CN112089742A

CN112089742A - A herba Blumeae Balsamiferae extract with anti-influenza virus effect, and its preparation method

Info

Publication number: CN112089742A
Application number: CN202011068713.3A
Authority: CN
Inventors: 柴立; 贾金艳; 李霞; 高滔; 朱燕
Original assignee: Guizhou Holy Pharmaceutical Co ltd
Current assignee: Guizhou Holy Pharmaceutical Co ltd
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2020-12-18
Also published as: CN114306404A; CN114306404B

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a blumea balsamifera extract with an anti-influenza virus effect and a preparation method thereof. The blumea balsamifera extract is an ethanol extract of blumea balsamifera, and the mass percentage of total flavonoids in the extract is more than or equal to 18.56 percent and less than or equal to 38.48 percent. The preparation method of the blumea balsamifera extract adopts blumea balsamifera as a raw material, 50-95% ethanol as a solvent and supercritical CO₂Extracting by extraction, cold soaking, ultrasonic extraction, cold soaking and ultrasonic extraction, percolation, and reflux. The blumea balsamifera ethanol extract has the effect of resisting various influenza viruses. Experiments show thatThe blumea balsamifera ethanol extract has good inhibition effect on influenza A virus and influenza B virus.

Description

A herba Blumeae Balsamiferae extract with anti-influenza virus effect, and its preparation method

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a blumea balsamifera extract with an anti-influenza virus effect and a preparation method thereof.

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

The invention aims to provide a blumea balsamifera extract with anti-influenza virus effect, which is a blumea balsamifera ethanol extract.

The second purpose of the invention is to provide a preparation method of the blumea balsamifera extract.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the blumea balsamifera extract with the anti-influenza virus effect is an ethanol extract of blumea balsamifera.

In some embodiments of the invention, the ethanol extract contains 18.56-38.48% of total flavonoids by mass; preferably 24-29%.

In some embodiments of the present invention, the total flavonoids of blumea balsamifera include blumea balsamifera, chrysoeriol, and pademamectin.

The preparation method of the blumea balsamifera extract with the anti-influenza virus effect comprises the steps of taking blumea balsamifera as a raw material, taking 50-95% ethanol as a solvent, and adopting supercritical CO₂Extracting by extraction, cold soaking, ultrasonic extraction, cold soaking and ultrasonic extraction, percolation, or reflux.

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 invention, the feed solution is a 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 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 firstly researches the components of the blumea balsamifera except volatile oil, and ethanol is used as a solvent to prepare the blumea balsamifera ethanol extract. The invention unexpectedly discovers that 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.

The preparation method is simple, simple to operate, controllable in quality and suitable for industrial production.

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.

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 supercritical CO₂The method for preparing the blumea balsamifera extract comprises the following steps:

taking appropriate amount of Blumea balsamifera (L.) DC coarse powder, and placing in CO₂In the supercritical extraction device, 95% ethanol is used as entrainer, and the dosage of the entrainer is 6 times of that of the raw material (volume/mass, when the mass is g, the volume is mL).

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 11

Examples 6-11 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

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%, and the relative cell viability was defined as the value measured for each group of cells/value measured for the blank group of cells × 100%

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. Are added separately100 μ L of different concentrations of the extract solution was used as an extract group, each concentration was repeated in 3 wells, and a positive control group (OSV, 100 μ M/mL) and a blank control group (DMSO, 1%) were set in an incubator (37 ℃, 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

Statistical analysis is carried out on experimental data by adopting SPSS 21.0 software, and quantitative data adopts mean +/-standard deviation

Showing that the data comparison among the groups adopts one-factor 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 in the data in Table 1, when the extract concentration was 0.78. mu.g/mL,Cell viability at 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 was not statistically significant (P) relative to the blank group>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

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

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 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 the experiment is a strain isolated from the influenza B/Darwin laboratory and numbered 58/2019 in the laboratory (belonging to the Yamagata strain of 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

5. Grouping and dose design

(1) Dissolving herba Blumeae Balsamiferae extract with DMSO, and diluting with culture medium to 50 μ g/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

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 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

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

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

7. Data processing

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: influenza virus attenuated live strain B/Sichuan high new/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 new/531/2018 (Victoria) virus strain adopted in the experiment is a virus strain (Victoria strain belonging to influenza B) separated from/in 531/2018 years of a laboratory of type B/Sichuan high new.

2. Cell: MDCK cells (Madin-Daby cancer cells)

3. Virus culture and virulence determination

4. Adding chemicals for treatment

5. Grouping and dose design

(3) negative control: blank DMSO.

6. Cytopathy and cell viability examination

Experiment adopts

7. Data processing

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

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 virus vaccine strain B/Sichuan high new/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 Hokko/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. 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^-4.37. The MOI of this experiment is 0.0005.

4. Adding chemicals for treatment

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;

(3) negative control: 1% DMSO.

6. Cytopathic effect and cell viability assay

Experiment adopts

7. Data processing

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.

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

1. A blumea balsamifera extract with anti-influenza virus effect is characterized in that the extract is an ethanol extract of blumea balsamifera.

2. The blumea balsamifera extract with the effect of resisting the influenza viruses as claimed in claim 1, wherein the mass percentage of the total flavonoids in the ethanol extract is more than or equal to 18.56% and less than or equal to 38.48%; preferably 24-29%.

3. The method for preparing blumea balsamifera extract with anti-influenza virus effect as claimed in claim 1 or 2, wherein the ethanol extract is prepared by using blumea balsamifera as raw material, 50-95% ethanol as solvent, and adopting super-ethanolCritical CO₂Extracting by extraction, cold soaking, ultrasonic extraction, cold soaking and ultrasonic extraction, percolation, or reflux.

4. The method for preparing blumea balsamifera extract with anti-influenza virus effect according to claim 3, wherein the extraction method 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.

5. The method for preparing blumea balsamifera extract with anti-influenza virus effect according to claim 3, wherein 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 time is 60-180min, preferably 120 min;

6. The preparation method of the blumea balsamifera extract with the anti-influenza virus effect as claimed in claim 3, wherein the extraction method comprises the following steps when the extraction method is a cold soaking method: taking a blumea balsamifera raw material, and mixing the raw material with the liquid material ratio of 1: 5-40 adding 60-95% ethanol, cold soaking for 2-72 hr, filtering, collecting filtrate, and volatilizing ethanol to obtain herba Blumeae Balsamiferae extract;

preferably, the feed liquid is 1: 10-30, more preferably 1: 20;

preferably, the cold soak time is 8-60 hours, more preferably 48 hours;

preferably, the ethanol concentration is 75-95%, more preferably 95%.

7. The preparation method of the blumea balsamifera extract with the anti-influenza virus effect as claimed in claim 3, wherein 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, the number of extractions is 1-3, more preferably 2;

preferably, the ultrasonic temperature is preferably normal temperature;

8. The method for preparing blumea balsamifera extract with anti-influenza virus effect according to claim 3, wherein the method comprises the following steps when the method is a cold soaking and ultrasonic extraction method: 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, the sonication time is 0.5 to 4 hours, preferably 2 hours;

preferably, the ultrasonic temperature is preferably normal temperature.

9. The method for preparing blumea balsamifera extract with anti-influenza virus effect according to claim 3, which is a percolation method and comprises the following steps: 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, the percolate flow rate is 2-3 ml/min.

10. The method for preparing blumea balsamifera extract with anti-influenza virus effect according to claim 3, wherein the method comprises the following steps when the method is a reflux method: 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;