, finally in a paste form, 9.3g inula flower water extract was obtained.
Taking 50g of dried inula flower overground part (flower is removed), adding 15 times of water by mass, decocting for 40 minutes, filtering by 4 layers of gauze, adding 10 times of water to residues, decocting for 20 minutes, and combining filtrates to form a paste to obtain 8.7g of inula flower overground part water extract.
(2) Ethanol extract
Taking 50g of dried inula flower, adding 14 times of 95 v/v% ethanol solution by mass, decocting for 20 minutes, filtering with 4 layers of gauze, adding 6 times of 95 v/v% ethanol solution by mass to the residue, decocting for 15 minutes, and combining the filtrates to form a paste to obtain 2.6g of inula flower alcohol extract.
Taking 50g of the overground part of the dried inula flower (flower is removed), adding 12 times of 95 v/v% ethanol solution by mass, decocting for 30 minutes, filtering by 4 layers of gauze, adding 10 times of 95 v/v% ethanol solution by mass to the residue, decocting for 15 minutes, and combining the filtrates to form a paste to obtain 1.5g of the overground part ethanol extract of the inula flower.
Inhibition of cytopathic effects
At 3X 104One/well inoculated MDCK cells in 96-well cell plates (100. mu.L per well), 5% CO at 37 ℃2Incubating for 24h, and performing subsequent tests when the cell density reaches 80%;
1) removing cell culture supernatant, adding 50 μ L gradient concentration drug virus culture medium to treat cells, and adopting Inula japonica water extract, Inula japonica alcohol extract, Inula japonica water extract, Kochiae fructus alcohol extract, Kochiae fructus water extract, Compound preparation alcohol extract, Compound preparation dichloromethane extract and Compound preparation petroleum ether extract of examples 1, 2 and 3 to make their final concentrations respectively as in the following tables 1-14.
2) At the same time, 50. mu.L of influenza virus infection was added to the drug group. Influenza viruses of H1N1 type (a/17/California/2009/38 strain, hereinafter abbreviated to H1N1(California)) (MOI ═ 0.005), influenza viruses of H1N1 type (a/New-York/61/2015-CDC-LV16A strain, hereinafter abbreviated to H1N1(New-York)) (MOI ═ 0.005), influenza viruses of H3N2 type (a/17/HongKong/2014/8296 strain, hereinafter abbreviated to H3N2 (HongKong)) (MOI ═ 0.04) were used as the virus culture medium, respectively; setting cell control group without virus and virus control group without drug, setting 3 multiple wells per concentration, placing into a chamber containing 5% CO at 35 deg.C2The incubator is infected for 1h, and the cell plate is shaken once every half an hour;
3) after 1h, each well is washed once by using 100 mu L of sterile PBS buffer solution, and then a virus culture medium with the final concentration corresponding to the medicine is added;
4) CellTiter-
Transferring the detection reagent of 100 mu L into a porous plate after 5min, and detecting a fluorescence signal under 560nm exciting light by using the multi-labeled porous plate;
5) from the fluorescence signal, the inhibition rate of cytopathic effect at each drug concentration was calculated as [ (fluorescence value of infected and administered group-fluorescence value of infected control group)/fluorescence value of cell control group ] × 100%.
The anti-influenza test results are shown in tables 1-8: the inula flower water extract, the inula flower alcohol extract, the inula flower overground part water extract and the inula flower overground part alcohol extract have strong inhibition effects on cytopathic effects caused by the influenza A H1N1(California) and H1N1(New-York) infection, but have weak inhibition effects on the cytopathic effects caused by the influenza A H3N2 (Hongkong). Similarly, the aqueous extract, alcoholic extract, aqueous extract and alcoholic extract of the aerial parts of the broom cypress fruit have strong inhibitory effects on the cytopathic effect caused by the infection of H1N1(California) and H1N1(New-York) influenza viruses, but have weak inhibitory effects on the cytopathic effect caused by the infection of H3N2(Hongkong) influenza viruses.
TABLE 1 inhibition of cytopathic effects of Inula japonica water extract on H1N1(California), H1N1(New-York), H3N2(HongKong) influenza virus infection
TABLE 2 alcohol extract of Inula japonica has inhibitory effect on H1N1(California), H1N1(New-York), H3N2(HongKong) influenza virus infection induced cytopathic effect
TABLE 3 inhibition of the cytopathic effect of H1N1(California), H1N1(New-York) and H3N2(HongKong) influenza virus infection by water extracts of aerial parts of Inula japonica
TABLE 4 inhibitory Effect of alcohol extracts of aerial parts of Inula japonica on the cytopathic Effect of H1N1(California), H1N1(New-York), H3N2(Hongkong) influenza Virus infection
TABLE 5 inhibition of the cytopathic effects of H1N1(California), H1N1(New-York), H3N2(HongKong) influenza virus infection by aqueous extracts of Kochia scoparia
TABLE 6 inhibitory Effect of alcohol extracts of Kochia scoparia on the cytopathic Effect of H1N1(California), H1N1(New-York), H3N2(HongKong) influenza Virus infection
TABLE 7 inhibition of the cytopathic effect of H1N1(California), H1N1(New-York), H3N2(HongKong) influenza virus infection by water extract of aerial parts of Kochia scoparia
TABLE 8 inhibitory Effect of the alcohol extract of aerial parts of Kochia scoparia on the cytopathic Effect of H1N1(California), H1N1(New-York), H3N2(Hongkong) influenza Virus infection
The anti-influenza test results are shown in tables 9-14: the compound preparation water extract and compound preparation alcohol extract have excellent anti-H1N 1(California), anti-H1N 1(Nork York) and anti-H3N 2(Hong Kong) effects; the dichloromethane extract and petroleum ether extract of the compound preparation are weak in resistance to H1N1(California), H1N1(Nork York) and H3N2(Hong Kong).
TABLE 9 inhibition of H1N1(California) influenza virus infection induced cytopathic effect by compound preparation water extract
TABLE 10 inhibitory Effect of Compound preparation Water extract on cytopathic Effect caused by H1N1(New-York) influenza Virus infection
TABLE 11 inhibition of cytopathic effects of aqueous extracts of the Compound preparation against H3N2(HongKong) influenza Virus infection
TABLE 12 inhibitory Effect of alcoholic extracts of the Compound preparation on the cytopathic Effect of H1N1(California) influenza Virus infection
TABLE 13 inhibitory Effect of alcohol extracts of Compound preparations on cytopathic Effect caused by H1N1(New-York) influenza Virus infection
TABLE 14 inhibitory Effect of alcoholic extracts of Complex preparations on cytopathic Effect caused by H3N2(HongKong) influenza Virus infection
Example 4 drug inhibition of influenza Virus nucleic acid replication
1) At 2X 105One well was seeded with MDCK cells in 24 well cell plates (500. mu.L culture medium per well) at 37 ℃ in 5% CO2Incubating for 24h, and performing subsequent tests when the cell density reaches 80%;
2) the supernatant was aspirated off, and 250 μ L of virus medium containing the drug was added to give final drug concentrations: water extract of Inula flower: 200 mu g/mL; water extract of aerial parts of inula flower: 100 mu g/mL; ethanol extract of aerial parts of inula flower: 100 mu g/mL; water extract of aerial parts of broom cypress fruit: 50 mu g/mL; alcohol extraction of the overground part of the broom cypress fruit: 200 mu g/mL; water extract of compound preparation: 100 mu g/mL; alcohol extract of compound preparation: 100 mu g/mL;
3) meanwhile, a virus culture medium containing 250 μ L H1N1(California) (MOI 0.005), H1N1(New-York) (MOI 0.005), and H3N2(HongKong) (MOI 0.04) was added to the administration group; setting a control group without virus infection and a virus contrast without drug treatment, setting 3 parallel controls for different drugs, and placing the drugs at 35 ℃ and containing 5% CO2The incubator is infected for 1h, and the cell plate is shaken once every half an hour;
4) after 1h, each well was washed once with 100. mu.L sterile PBS buffer, and 500. mu.L of the culture medium containing the drug (final concentration as above) was added; after 12h, discarding the supernatant and collecting cells;
5) viral RNA was extracted using an RNA extraction kit for virus detection (tiangen) according to the instructions;
6) one Step was used as per the instructions
PrimeScriptTMRT-PCR Kit II (TaKaRa) and influenza virus NP segment specific primers quantitatively detect the content of enterovirus 71 type RNA in cells, and the content is relatively quantified with a virus control group.
The results of the nucleic acid experiments are shown in tables 15-17: the inula flower water extract, inula flower alcohol extract, inula flower overground water extract, inula flower overground alcohol extract, fructus kochiae overground water extract and fructus kochiae overground alcohol extract are resistant to H1N1(California) and are weak in resistance to H1N1(Nork York) and H3N2(Hong Kong). The compound preparation water extract and compound preparation alcohol extract have strong resistance to H1N1(California), H1N1(Nork York) and H3N2(Hong Kong).
TABLE 15 inhibitory Effect of Individual herb extracts on viral genomic RNA replication in H1N1(California) influenza Virus-infected cells
TABLE 16 inhibition of viral genome RNA replication in H1N1(California), H1N1(New-York), H3N2(Hongkong) influenza virus infected cells by water extract of the combination preparation
TABLE 17 inhibitory Effect of alcoholic extracts of the Complex formulation on viral genomic RNA replication in H1N1(California), H1N1(New-York), H3N2(Hongkong) influenza virus-infected cells
Comparative example 1
The inventor replaces the samphire of the compound preparation in the example 2 with gardenia to perform anti-influenza virus test, and finds that the anti-influenza virus test has weaker resistance to H3N2 and has obvious effect difference with the anti-H3N 2 effect of the water extract or alcohol extract of the compound preparation in the example 2.
The inventor also carries out other replacement tests with similar drug properties, and the anti-H3N 2 effect is weak.
Comparative example 2
Based on the compound preparation in the embodiment 2, a certain amount of the wucao leaf, the mongolian milkvetch root, the sea buckthorn and the trollius chinensis are added, the same drug concentration is found, the effect difference of the compound preparation water extract or alcohol extract with the effect of resisting H1N1 and H3N2 is obvious, and the H1N1 resistance and the H3N2 resistance are weaker.
Comparative example 3
The dried inula flower, the dried fruit of fructus kochiae, the overground part of the agriophyllum squarrosum and the seeds in the example 2 are mixed according to the mass ratio of 4:2:4 or 1:1:1 to prepare the compound preparation, and the compound preparation is subjected to an anti-influenza virus test, so that the anti-H1N 1 and the anti-H3N 2 of the compound preparation are weak, and the compound preparation has obvious effect difference with the water extract or the alcohol extract of the compound preparation in the example 2 in resisting H1N1 and H3N 2.
The above embodiments are preferred embodiments of the present disclosure, but the embodiments of the present disclosure are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present disclosure should be regarded as equivalent replacements within the scope of the present disclosure.
Claims (4)
1. The anti-influenza virus pharmaceutical composition is characterized by being prepared from the following raw materials in parts by weight:
45-55 parts of sargentgloryvine root, 25-35 parts of inula flower and 15-25 parts of fructus kochiae;
the overground part and seeds of the sarcandra bungeana, the flowers of the inula flower and the fruits of the broom cypress fruit are adopted as the medicine.
2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is prepared from the following raw materials in parts by weight:
50 parts of sargentgloryvine root, 30 parts of inula flower and 20 parts of broom cypress fruit.
3. A process for the preparation of a pharmaceutical composition according to claim 1 or 2, characterized in that it comprises the following steps:
mixing the raw materials in a certain weight proportion, and extracting with ethanol solution or water for several times to obtain extractive solution, i.e. the pharmaceutical composition.
4. A pharmaceutical preparation against influenza virus, characterized by being prepared from the pharmaceutical composition of claim 1 or 2 and pharmaceutically acceptable excipients.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005067948A2 (en) * | 2004-01-13 | 2005-07-28 | Jinhua Wang | Biological medicine for treating various diseases caused by various virus, bacterial infection |
| CN108030796A (en) * | 2017-12-18 | 2018-05-15 | 王秀兰 | A kind of anaesthetic of resisiting influenza virus and preparation method thereof |
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| JP2002241310A (en) * | 2001-02-21 | 2002-08-28 | Taisho Pharmaceut Co Ltd | Locally applying composition |
| JP2013112644A (en) * | 2011-11-29 | 2013-06-10 | Obihiro Univ Of Agriculture & Veterinary Medicine | Anti-influenza virus agent and production method of the same |
| CN103599148B (en) * | 2013-11-15 | 2016-06-15 | 沈阳药科大学 | Husky fluffy extractive of general flavone and its preparation method and application |
| KR102067328B1 (en) * | 2018-02-09 | 2020-01-16 | 전남대학교산학협력단 | Compositions for Anti-Influenza Virus comprising Lichen Extract |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005067948A2 (en) * | 2004-01-13 | 2005-07-28 | Jinhua Wang | Biological medicine for treating various diseases caused by various virus, bacterial infection |
| CN108030796A (en) * | 2017-12-18 | 2018-05-15 | 王秀兰 | A kind of anaesthetic of resisiting influenza virus and preparation method thereof |
Non-Patent Citations (2)
| Title |
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| 地肤子皂苷抗变态反应作用及其量效关系的研究;刘建萍等;《江苏农业科学》;20071231(第5期);第177页左栏倒数第1段和右栏第2段，第178页右栏倒数第1段 * |
| 肥大细胞在抗感染免疫作用中的研究进展;王晓曼等;《医学研究与教育》;20140430;第31卷(第2期);第89页第1和3段 * |
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