CN114306405A - New use of herba Blumeae Balsamiferae extract in preparing medicine for resisting influenza virus - Google Patents

New use of herba Blumeae Balsamiferae extract in preparing medicine for resisting influenza virus Download PDF

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CN114306405A
CN114306405A CN202110082036.9A CN202110082036A CN114306405A CN 114306405 A CN114306405 A CN 114306405A CN 202110082036 A CN202110082036 A CN 202110082036A CN 114306405 A CN114306405 A CN 114306405A
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virus
extract
blumea balsamifera
influenza
cells
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CN114306405B (en
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柴立
陈真
俞飞
李霞
贾金艳
温荣城
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Guizhou Holy Pharmaceutical Co ltd
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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
    • 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
    • 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
    • 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
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a new application of blumea balsamifera extract in preparation of anti-influenza virus medicines. The invention relates to a new application of blumea balsamifera extract in preparation of anti-influenza virus drugs, wherein the extract is an ethanol extract of blumea balsamifera. The blumea balsamifera ethanol extract has the effect of resisting various influenza viruses. Experiments show that the blumea balsamifera ethanol extract has good inhibition effect on influenza A virus and influenza B virus, and the antiviral activity and the dosage are in positive correlation within a certain dosage.

Description

New use of herba Blumeae Balsamiferae extract in preparing medicine for resisting influenza virus
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a new application of blumea balsamifera extract in preparation of anti-influenza virus medicines.
Background
Blumea balsamifera is leaves and twigs of blumea plants of Compositae, has the effects of dispelling wind, removing dampness and the like, has a long medication history in minority regions of Li nationality, Miao nationality, Zhuang nationality and the like, and is an important folk medicine. Meanwhile, blumea balsamifera is also one of the important sources for obtaining blumea balsamifera tablets.
The blumea balsamifera is rich in volatile oil components, and the utilization of the blumea balsamifera in the prior art is mostly based on the volatile oil components. For example, patent No. 200810044799.9 entitled "oily composition containing 1, 8-cineole and blumea balsamifera extract and its use" discloses oily substances extracted from Litsea cubeba and blumea balsamifera and its application in oral cavity injury, pharyngeal infection, swelling and pain, etc. Patent No. 98112092.X liquid spray for treating oral and throat diseases and its preparation method disclose liquid spray prepared from blumea balsamifera oil and rice paper oil, and its application in treating oral and throat diseases. Patent No. 200610200223.8 entitled "A bacteriostatic agent for oral cleaning" and its preparation method disclose a bacteriostatic agent for oral cleaning, which is prepared from blumea balsamifera oil and peppermint oil, and has dual functions of toothpaste and mouthwash.
Blumea balsamifera contains a large amount of other compounds besides volatile oil components. In the prior art, no research report is available on the effects of other components of blumea balsamifera except volatile oil. Therefore, the research on other components of blumea balsamifera except for volatile oil becomes a problem to be solved by those skilled in the art.
Disclosure of Invention
One of the purposes of the invention is to provide a new application of blumea balsamifera extract in preparation of anti-influenza virus drugs, wherein the blumea balsamifera extract is a blumea balsamifera ethanol extract.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention relates to a new application of blumea balsamifera extract in preparing anti-influenza virus drugs, wherein the extract is an ethanol extract of blumea balsamifera.
In some embodiments of the present invention, the content of total flavonoids in the ethanol extract is greater than or equal to 18.56% and less than or equal to 38.48% by mass; preferably 24-29%.
In some embodiments of the present invention, the total flavonoids of blumea balsamifera include blumea balsamifera, chrysoeriol, and pademamectin.
In some embodiments of the invention, the influenza virus is an influenza a virus, or/and an influenza B virus.
In some embodiments of the invention, the influenza a virus comprises H1N1 virus, or/and H3N2 virus.
In some embodiments of the invention, the influenza B virus comprises Yamagata, or/and Victoria virus.
Preferably, the Yamagata virus comprises B/Darwin/58/2019(Yamagata) plants, B/Phuket/3073/2013(B/Yamagata linkage) plants;
preferably, the Victoria virus comprises B/Sichuan homonov/531/2018 (Victoria) plants, B/Brisbane/60/2008/NYMC BX-35(B/Victoria linkage) plants.
In some embodiments of the present invention, the ethanol extract of blumea balsamifera is prepared by using blumea balsamifera as raw material, 50-95% ethanol as solvent, and supercritical CO2Extracting by extraction, cold soaking, ultrasonic extraction, cold soaking and ultrasonic extraction, percolation or reflux.
In some embodiments of the present invention, the ethanol extract of blumea balsamifera is obtained by extracting blumea balsamifera as a raw material with 50-95% ethanol as a solvent.
In some embodiments of the invention, the extraction process is supercritical CO2In the extraction method, the entrainer is ethanol which is 3-10 times of the raw material and accounts for 60-95%; the extraction temperature is 30-65 ℃, and the extraction pressure is 20.0-38.0 MPa; the temperature of the first separation kettle is 35-65 ℃, and the pressure of the first separation kettle is 5-10 MPa; the temperature of the second separation kettle is 35-65 ℃, and the pressure of the second separation kettle is 3-8 MPa; the extraction time is 30-240 min; the ethanol is volatilized from the obtained product to obtain the blumea balsamifera extract.
In some embodiments of the invention, the entrainer is 75-95% ethanol, preferably 95% ethanol; the dosage of the compound is 4 to 8 times of the raw material, preferably 5 to 6 times;
or/and the extraction temperature is 35-50 ℃, preferably 45 ℃; the extraction pressure is 22.0-26.0 MPa;
or/and the temperature of the first separation kettle is 40-55 ℃, and the pressure is 5-8 MPa; preferably, the temperature of the first separation kettle is 45 ℃ and the pressure is 6 MPa;
or/and the temperature of the second separation kettle is 40-55 ℃, and the pressure is 4-6 MPa; preferably, the temperature of the second separation kettle is 45 ℃ and the pressure is 5 Mpa;
or/and the extraction time is 60-180min, preferably 120 min;
preferably, the pressure of the second separation kettle is lower than that of the first separation kettle.
In some embodiments of the present invention, when the extraction process is a cold leaching process, the method comprises the steps of: taking a blumea balsamifera raw material, and mixing the raw material with the liquid material ratio of 1: adding 60-95% ethanol 5-40, cold soaking for 2-72 hr, filtering, collecting filtrate, and volatilizing ethanol to obtain herba Blumeae Balsamiferae extract.
In some embodiments of the present invention, the feed-to-liquid ratio is 1: 10-30, preferably 1: 20;
or/and the cold soaking time is 8-60 hours, preferably 48 hours;
or/and the concentration of ethanol is 75-95%, preferably 95%.
In some embodiments of the present invention, when the extraction method is an ultrasonic extraction method, the method comprises the following steps: adding appropriate amount of herba Blumeae Balsamiferae raw material into 60-95% ethanol, ultrasonic treating, collecting filtrate, and volatilizing ethanol to obtain herba Blumeae Balsamiferae extract;
preferably, 75-95% ethanol is added for ultrasonic treatment, more preferably, 95% ethanol is added for ultrasonic treatment;
preferably, the feed-liquid ratio of the blumea balsamifera raw material to the ethanol is 1: 5-40, more preferably 1: 10-30, more preferably 1: 20;
preferably, the number of extractions is 1-3, more preferably 2;
preferably, the ultrasonic temperature is preferably normal temperature;
preferably, the ultrasound time for each time is 10-120min, more preferably 60 min.
In some embodiments of the invention, where the method is a cold soaking and ultrasonic extraction method, the method comprises the steps of: taking blumea balsamifera raw material, adding 60-95% ethanol for cold soaking, performing ultrasonic treatment, filtering, collecting filtrate, and volatilizing ethanol to obtain blumea balsamifera extract;
preferably, adding 75-95% ethanol for cold soaking, more preferably, adding 95% ethanol for cold soaking;
preferably, the feed-liquid ratio of the blumea balsamifera raw material to the ethanol is 1: 5-40, more preferably 1: 10-30, more preferably 1: 20;
preferably, the cold soaking time is 2 to 72 hours, more preferably 8 to 60 hours, and further preferably 48 hours;
preferably, the sonication time is 0.5 to 4 hours, preferably 2 hours;
preferably, the ultrasonic temperature is preferably normal temperature.
In some embodiments of the invention, where the process is a reflux process, it comprises the steps of: taking blumea balsamifera raw material, adding 50-70% ethanol, heating and refluxing, keeping slightly boiling, extracting, filtering, collecting filtrate, volatilizing ethanol to obtain blumea balsamifera extract;
preferably, 60% ethanol is added and heated under reflux;
preferably, the number of extractions is 1-3, more preferably 1 or 2;
preferably, each extraction time is 0.5-4 hours, more preferably 2 hours;
preferably, the feed-liquid ratio of the blumea balsamifera raw material to the ethanol is 1: 5-40, more preferably 1: 10-30, more preferably 1: 20.
in some embodiments of the invention, where the method is a percolation method, the method comprises the steps of: taking blumea balsamifera raw material, adding 60-95% ethanol of 1-3 times of the volume of the raw material, stirring uniformly, sealing and standing for 1-5 hours to moisten and expand the blumea balsamifera raw material, filling into a cylinder, adding 60-95% ethanol of 10-15 times of the volume of the raw material, soaking for 24-48 hours, percolating at a percolate flow rate of 1-5ml/min, collecting percolate, and volatilizing ethanol to obtain blumea balsamifera extract;
preferably, 95 percent of ethanol with the volume 1 time that of the raw materials is added and evenly stirred, and the mixture is placed for 2 hours in a closed manner;
preferably, ethanol with the volume of 15 times that of the raw materials and 95 percent of the raw materials is added for soaking for 24 hours after the raw materials are loaded into the cylinder;
preferably, the percolate flow rate is 2-3 ml/min.
In some embodiments of the present invention, the total flavonoids are determined by uv-vis spectrophotometry, specifically:
(1) selection of the wavelength. A proper amount of sample solution is taken, and after the sample solution is developed by aluminum nitrate under the alkaline condition that 0.3mL of 5% sodium nitrite solution exists, the absorbance of the complex is measured in the wavelength range of 420-700 nm by taking a reagent as a blank reference solution, and the complex has the maximum absorption at the wavelength of 504 nm.
(2) And (6) drawing a standard curve. Precisely sucking rutin control solutions (0.10mg/mL)0.00, 1.00, 2.00, 3.00, 4.00 and 5.00mL into 10.00mL volumetric flasks, respectively adding 5% sodium nitrite solution 0.30mL, shaking up, standing for 6min, adding 10% aluminum nitrate solution 0.3mL, shaking up, standing for 6min, adding 4% sodium hydroxide solution 4.00mL, diluting with water to scale, shaking up, standing for 12min, taking the reagent as blank reference solution, and measuring absorbance at 504 nm.
(3) Preparing a test solution. Weighing 0.5mL of the product, placing the product in a 10mL measuring flask, respectively adding 0.3mL of 5% sodium nitrite solution, shaking up, standing for 6min, adding 0.3mL of 10% aluminum nitrate solution, shaking up, standing for 6min, adding 4mL of 4% sodium hydroxide solution, diluting with water to scale, shaking up, and standing for 12min to obtain the product.
(4) Measuring absorbance at 504nm, and calculating total flavone content.
In the invention, the Blumea balsamifera is leaves and twigs of Blumea DC.
In some embodiments of the present invention, Blumea balsamifera is leaf and twig of Blumea balsamifera dc, b.balsamifera dc, b.oblongifolia Kitam, or b.megacephala cc.
Compared with the prior art, the invention has the following beneficial effects:
the invention unexpectedly discovers that the blumea balsamifera ethanol extract, namely the naxaea balsamifera ethanol extract, has the effect of resisting various influenza viruses. Experiments show that the blumea balsamifera ethanol extract has good inhibition effect on influenza A virus and influenza B virus.
Drawings
FIG. 1 is a design drawing of a dosing treatment experiment.
FIG. 2 is a graph showing the results of the anti-influenza virus activity test of the blumea balsamifera extract of example 6, wherein blumea balsamifera refers to the blumea balsamifera extract, OSV is oseltamivir, which is a positive drug, and DMSO is a blank sample.
FIG. 3 is a graph showing the inhibition rate of H1N1 virus by various concentrations of the blumea balsamifera extract in example 7.
FIG. 4 is a graph showing the results of the activity test of the extract of blumea balsamifera in example 8 against Yamagata-based type B viruses.
FIG. 5 is a graph showing the results of the Yamagata-based activity assay for the anti-influenza B virus of the extract of blumea balsamifera obtained in example 9.
FIG. 6 is a graph showing the results of the inhibitory concentration of blumea balsamifera extract against influenza B virus Yamagata system at half maximal concentration in example 9.
FIG. 7 is a graph showing the results of the activity test of the blumea balsamifera extract against Victoria-based type B viruses in example 10.
FIG. 8 is a graph showing the results of the activity test of the blumea balsamifera extract against influenza B virus Victoria line in example 11.
FIG. 9 is a graph showing the results of the concentration of blumea balsamifera extract half-inhibitory against influenza B virus Victoria lines in example 11.
FIG. 10 is a graph showing the results of the activity test of the blumea balsamifera extract against influenza A virus H3N2 in example 12.
FIG. 11 is a graph showing the results of the half inhibitory concentration against influenza A virus H3N2 of the blumea balsamifera extract in example 12.
FIG. 12 is a graph showing the results of Yamagata series activity tests on the anti-oseltamivir-resistant influenza B viruses of the blumea balsamifera extract in example 13.
FIG. 13 is a graph showing the results of the Yamagata-based half maximal inhibitory concentration of influenza B viruses against oseltamivir resistance obtained in example 13.
FIG. 14 is a graph showing the results of the activity test of the I.blumea extract against oseltamivir-resistant influenza B virus Victoria series in example 14.
FIG. 15 is a graph showing the results of inhibiting concentration at half maximal of the anti-oseltamivir resistant influenza B virus Victoria series of the blumea balsamifera extract of example 14.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The ultrasonic power during ultrasonic extraction in the embodiment of the invention is 100W, and the frequency is 40 kHz.
Example 1
This example discloses the use of CO2The supercritical extraction method for preparing the blumea balsamifera extract comprises the following steps:
taking appropriate amount of Blumea balsamifera (L.) DC coarse powder, and placing in supercritical CO2In the extraction apparatus, 95% ethanol was used as an entrainer in an amount 6 times (volume/mass, g/mass, mL/volume) the amount of the raw material.
The extraction temperature is 45.0 ℃, and the extraction pressure is 22.0 MPa; the first separation kettle has the temperature of 50.0 ℃ and the pressure of 6 MPa; the temperature of the second separation kettle is 50.0 ℃, and the pressure is 5 MPa; the extraction time was 120 min. The ethanol is volatilized from the obtained product to obtain the blumea balsamifera extract, and the content of the total flavone is 28 wt%.
Example 2
The embodiment discloses a method for preparing blumea balsamifera extract by adopting a cold soaking method, which specifically comprises the following steps: taking blumea balsamifera (B.balsamifera (L.) DC.) raw material coarse powder, and mixing the coarse powder according to a material-liquid ratio of 1: adding 95% ethanol into 20, cold soaking for 48 hr, filtering, collecting filtrate, and volatilizing ethanol to obtain herba Blumeae Balsamiferae extract with total flavone content of 24 wt%.
Example 3
The embodiment discloses a blumea balsamifera extract prepared by an ultrasonic extraction method, which specifically comprises the following steps: taking appropriate amount of Blumea balsamifera (L.) DC) raw material, extracting for 2 times, adding 20 times of 95% ethanol each time, performing ultrasonic treatment at room temperature for 60min, filtering, collecting filtrate, and volatilizing ethanol to obtain Blumea balsamifera extract with total flavone content of 19 wt%.
Example 4
The embodiment discloses a method for preparing a blumea balsamifera extract by adopting a cold soaking and ultrasonic extraction method, which specifically comprises the following steps: taking a proper amount of blumea balsamifera (B.balsamifera (L.) DC.) raw material, and mixing the raw material according to a material-liquid ratio of 1: adding 95% ethanol into 20, cold soaking for 48 hr, ultrasonic treating for 2 hr, filtering, collecting filtrate, and volatilizing ethanol to obtain herba Blumeae Balsamiferae extract with total flavone content of 29 wt%.
Example 5
The embodiment discloses a method for preparing a blumea balsamifera extract by a reflux method, which specifically comprises the following steps: taking a proper amount of blumea balsamifera (B.balsamifera (L.) DC.) raw material, and mixing the raw material according to a material-liquid ratio of 1: adding 60% ethanol into 10, heating and reflux extracting, maintaining slight boiling, extracting for 2 hr, filtering, collecting filtrate, and volatilizing ethanol to obtain herba Blumeae Balsamiferae extract with total flavone content of 30 wt%.
Examples 6 to 14
Examples 6-14 are antiviral study tests performed on the blumea balsamifera extract of the present invention.
Example 6
This example discloses the evaluation of anti-influenza virus activity on a sample of blumea balsamifera extract of the present invention. The blumea balsamifera extract in this example was prepared according to the method of example 1.
The research and test method specifically comprises the following steps:
1. virus strain: attenuated live influenza virus strain A/WSN/33(H1N1)
2. Cell: MDCK cells (Madin-Daby Canine Kidney cells) were isolated and cultured in 9 months of 1958, and derived from the kidney of Cockstringy dogs. Currently, MDCK cell amplified virus has the characteristics of high infection rate, rapid proliferation, high stability and the like, so the cell is considered to be one of 3 cell lines which are most suitable for producing influenza A and B virus vaccines.
3. Adding chemicals for treatment
The administration mode is shown in the attached figure 1: adding different samples to be tested in each vertical row of a B2-G9 area, adding positive control oseltamivir in a B10-G10 area, and adding negative control DMSO in a B11-G11 area; virus was added in the three horizontal rows below the black line. Such administration allows for the detection of both the antiviral activity and the cytotoxicity of the sample.
4. Grouping and dose design
(1) Dissolving herba Blumeae Balsamiferae extract in DMSO, and adding 50 μ g/mL
(2) Positive drug control: oseltamivir (OSV), the dosage is designed to be 100 mu M/mL;
(3) negative control: blank DMSO.
5. Cytopathic effect
Influenza viruses can cause MDCK cells to develop lesions, known as the CPE effect, with a significant reduction in cell viability. The negative control DMSO group showed significant CPE effect after virus addition, while the samples were still good after the same amount of virus was added if they were effective in inhibiting influenza infection.
6. And (3) cell viability detection:
experiment adopts
Figure BDA0002909425160000071
The kit detects the cell activity. The CellTiter-Glo method is a homogeneous assay for detecting cell viability in culture by quantitative determination of ATP. ATP in the living cells directly participates in the reaction, the light generated by the reaction is in direct proportion to the number of the living cells, and the cell activity in the hole to be detected can be obtained by detecting the chemiluminescence value, so that whether the sample to be detected has the activity of inhibiting the influenza virus infected cells or not is reflected.
7. Data processing:
the cell viability corresponding to the chemiluminescence value of the blank (DMSO) group was defined as 100%,
relative cell viability ═ 100% of the values measured for each group of cells/the values measured for the cells of the blank control group
The inhibition rate is (1- (detection of cell action of extract-detection of virus action of extract)/(detection of blank group-detection of virus control) × 100%
The inhibition rate is the rate at which the compound acts to inhibit viral growth. Reflecting the antiviral activity of the compound.
The research and test results are as follows:
as shown in fig. 2, under the present experimental conditions, 50 μ g/mL blumea balsamifera extract exhibited significant anti-influenza virus activity, while also exhibiting slight cytotoxicity.
Example 7
This example discloses the evaluation of anti-influenza virus activity of various doses of blumea balsamifera extract prepared according to the method of example 1. The specific research method is as follows:
1 Material
1.1 reagents
DMEM medium (Gibco); DMSO (tianjingdong fine chemical reagent factory); oseltamivir (OSV, annagi corporation); TPCK-trypsin (Shanghai national Biotechnology Co., Ltd.); CellTiter-Glo kit ((Promega Corp., Madison, Wis., USA)).
1.2 instruments
QP-160 type CO2A constant temperature incubator (BIOBASE Co.); XDS-1 inverted phase contrast microscope (Shenzhen Star optical Instrument, Inc.); an L500 low speed centrifuge (hunan instrument laboratory development ltd); 1510 full-wavelength microplate reader (Thermofeisher Co.).
1.3 viruses and cells
An attenuated strain of influenza A virus A/WSN/33(H1N1) provided by institute of medical and biological research of Chinese academy of medical sciences; canine kidney cells (MDCK), provided by the institute of medical and biological research, academy of Chinese medical sciences.
2 method
2.1 preparation of test articles
The samples used in this example were the extracts of blumea balsamifera prepared by the method of example 1, the extracts were dissolved in DMSO to prepare stock solutions having concentrations of 10mg/mL, 5mg/mL, 2.5mg/mL, 1.25mg/mL, 0.63mg/mL, 0.32mg/mL, 0.16mg/mL, and 0.08mg/mL, and the stock solutions were diluted with media to prepare eight concentrations of 100. mu.g/mL, 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.13. mu.g/mL, 1.56. mu.g/mL, and 0.78. mu.g/mL, respectively, as the solutions to be tested for the extract group.
Dissolving OSV by DMSO, and diluting to 100 μ M/mL concentration by culture medium to obtain positive control group solution to be tested; and preparing a culture medium containing 1% DMSO, and using the culture medium as a blank control group test solution.
2.2 Virus culture and virulence determination
Inoculating the virus into allantoic cavities of 9-11 days old chick embryos, culturing for 72h at 35 ℃, and performing aseptic operation to obtain allantoic fluid. The precipitate was removed by centrifugation at 3000rpm for 15min and the supernatant was collected. Diluting the collected virus liquid to 10 times in series-1To 10-8A total of 8 dilutions were added to adherent 96-well MDCK cells in 8 wells per concentration, approximately 3 × 10 cells per well4Blank control group was added. MDCK cells in each well adsorb virus at 35 deg.C for 2 hr, replace with culture solution, and culture in incubator (37 deg.C, 5% CO)2) After 48h, the cytopathic effect was observed under a microscope, and the infection amount TCID of half of the cell cultures was calculated according to the Reed-Muench method50/mL＝10-6.25。
2.3 determination of the toxic Effect of the extracts on cells
MDCK cells were plated at 3X 10 per well4The individual cells were inoculated in a 96-well plate and placed in an incubator (37 ℃ C., 5% CO)2) In (1), after the cell monolayer was adsorbed to a 96-well plate, the supernatant was aspirated off. Adding 100 μ L of different concentrations of extract solution as extract groups, repeating the extraction with 3 wells for each concentration, setting positive control group (OSV, 100 μ M/mL) and blank control group (DMSO, 1%), placing in incubator (37 deg.C, 5% CO)2) After culturing for 36-40 h, detecting the cell activity by adopting a CellTiter-Glo kit.
Cell viability ═ 100% of assay values for each group of cells/assay value for the blank control group of cells
2.4 Effect of extracts on Activity of influenza Virus infected cells
Taking a 96-well plate with cells adsorbed in a single layer, and inoculating 100TCID50Virus solution, 100. mu.L per well, in an incubator (37 ℃, 5% CO)2) After adsorbing for 2h, the virus liquid is discarded. Adding extract solutions with different concentrations into each well as repeating 3 wells for each concentration, and setting positive drug group, virus control group and blank control group. Placing in an incubator (37 ℃, 5% CO)2) After culturing for 36-40 h, the cell viability of each group in the virus-containing state is measured. Calculating the inhibition rate of the extract on the virus, and adopting Probit regression analysis[14]The half inhibitory Concentration (50% Inhibiting Concentration, IC) was obtained50) And the Selection Index (SI) is calculated.
The rate of inhibition of the extract on the virus is 1- (detection of the effect of the extract on cells-detection of the effect of the extract on the virus)/(detection of blank group-detection of virus control)
SI＝TC50/IC50
2.5 statistical methods
The experimental data were statistically analyzed using SPSS 21.0 software, the quantitative data were expressed as mean + -standard deviation (x + -S), and the data between groups were compared using one-way analysis of variance. The inspection level α was set to 0.05 unless otherwise specified.
3 results
3.1 determination of the cytotoxic Effect of the extract
As shown by the data in Table 1, cell viability was not statistically significant (P) from the blank group when the extract concentrations were 0.78. mu.g/mL, 1.56. mu.g/mL, 3.13. mu.g/mL, 6.25. mu.g/mL, 12.5. mu.g/mL, 25. mu.g/mL, and 50. mu.g/mL>0.05); when the extract concentration was increased to 100. mu.g/mL, the cell viability was reduced to 87.42%, lower than that of the blank control group (P)<0.05) at which the extract shows slight cytotoxicity, but the median Toxic Concentration (TC) of the extract to the cells can also be inferred50) Should be greater than 100. mu.g/mL.
TABLE 1 Effect of different concentrations of extracts on MDCK cell viability
Figure BDA0002909425160000091
Note: p <0.05, P <0.01 compared to placebo;
3.2 in vitro antiviral Effect assay results of extracts
As shown in the data in Table 2, when the concentration of the extract is 0.78. mu.g/mL, 1.56. mu.g/mL, 3.13. mu.g/mL, the cell viability of the extract group is not statistically significant (P >0.05) compared with the virus control group, indicating that the extract has no inhibitory effect on influenza virus at these concentrations; when the concentration of the extract is 6.25 mu g/mL, 12.5 mu g/mL, 25 mu g/mL, 50 mu g/mL and 100 mu g/mL, the cell activity of the extract group is higher than that of a virus control group, and the difference has statistical significance (P <0.05), which indicates that the extract has an inhibiting effect on influenza virus at the concentrations.
TABLE 2 Effect of different concentrations of extracts on Virus inhibition
Figure BDA0002909425160000101
Note: 1) Δ P <0.05, Δ Δ P <0.01 compared to the virus control group; compared with the positive control group, # P <0.05, # P < 0.01. 2) The inhibition ratios of more than 100% are all expressed as 100%.
3.3 half inhibitory concentration of extract on Virus
Half maximal Inhibitory Concentration (IC) of the extract on the virus was calculated by Probit regression analysis50) It was 8.71. mu.g/mL, as shown in FIG. 3. SI ═ TC50/IC50Due to TC50Should be greater than 100. mu.g/mL, so the extract selection index SI should be greater than 11.48. (IC of OSV under this screening System50Is 7.12. mu.g/mL)
Example 8
This example discloses the preliminary evaluation of antiviral activity of the blumea balsamifera extract of the present invention against Yamagata-based influenza B viruses. The blumea balsamifera extract in this example was prepared according to the method of example 2. The research and test method specifically comprises the following steps:
1. virus strain: live attenuated influenza strain B/Darwin/58/2019(Yamagata)
Influenza B viruses are classified into two major lineages, the Yamagata line and the Victoria line, according to their antigenic properties and the nucleotide sequence of HA1 region. The B/Darwin/58/2019(Yamagata) virus strain used in this experiment was a strain isolated from/obtained by the influenza B/Darwin laboratory under the No. 58/2019 (a Yamagata strain belonging to influenza B).
2. Cell: MDCK cells (Madin-Daby cancer cells)
3. Virus culture and virulence determination
Inoculating the virus into allantoic cavities of 9-11 days old chick embryos, culturing for 72h at 35 ℃, and performing aseptic operation to obtain allantoic fluid. The precipitate was removed by centrifugation at 3000rpm for 15min and the supernatant was collected. Diluting the collected virus liquid to 10 times in series-1To 10-8A total of 8 dilutions were added to adherent 96-well MDCK cells in 8 wells per concentration, approximately 3 × 10 cells per well4Blank control group was added. MDCK cells in each well adsorb virus at 35 deg.C for 2 hr, replace with culture solution, and culture in incubator (37 deg.C, 5% CO)2) Cytopathic effects were observed under a microscope after 48 h.
4. Adding chemicals for treatment
The administration mode is shown in the attached figure 1: adding different samples to be tested in each vertical row of a B2-G9 area, adding positive control oseltamivir in a B10-G10 area, and adding negative control DMSO in a B11-G11 area; virus was added in the three horizontal rows below the black line. Such administration allows for the detection of both the antiviral activity and the cytotoxicity of the sample.
5. Grouping and dose design
(1) Dissolving herba Blumeae Balsamiferae extract with DMSO, and diluting with culture medium to 50 μ g/mL;
(2) positive drug control: oseltamivir (OSV), the dosage is designed to be 100 mu M/mL;
(3) negative control: blank DMSO.
6. Cytopathy and cell viability examination
Influenza viruses can cause MDCK cells to develop lesions, known as the CPE effect, with a significant reduction in cell viability. The negative control DMSO group showed significant CPE effect when added with virus, while the compound was effective in inhibiting infection by influenza virus, so that the cell status remained good when the same amount of virus was added.
Experiment adopts
Figure BDA0002909425160000111
The kit detects the cell activity. The cell viability corresponding to the chemiluminescence value of the blank (DMSO) group was defined as 100%,
relative cell viability ═ 100% of the values measured for each group of cells/the values measured for the cells of the blank control group
7. Data processing
The inhibition rate is the rate at which the compound acts to inhibit viral growth. Reflecting the antiviral activity of the compound.
The inhibitory rate of the extract on virus is 1- (detection value of the effect of the extract on cells-detection value of the effect of the extract on virus)/(detection value of blank group-detection value of virus control) × 100%
The research and test results are as follows:
the effect of the blumea balsamifera extract on MDCK cell activity and the results of activity test against Yamagata-line influenza B virus are shown in table 3 and fig. 4. Under the experimental condition, the blumea balsamifera extract does not affect the activity of MDCK cells at the dose of 50 mu g/mL, and simultaneously shows an inhibition effect on Yamagata-series B-type influenza viruses, and the inhibition rate is 91.84%. The Yamagata influenza B virus adopted in the experiment has certain drug resistance to Oseltamivir (OSV), and the OSV inhibition rate under the same experiment condition is only 40.61%.
TABLE 3 comparison of the Blumeae Balsamiferae extract to Yamagata-series B-type influenza virus test
Figure BDA0002909425160000121
And (3) knotting:
under the experimental conditions, the blumea balsamifera extract has antiviral activity to Oseltamivir resistant Yamagata series B influenza virus, and the inhibition rate of 50 mu g/mL blumea balsamifera extract to the virus is 91.84%.
Example 9
This example discloses the evaluation of antiviral activity of the extracts of blumea balsamifera of the present invention against the Yamagata line of influenza B virus at different doses. The blumea balsamifera extract in this example was prepared according to the method of example 1. The research and test method specifically comprises the following steps:
1. virus strain:
influenza vaccine strain B/Darwin/58/2019(Yamagata)
Note: influenza B viruses are classified into two major lineages, the Yamagata line and the Victoria line, according to their antigenic properties and the nucleotide sequence of HA1 region. The B/Darwin/58/2019(Yamagata) strain was used in this experiment.
2. Cell: MDCK cells (Madin-Daby cancer cells)
3. Virus culture and virulence determination
Inoculating the virus into allantoic cavities of 9-11 days old chick embryos, culturing for 72h at 35 ℃, and performing aseptic operation to obtain allantoic fluid. The precipitate was removed by centrifugation at 3000rpm for 15min and the supernatant was collected. Diluting the collected virus liquid to 10 times in series-1To 10-8A total of 8 dilutions were added to adherent 96-well MDCK cells in 8 wells per concentration, approximately 3 × 10 cells per well4Blank control group was added. MDCK cells in each well adsorb virus at 35 deg.C for 2 hr, replace with culture solution, and culture in incubator (37 deg.C, 5% CO)2) Cytopathic effects were observed under a microscope after 48 h. According to the calculation of the amount of infection TCID of half of the cell cultures by the Reed-Muench method50/mL＝10-5.73. The MOI of this experiment is 0.001.
4. Adding chemicals for treatment
The administration mode is shown in the attached figure 1: adding different samples to be tested in each vertical row of a B2-G9 area, adding positive control oseltamivir in a B10-G10 area, and adding negative control DMSO in a B11-G11 area; virus was added in the three horizontal rows below the black line. Such administration allows for the detection of both the antiviral activity and the cytotoxicity of the sample.
5. Grouping and dose design
(1) Dissolving herba Blumeae Balsamiferae extract with DMSO, and diluting with culture medium at gradient of 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 μ g/mL for 8 concentrations;
(2) positive control: oseltamivir (OSV), the dosage is designed to be 100 mu M/L;
(3) negative control: 1% DMSO.
6. Cytopathy and cell viability examination
Influenza viruses can cause MDCK cells to develop lesions, known as the CPE effect, with a significant reduction in cell viability. The negative control DMSO group can see obvious CPE effect after adding virus, and if the compound can effectively inhibit infection of influenza virus, the cell state is still good after adding the same amount of virus.
Experiment adopts
Figure BDA0002909425160000131
The kit detects the cell activity. The cell viability corresponding to the chemiluminescence value of the blank (DMSO) group was defined as 100%,
relative cell viability ═ 100% of the values measured for each group of cells/the values measured for the cells of the blank control group
7. Data processing
The inhibition rate is the rate at which the compound acts to inhibit viral growth. Reflecting the antiviral activity of the compound.
The inhibitory rate of the extract on virus is 1- (detection value of the effect of the extract on cells-detection value of the effect of the extract on virus)/(detection value of blank group-detection value of virus control) × 100%
The rate of inhibition of the extract on the virus was calculated and the half Inhibitory Concentration (IC) was determined by analysis using GraphPad Prism software50)。
The research and test results are as follows:
the results of the Yamagata-series activity test of blumea balsamifera extract against influenza B virus are shown in FIG. 5, with oseltamivir (OSV, 100. mu.M) as a positive control and DMSO as a negative control. When the concentration of the blumea balsamifera extract is more than 25 mu g/mL, the blumea balsamifera extract has the effect of remarkably inhibiting virus from infecting host cells.
By dilution at a multiple ratio, antiviral half maximal Inhibitory Concentration (IC) of herba Blumeae Balsamiferae extract under the condition is obtained50) It was 10.14. mu.g/mL (as shown in FIG. 6).
And (3) knotting:
under the experimental condition, the activity of the blumea balsamifera extract resisting the B-type influenza virus Yamagata series is positively correlated with the dosage, and when the dosage concentration is more than 25 mu g/mL, the blumea balsamifera extract has the activity of obviously inhibiting virus infection host cells and half Inhibition Concentration (IC)50) 10.14. mu.g/mL.
Example 10
This example discloses the evaluation of the antiviral activity of the blumea balsamifera extract of the present invention against influenza B virus Victoria line. The blumea balsamifera extract in this example was prepared according to the method of example 3. The research and test method specifically comprises the following steps:
the research and test method comprises the following steps:
1. virus strain: live attenuated influenza B/Sichuan high novel strain 531/2018(Victoria)
Influenza B viruses are classified into two major lineages, the Yamagata line and the Victoria line, according to their antigenic properties and the nucleotide sequence of HA1 region. The B/Sichuan high and new/531/2018 (Victoria) virus strain used in the experiment is a 531/2018 year-numbered strain (Victoria strain belonging to influenza B) isolated from/in the influenza B/Sichuan high and new laboratory.
2. Cell: MDCK cells (Madin-Daby cancer cells)
3. Virus culture and virulence determination
Inoculating the virus into allantoic cavities of 9-11 days old chick embryos, culturing for 72h at 35 ℃, and performing aseptic operation to obtain allantoic fluid. The precipitate was removed by centrifugation at 3000rpm for 15min and the supernatant was collected. Diluting the collected virus liquid to 10 times in series-1To 10-8A total of 8 dilutions were added to adherent 96-well MDCK cells in 8 wells per concentration, approximately 3 × 10 cells per well4Blank control group was added. MDCK cells in each well adsorb viruses for 2h at 35 ℃, then the culture solution is used for replacement,placing in an incubator for continuous culture (37 ℃, 5% CO)2) Cytopathic effects were observed under a microscope after 48 h.
4. Adding chemicals for treatment
The administration mode is shown in the attached figure 1: adding different samples to be tested in each vertical row of a B2-G9 area, adding positive control oseltamivir in a B10-G10 area, and adding negative control DMSO in a B11-G11 area; virus was added in the three horizontal rows below the black line. Such administration allows for the detection of both the antiviral activity and the cytotoxicity of the sample.
5. Grouping and dose design
(1) Dissolving herba Blumeae Balsamiferae extract with DMSO, and diluting with culture medium to 50 μ g/mL;
(2) positive drug control: oseltamivir (OSV), the dosage is designed to be 100 mu M/mL;
(3) negative control: blank DMSO.
6. Cytopathy and cell viability examination
Influenza viruses can cause MDCK cells to develop lesions, known as the CPE effect, with a significant reduction in cell viability. The negative control DMSO group showed significant CPE effect when added with virus, while the compound was effective in inhibiting infection by influenza virus, so that the cell status remained good when the same amount of virus was added.
Experiment adopts
Figure BDA0002909425160000152
The kit detects the cell activity. The cell viability corresponding to the chemiluminescence value of the blank (DMSO) group was defined as 100%,
relative cell viability ═ 100% of the values measured for each group of cells/the values measured for the cells of the blank control group
7. Data processing
The inhibition rate is the rate at which the compound acts to inhibit viral growth. Reflecting the antiviral activity of the compound.
The inhibitory rate of the extract on virus is 1- (detection value of the effect of the extract on cells-detection value of the effect of the extract on virus)/(detection value of blank group-detection value of virus control) × 100%
The research and test results are as follows:
the effect of the blumea balsamifera extract on MDCK cell activity and the results of activity test against Victoria line type B influenza virus are shown in table 4 and fig. 7. Under the experimental condition, the blumea balsamifera extract does not affect the activity of MDCK cells at a dose of 50 mu g/mL, and simultaneously shows an inhibition effect on Victoria system B type influenza viruses, and the inhibition rate is 88.16%. The Victoria influenza B virus used in this experiment was resistant to Oseltamivir (OSV), and OSV did not exhibit antiviral effect under the same experimental conditions.
TABLE 4 Blumeae Balsamiferae extract tested against Victoria series B influenza virus
Figure BDA0002909425160000151
And (3) knotting:
under the experimental condition, the blumea balsamifera extract has antiviral activity to the Oseltamivir resistant Victoria type B influenza virus, and the inhibition rate of 50 mu g/mL blumea balsamifera extract to the virus is 88.16%.
Example 11
This example discloses the evaluation of antiviral activity of various doses of the blumea balsamifera extract of the present invention against influenza B virus Victoria line. The blumea balsamifera extract in this example was prepared by the method of example 1. The research and test method specifically comprises the following steps:
1. virus strain: influenza vaccine strain B/Sichuan high novel/531/2018 (Victoria)
Influenza B viruses are classified into two major lineages, the Yamagata line and the Victoria line, according to their antigenic properties and the nucleotide sequence of HA1 region. The B/Sichuan Homophilum/531/2018 (Victoria) strain was used in this experiment.
2. Cell: MDCK cells (Madin-Daby cancer cells)
3. Virus culture and virulence determination
Inoculating the virus into allantoic cavities of 9-11 days old chick embryos, culturing for 72h at 35 ℃, and performing aseptic operation to obtain allantoic fluid. Centrifuging at 3000rpm for 15min to remove precipitate, collectingAnd (5) clear liquid. Diluting the collected virus liquid to 10 times in series-1To 10-8A total of 8 dilutions were added to adherent 96-well MDCK cells in 8 wells per concentration, approximately 3 × 10 cells per well4Blank control group was added. MDCK cells in each well adsorb virus at 35 deg.C for 2 hr, replace with culture solution, and culture in incubator (37 deg.C, 5% CO)2) Cytopathic effects were observed under a microscope after 48 h. According to the calculation of the amount of infection TCID of half of the cell cultures by the Reed-Muench method50/mL＝10-4.37. The MOI of this experiment is 0.0005.
4. Adding chemicals for treatment
The administration mode is shown in the attached figure 1: adding different samples to be tested in each vertical row of a B2-G9 area, adding positive control oseltamivir in a B10-G10 area, and adding negative control DMSO in a B11-G11 area; virus was added in the three horizontal rows below the black line. Such administration allows for the detection of both the antiviral activity and the cytotoxicity of the sample.
5. Grouping and dose design
(1) Dissolving herba Blumeae Balsamiferae extract with DMSO, and diluting with culture medium at gradient of 8 concentrations such as 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 μ g/mL;
(2) positive control: oseltamivir (OSV), the dosage is designed to be 100 mu M/L;
(3) negative control: 1% DMSO.
6. Cytopathic effect and cell viability assay
Influenza viruses can cause MDCK cells to develop lesions, known as the CPE effect, with a significant reduction in cell viability. The negative control DMSO group can see obvious CPE effect after adding virus, and if the compound can effectively inhibit infection of influenza virus, the cell state is still good after adding the same amount of virus.
Experiment adopts
Figure BDA0002909425160000161
The kit detects the cell activity. The cell viability corresponding to the chemiluminescence value of the blank (DMSO) group was defined as 100%,
relative cell viability ═ 100% of the values measured for each group of cells/the values measured for the cells of the blank control group
7. Data processing
The inhibition rate is the rate at which the compound acts to inhibit viral growth. Reflecting the antiviral activity of the compound.
The inhibitory rate of the extract on virus is 1- (detection value of the effect of the extract on cells-detection value of the effect of the extract on virus)/(detection value of blank group-detection value of virus control) × 100%
The rate of inhibition of the extract on the virus was calculated and the half Inhibitory Concentration (IC) was determined by analysis using GraphPad Prism software50)。
The research and test results are as follows:
the results of the activity test of the blumea balsamifera extract against Victoria series B virus are shown in FIG. 8, with oseltamivir (OSV, 100. mu.M) as a positive control and DMSO as a negative control. When the concentration of the blumea balsamifera extract is more than 25 mu g/mL, the blumea balsamifera extract has the activity of obviously inhibiting the Victoria line B type virus from infecting host cells.
The half Inhibitory Concentration (IC) of blumea balsamifera extract against Victoria B-type virus under the condition is obtained by dilution in multiple proportion50) It was 3.17. mu.g/mL (as shown in FIG. 9).
And (3) knotting:
under the experimental condition, the anti-B influenza virus Victoria system activity of the blumea balsamifera extract is positively correlated with the dosage, and when the dosage concentration is more than 25 mu g/mL, the blumea balsamifera extract has the advantages of obviously inhibiting the activity of virus infected host cells and half Inhibiting Concentration (IC)50) It was 3.17. mu.g/mL.
Example 12
This example discloses the evaluation of the antiviral activity of various doses of blumea balsamifera extract samples of the present invention against influenza a virus H3N 2. The blumea balsamifera extract in this example was prepared according to the method of example 1. The research and test method specifically comprises the following steps:
1. virus strain:
influenza vaccine strain A/Hong Kong/4801/2014(H3N2)
2. Cell: MDCK cells (Madin-Daby cancer cells)
3. Virus culture and virulence determination
Inoculating the virus into allantoic cavities of 9-11 days old chick embryos, culturing for 72h at 35 ℃, and performing aseptic operation to obtain allantoic fluid. The precipitate was removed by centrifugation at 3000rpm for 15min and the supernatant was collected. Diluting the collected virus liquid to 10 times in series-1To 10-8A total of 8 dilutions were added to adherent 96-well MDCK cells in 8 wells per concentration, approximately 3 × 10 cells per well4Blank control group was added. MDCK cells in each well adsorb virus at 35 deg.C for 2 hr, replace with culture solution, and culture in incubator (37 deg.C, 5% CO)2) Cytopathic effects were observed under a microscope after 48 h.
4. Adding chemicals for treatment
The administration mode is shown in the attached figure 1: adding different samples to be tested in each vertical row of a B2-G9 area, adding positive control oseltamivir in a B10-G10 area, and adding negative control DMSO in a B11-G11 area; virus was added in the three horizontal rows below the black line. Such administration allows for the detection of both the antiviral activity and the cytotoxicity of the sample.
5. Grouping and dose design
(1) Dissolving herba Blumeae Balsamiferae extract with DMSO, and diluting with culture medium at gradient of 8 concentrations such as 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 μ g/mL;
(2) positive control: oseltamivir (OSV), the dosage is designed to be 100 mu M/L;
(3) negative control: 1% DMSO.
6. Cytopathic effect and cell viability assay
Influenza viruses can cause MDCK cells to develop lesions, known as the CPE effect, with a significant reduction in cell viability. The negative control DMSO group can see obvious CPE effect after adding virus, and if the compound can effectively inhibit infection of influenza virus, the cell state is still good after adding the same amount of virus.
Experiment adopts
Figure BDA0002909425160000181
The kit detects the cell activity. Blank control (DM)SO) set was defined as 100% of the cell viability corresponding to the chemiluminescence values,
relative cell viability ═ 100% of the values measured for each group of cells/the values measured for the cells of the blank control group
7. Data processing
The inhibition rate is the rate at which the compound acts to inhibit viral growth. Reflecting the antiviral activity of the compound.
The inhibitory rate of the extract on virus is 1- (detection value of the effect of the extract on cells-detection value of the effect of the extract on virus)/(detection value of blank group-detection value of virus control) × 100%
The rate of inhibition of the extract on the virus was calculated and the half Inhibitory Concentration (IC) was determined by analysis using GraphPad Prism software50)。
The research and test results are as follows:
the results of the activity test of blumea balsamifera extract against influenza a virus H3N2 are shown in fig. 10, with oseltamivir (OSV, 100 μ M) as a positive control and DMSO as a negative control. When the concentration of the blumea balsamifera extract is more than 6.25 mu g/mL, the blumea balsamifera extract has the activity of remarkably inhibiting the influenza A virus H3N2 from infecting host cells. When the dosage concentration of the blumea balsamifera extract is 6.25 mu g/mL, 12.5 mu g/mL, 25 mu g/mL and 50 mu g/mL, the inhibition rates of the blumea balsamifera extract on an influenza A virus A/Hong Kong/4801/2014(H3N2) strain are 60.48%, 29.36%, 106.28% and 113.03% respectively, the A/Hong Kong/4801/2014(H3N2) strain adopted in the experiment has certain drug resistance on Oseltamivir (OSV), and the inhibition rate of the OSV at 100 mu M under the same experiment condition is only 50.97%.
The half Inhibitory Concentration (IC) of blumea balsamifera extract against influenza A virus H3N2 under the condition is obtained by dilution in multiple proportion50) 11.38. mu.g/mL, as shown in FIG. 11.
And (3) knotting:
under the experimental condition, the blumea balsamifera extract has antitoxic activity to the oseltamivir-resistant influenza A virus A/Hong Kong/4801/2014(H3N2), the activity is positively correlated with the dosage, and when the dosage concentration is more than 6.25 mug/mL, the blumea balsamifera extract has the activity of obviously inhibiting virus from infecting host cells and half the Inhibitory Concentration (IC)50) Is 11.38μg/mL。
Example 13
This example discloses the evaluation of antiviral activity of various doses of blumea balsamifera extract against oseltamivir-resistant influenza B virus Yamagata line. The blumea balsamifera extract in this example was prepared according to the method of example 1. The research and test method specifically comprises the following steps:
1. virus strain:
influenza vaccine strain B/Phuket/3073/2013(B/Yamagata linkage)
2. Cell: MDCK cells (Madin-Daby cancer cells)
3. Virus culture and virulence determination
Inoculating the virus into allantoic cavities of 9-11 days old chick embryos, culturing for 72h at 35 ℃, and performing aseptic operation to obtain allantoic fluid. The precipitate was removed by centrifugation at 3000rpm for 15min and the supernatant was collected. Diluting the collected virus liquid to 10 times in series-1To 10-8A total of 8 dilutions were added to adherent 96-well MDCK cells in 8 wells per concentration, approximately 3 × 10 cells per well4Blank control group was added. MDCK cells in each well adsorb virus at 35 deg.C for 2 hr, replace with culture solution, and culture in incubator (37 deg.C, 5% CO)2) Cytopathic effects were observed under a microscope after 48 h.
4. Adding chemicals for treatment
The administration mode is shown in the attached figure 1: adding different samples to be tested in each vertical row of a B2-G9 area, adding positive control oseltamivir in a B10-G10 area, and adding negative control DMSO in a B11-G11 area; virus was added in the three horizontal rows below the black line. Such administration allows for the detection of both the antiviral activity and the cytotoxicity of the sample.
5. Grouping and dose design
(1) Dissolving herba Blumeae Balsamiferae extract with DMSO, and diluting with culture medium at gradient of 8 concentrations such as 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 μ g/mL;
(2) positive control: oseltamivir (OSV), the dosage is designed to be 100 mu M/L;
(3) negative control: 1% DMSO.
6. Cytopathy and cell viability examination
Influenza viruses can cause MDCK cells to develop lesions, known as the CPE effect, with a significant reduction in cell viability. The negative control DMSO group can see obvious CPE effect after adding virus, and if the compound can effectively inhibit infection of influenza virus, the cell state is still good after adding the same amount of virus.
Experiment adopts
Figure BDA0002909425160000201
The kit detects the cell activity. The cell viability corresponding to the chemiluminescence value of the blank (DMSO) group was defined as 100%,
relative cell viability ═ 100% of the values measured for each group of cells/the values measured for the cells of the blank control group
7. Data processing
The inhibition rate is the rate at which the compound acts to inhibit viral growth. Reflecting the antiviral activity of the compound.
The inhibitory rate of the extract on virus is 1- (detection value of the effect of the extract on cells-detection value of the effect of the extract on virus)/(detection value of blank group-detection value of virus control) × 100%
The rate of inhibition of the extract on the virus was calculated and the half Inhibitory Concentration (IC) was determined by analysis using GraphPad Prism software50)。
The research and test results are as follows:
the results of Yamagata-based activity test of osmia extract against oseltamivir resistant influenza B virus are shown in fig. 12, with oseltamivir (OSV, 100 μ M) as a positive control and DMSO as a negative control. When the concentration of the blumea balsamifera extract is more than 12.5 mu g/mL, the blumea balsamifera extract has the activity of remarkably inhibiting viruses from infecting host cells. The inhibition rates of the blumea balsamifera extract on B/Phuket/3073/2013(B/Yamagata link) strains of influenza B viruses are 71.17%, 75.57% and 97.94% respectively when the dose concentrations of the blumea balsamifera extract are 12.5 mu g/mL, 25 mu g/mL and 50 mu g/mL, the B/Phuket/3073/2013(B/Yamagata link) strains adopted in the experiment have certain drug resistance on Oseltamivir (OSV), and the OSV inhibition rate of 100 mu M is only 62.64% under the same experiment conditions.
By dilution at a multiple ratio, antiviral half maximal Inhibitory Concentration (IC) of herba Blumeae Balsamiferae extract under the condition is obtained50) It was 4.66. mu.g/mL, as shown in FIG. 13.
And (3) knotting:
under the experimental condition, the blumea balsamifera extract has the anti-virus activity and the anti-virus activity on the oseltamivir resistant Yamagata series B influenza virus B/Phuket/3073/2013(B/Yamagata linkage), the activity of the blumea balsamifera extract is positively correlated with the dosage, and when the dosage concentration is more than 12.5 mu g/mL, the blumea balsamifera extract has the activity of obviously inhibiting virus from infecting host cells and half the Inhibitory Concentration (IC)50) It was 4.66. mu.g/mL.
Example 14
This example discloses antiviral experiments of influenza B virus Victoria lines isolated from Brisbane experiments with different doses of blumea balsamifera extract, and the specific study and test methods are as follows:
1. virus strain:
influenza vaccine strain B/Brisbane/60/2008/NYMC BX-35(B/Victoria line)
2. Cell: MDCK cells (Madin-Daby cancer cells)
3. Virus culture and virulence determination
Inoculating the virus into allantoic cavities of 9-11 days old chick embryos, culturing for 72h at 35 ℃, and performing aseptic operation to obtain allantoic fluid. The precipitate was removed by centrifugation at 3000rpm for 15min and the supernatant was collected. Diluting the collected virus liquid to 10 times in series-1To 10-8A total of 8 dilutions were added to adherent 96-well MDCK cells in 8 wells per concentration, approximately 3 × 10 cells per well4Blank control group was added. MDCK cells in each well adsorb virus at 35 deg.C for 2 hr, replace with culture solution, and culture in incubator (37 deg.C, 5% CO)2) Cytopathic effects were observed under a microscope after 48 h.
4. Adding chemicals for treatment
The administration mode is shown in the attached figure 1: adding different samples to be tested in each vertical row of a B2-G9 area, adding positive control oseltamivir in a B10-G10 area, and adding negative control DMSO in a B11-G11 area; virus was added in the three horizontal rows below the black line. Such administration allows for the detection of both the antiviral activity and the cytotoxicity of the sample.
5. Grouping and dose design
(1) Dissolving herba Blumeae Balsamiferae extract with DMSO, and diluting with culture medium at gradient of 8 concentrations such as 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 μ g/mL;
(2) positive control: oseltamivir (OSV), the dosage is designed to be 100 mu M/L;
(3) negative control: 1% DMSO.
6. Cytopathic effect and cell viability assay
Influenza viruses can cause MDCK cells to develop lesions, known as the CPE effect, with a significant reduction in cell viability. The negative control DMSO group can see obvious CPE effect after adding virus, and if the compound can effectively inhibit infection of influenza virus, the cell state is still good after adding the same amount of virus.
Experiment adopts
Figure BDA0002909425160000211
The kit detects the cell activity. The cell viability corresponding to the chemiluminescence value of the blank (DMSO) group was defined as 100%,
relative cell viability ═ 100% of the values measured for each group of cells/the values measured for the cells of the blank control group
7. Data processing
The inhibition rate is the rate at which the compound acts to inhibit viral growth. Reflecting the antiviral activity of the compound.
The inhibitory rate of the extract on virus is 1- (detection value of the effect of the extract on cells-detection value of the effect of the extract on virus)/(detection value of blank group-detection value of virus control) × 100%
The rate of inhibition of the extract on the virus was calculated and the half Inhibitory Concentration (IC) was determined by analysis using GraphPad Prism software50)。
The research and test results are as follows:
the results of the activity test of the Osmunia blumea extract against Oseltamivir-resistant virus type B virus Victoria series are shown in FIG. 14, with Oseltamivir (OSV, 100. mu.M) as a positive control and DMSO as a negative control. When the concentration of the blumea balsamifera extract is more than 3.125 mug/mL, the activity of remarkably inhibiting host cell infection by the Oseltamivir resistant Victoria B-type virus is achieved. The inhibition rates of the blumea balsamifera extract on B/Brisbane/60/2008/NYMC BX-35(B/Victoria link) strains of influenza B viruses are 44.83%, 68.50%, 77.22%, 121.86% and 114.02% respectively when the dose concentrations of the blumea balsamifera extract are 3.125 mu g/mL, 6.25 mu g/mL, 12.5 mu g/mL, 25 mu g/mL and 50 mu g/mL, and the B/Brisbane/60/2008/NYMC BX-35(B/Victoria link) strains adopted in the experiment have certain drug resistance to Oseltamivir (OSV), and the inhibition rate of the OSV of 100 mu M under the same experiment conditions is only 11.78%.
The half Inhibition Concentration (IC) of the blumea balsamifera extract against the Oseltamivir resistant Victoria B type virus under the condition is obtained by dilution in multiple proportion50) It was 4.29. mu.g/mL (as shown in FIG. 15).
And (3) knotting:
under the experimental condition, the blumea balsamifera extract has the anti-virus activity and the anti-virus activity to the oseltamivir resistant Yamagata series B influenza virus B/Brisbane/60/2008/NYMC BX-35(B/Victoria line), the activity of the blumea balsamifera extract is positively correlated with the dosage, and when the dosage concentration is more than 3.125 mu g/mL, the blumea balsamifera extract has the activity of obviously inhibiting virus from infecting host cells and half the Inhibitory Concentration (IC)50) It was 4.29. mu.g/mL.
Finally, it should be noted that: the above embodiments are only preferred embodiments of the present invention to illustrate the technical solutions of the present invention, but not to limit the technical solutions, and certainly not to limit the patent scope of the present invention; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention; that is, the technical problems to be solved by the present invention, which are not substantially changed or supplemented by the spirit and the concept of the main body of the present invention, are still consistent with the present invention and shall be included in the scope of the present invention; in addition, the technical scheme of the invention is directly or indirectly applied to other related technical fields, and the technical scheme is included in the patent protection scope of the invention.

Claims (7)

1. The new application of the blumea balsamifera extract in preparing the anti-influenza virus medicine is characterized in that the extract is an ethanol extract of blumea balsamifera.
2. The new use as claimed in claim 1, wherein the mass percentage of the total flavonoids in the ethanol extract is greater than or equal to 18.56% and less than or equal to 38.48%; preferably 24-29%.
3. The new use according to claim 1, characterized in that the influenza virus is influenza a virus, or/and influenza B virus.
4. The novel use according to claim 3, wherein said influenza A virus comprises the H1N1 virus.
5. The novel use according to claim 3, wherein the influenza B virus comprises Yamagata or/and Victoria virus.
6. The new use as claimed in claim 1, wherein the ethanol extract of blumea balsamifera is prepared from blumea balsamifera as raw material, 50-95% ethanol as solvent, and supercritical CO2Extracting by extraction, cold soaking, ultrasonic extraction, cold soaking and ultrasonic extraction, percolation, or reflux.
7. The new use as claimed in claim 6, wherein the ethanol extract of blumea balsamifera is obtained by extracting blumea balsamifera as a raw material with 50-95% ethanol as a solvent.
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