CN115501288B - Extraction method of callicarpa nudiflora extract and application of extract in resisting H1N1 virus - Google Patents
Extraction method of callicarpa nudiflora extract and application of extract in resisting H1N1 virus Download PDFInfo
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Abstract
The invention relates to the technical field of medicines, in particular to an extraction method of a callicarpa nudiflora extract and application of the extract in resisting H1N1 virus, which are characterized in that callicarpa nudiflora medicinal materials are crushed and sieved, ethanol is added, and the mixture is soaked, refluxed, extracted and filtered to obtain first filtrate and first filter residues; adding ethanol into the first filter residue, carrying out reflux extraction and filtering to obtain a second filtrate and a second filter residue; adding water into the second filter residue, carrying out reflux extraction and filtering to obtain a third filtrate; and combining the first filtrate, the second filtrate and the third filtrate to obtain concentrated solution, respectively extracting the concentrated solution with petroleum ether and ethyl acetate, evaporating, concentrating and volatilizing in water bath to obtain the callicarpa nudiflora ethyl acetate extract. Therefore, the invention can effectively lighten the lung tissue injury caused by H1N1 infection, inhibit excessive inflammatory reaction, strengthen the immunity, obviously reduce the virus load of the lung tissue and inhibit the proliferation of the virus in the lung.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to an extraction method of a callicarpa nudiflora extract and application of the extract in resisting H1N1 viruses.
Background
Influenza A Virus (IAV) infection is a common cause of pneumonia-related death cases, and severe IAV infection causes bilateral pulmonary infiltration and hypoxia, i.e., acute respiratory distress syndrome, which is one of the main causes of death by IAV. The total incidence of acute respiratory distress syndrome caused by seasonal IAV infection is estimated to be 2.7 cases per 10 million years. Typical symptoms of H1N1 infected individuals include persistent or recurrent fever, cough, sometimes accompanied by sore throat and nasal leakage, dyspnea, etc., and epidemic H1N1 infection is also manifested as gastrointestinal symptoms (including nausea, vomiting, and diarrhea), etc.
Currently, drugs approved for clinical prophylaxis and treatment of influenza include M2 channel inhibitors (such as amantadine and rimantadine) and NA inhibitors, and M2 channel inhibitors are no longer recommended for treatment of influenza due to their broad resistance, and reports of NA inhibitor virus resistance such as Guan Aosi tavir are also rapidly increasing.
How to effectively reduce the damage of lung tissues caused by H1N1 infection, inhibit excessive inflammatory reaction, enhance immunity, obviously reduce the viral load of the lung tissues and inhibit the proliferation of viruses in the lung becomes a technical problem needing breakthrough.
In summary, it is clear that the prior art has inconvenience and defects in practical use, so that improvement is needed.
Disclosure of Invention
In view of the above-mentioned drawbacks, the present invention aims to provide a method for extracting beautyberry extract and application of the extract in resisting H1N1 virus, which can effectively reduce lung tissue injury caused by H1N1 infection, inhibit excessive inflammatory reaction, enhance immunity, remarkably reduce virus load of lung tissue and inhibit proliferation of virus in lung.
In order to achieve the above object, the present invention provides a method for extracting callicarpa nudiflora extract, comprising the steps of:
step one extraction of Callicarpa nudiflora
Pulverizing and sieving beautyberry medicinal materials, adding 94-96% ethanol, wherein the mass ratio of beautyberry medicinal material powder to ethanol is 1: 7-9, soaking, reflux-extracting and filtering to obtain a first filtrate and a first filter residue; adding 70-80% ethanol into the first filter residue, wherein the mass ratio of the first filter residue to the ethanol is 1: 7-9, reflux-extracting and filtering to obtain a second filtrate and a second filter residue; adding water into the second filter residue, wherein the mass ratio of the second filter residue to the water is 1: 7-9, reflux-extracting and filtering to obtain a third filtrate.
And combining the first filtrate, the second filtrate and the third filtrate, and evaporating and concentrating to obtain concentrated solution.
Step two, extraction of the effective components
Petroleum ether is added into the concentrated solution, and the volume ratio of the petroleum ether to the concentrated solution is 1: 0.8-1.2, extracting with petroleum ether, separating, and retaining water phase.
Adding ethyl acetate into the petroleum ether extracted water phase, wherein the volume ratio of the ethyl acetate to the petroleum ether extracted water phase is 1: 0.8-1.2, extracting and separating by using ethyl acetate, retaining an ethyl acetate extraction layer to obtain an ethyl acetate extract, evaporating the ethyl acetate extract, volatilizing in water bath to obtain the callicarpa nudiflora ethyl acetate extract.
According to the extraction method of the callicarpa nudiflora extract, the screening granularity of the crushing and screening is 30-50 meshes.
According to the extraction method of the callicarpa nudiflora extract, the evaporation concentration adopts a rotary evaporator.
According to the extraction method of the callicarpa nudiflora extract, the extraction times of petroleum ether and ethyl acetate are 3 times.
According to the extraction method of the callicarpa nudiflora extract, the soaking time is 30-35 minutes.
According to the extraction method of the callicarpa nudiflora extract, the reflux extraction time is 110-130 minutes.
According to the extraction method of the callicarpa nudiflora extract, the optimal mass ratio of callicarpa nudiflora medicinal material powder to ethanol, the optimal mass ratio of the first filter residue to ethanol and the optimal mass ratio of the second filter residue to water are all 1:8.
According to the extraction method of the callicarpa nudiflora extract, the optimal volume ratio of petroleum ether to concentrated solution and the optimal volume ratio of ethyl acetate to petroleum ether extracted water phase are 1:1.
application of beautyberry ethyl acetate extract in resisting H1N1 virus is provided.
The invention aims to provide an extraction method of beautyberry extract and application of the extract in resisting H1N1 virus, which comprises the steps of taking beautyberry medicinal material, crushing, repeatedly extracting in ethanol, evaporating and concentrating, extracting in petroleum ether and ethyl acetate respectively, evaporating and concentrating, volatilizing in water bath to obtain beautyberry ethyl acetate extract; the ethyl acetate extract of the callicarpa nudiflora can effectively relieve lung tissue injury caused by H1N1 infection, inhibit excessive inflammatory reaction, enhance immunity, obviously reduce the viral load of lung tissues, inhibit the proliferation of viruses in the lung, and can play a role in resisting H1N1 from multiple ways of directly inhibiting viruses, resisting inflammation, enhancing immunity and the like. In summary, the beneficial effects of the invention are as follows: can effectively relieve lung tissue injury caused by H1N1 infection, inhibit excessive inflammatory reaction, enhance immunity, remarkably reduce virus load of lung tissue, and inhibit proliferation of virus in lung.
Drawings
FIG. 1 is a graph showing the anti-H1N 1 action of an ethyl acetate extract of Callicarpa nudiflora;
FIG. 2 is a graph showing the effect of ethyl acetate extract of Callicarpa nudiflora on slowing down the weight loss trend of mice;
FIG. 3 is a graph showing the effect of ethyl acetate extract of Callicarpa nudiflora on reducing lung index of mice;
FIG. 4 is a graph showing the effect of ethyl acetate extract of Callicarpa nudiflora on reducing mouse TNF- α;
FIG. 5 is a graph showing the effect of ethyl acetate extract of Callicarpa nudiflora on reducing IL-1β in mice;
FIG. 6 is a graph showing the effect of ethyl acetate extract of Callicarpa nudiflora on reducing IFN-gamma in mice;
FIG. 7 is a graph showing the effect of ethyl acetate extract of Callicarpa nudiflora on reducing pulmonary H1N1 viral load in mice;
FIG. 8 is a graph showing the effect of ethyl acetate extract of Callicarpa nudiflora on increasing serum IgG levels in mice;
FIG. 9 is a graph showing the effect of ethyl acetate extract of Callicarpa nudiflora on increasing spleen index in mice;
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention provides a preparation method of an effective part of beautyberry for resisting H1N1, which comprises the following steps:
step one extraction of Callicarpa nudiflora
Pulverizing Callicarpa nudiflora, sieving with a 30-50 mesh sieve to obtain Callicarpa nudiflora powder, adding 94-96% ethanol (the mass ratio of the Callicarpa nudiflora powder to the ethanol is 1:7-9) into the Callicarpa nudiflora powder, soaking for 30-35 min, extracting under reflux for 110-130 min, and filtering to obtain a first filtrate and a first filter residue; adding 70-80% ethanol into the first filter residue (the mass ratio of the first filter residue to the ethanol is 1:7-9), extracting for 110-130 minutes under reflux, and filtering to obtain a second filtrate and a second filter residue; adding water into the second filter residue (the mass ratio of the second filter residue to the water is 1:7-9), extracting for 110-130 minutes by reflux, and filtering to obtain a third filtrate and a third filter residue.
And combining the first filtrate, the second filtrate and the third filtrate, and evaporating and concentrating by using a rotary evaporator to obtain concentrated solution.
Step two, extraction of the effective components
Adding petroleum ether (the volume ratio of petroleum ether to the concentrated solution is 1:0.8-1.2) into the concentrated solution, extracting for 3-4 times by using petroleum ether, separating, and retaining a water phase;
and adding ethyl acetate (the volume ratio of the ethyl acetate to the petroleum ether extracted water phase is 1:0.8-1.2) into the first water phase, extracting for 3-4 times by using ethyl acetate, separating, and retaining an ethyl acetate extraction layer to obtain an ethyl acetate extract. Recovering ethyl acetate from the ethyl acetate extract by using a rotary evaporator, volatilizing in a water bath, and obtaining the beautyberry ethyl acetate extract.
Preferably, the optimal mass ratio of the beautyberry medicinal material powder to the ethanol, the optimal mass ratio of the first filter residue to the ethanol and the optimal mass ratio of the second filter residue to the water are all 1:8.
preferably, the optimal volume ratio of petroleum ether to concentrated solution and the optimal volume ratio of ethyl acetate to petroleum ether extracted aqueous phase are all 1:1.
in order to verify the medicinal value of the beautyberry ethyl acetate extract, the prepared beautyberry ethyl acetate extract is subjected to in vitro H1N1 resistance test and in vivo H1N1 resistance test.
In vitro anti-H1N 1 Performance test (see FIG. 1)
Virus strain: influenza A virus H1N1 murine lung adapted strain (A/PR/8/1934).
Resuscitating and passaging of host cell MDCK
The MDCK cells stored in a refrigerator at the temperature of minus 80 ℃ are placed in warm water at the temperature of 37 ℃ for thawing, centrifugating (1000 r/min) for 5min, removing supernatant, transferring the cells into a cell culture flask, adding 5mL of cell culture solution, placing the cell culture flask in a cell culture box, culturing until a monolayer cell grows to 70-80% of the bottom area of the culture flask, washing the cells with PBS, rinsing once with 0.5mL of pancreatin, and then adding 1mL of pancreatin into the culture box for incubation for 6-8 min. And adding 2mL of cell culture solution into the cells subjected to enzymolysis, blowing uniformly, discarding 1/3-1/2 of the culture solution, supplementing the cell culture solution to 5mL, and completing one cell passage. After three passages, the cells were seeded in 96-well plates at a concentration of 1.0X10 4 Individual cells/wells
Determination of drug toxicity
The four extracts and oseltamivir phosphate were inoculated into MDCK cells at different concentrations and cultured 48, h. Four extracts and oseltamivir phosphate were tested for 50% cytotoxic concentration (CC 50) on MDCK cells using MTT kit. The maximum drug concentration with a cell viability greater than 90% was selected as the non-cytotoxic concentration of the drug. The CC50 of the ethyl acetate extract against MDCK cells was measured to be 0.86mg/mL.
anti-H1N 1 Effect study
Taking 96-well plate, inoculating 1×10 concentration in each well 5 cell/mL of cell suspension 100. Mu.L, cultured for 24h (37 ℃,5% CO) 2 ). The process of H1N1 infection of MDCK cells was performed in Opti-MEM medium containing 1000U/mL penicillin, 100. Mu.g/mL streptomycin and 2. Mu.g/mLTPCK-pancreatin. Cell plates with cells grown to 80% or more were discarded and washed twice with PBS, and 100. Mu.L of each extract or oseltamivir phosphate solution and 100. Mu.L of 1N1 virus dilution (TCID 50 of 1X 10) were added to each well 6 Is diluted 100 times) to obtain the virus stock. After 48H of incubation, the cell viability was measured with the MTT kit and the half inhibition concentrations of the four extracts and oseltamivir phosphate on H1N1 were calculated according to the Reed-Muench method. The EC50 of the ethyl acetate extract for H1N1 was determined to be 0.051mg/mL and the TI (CC 50/EC 50) value was determined to be 16.86.
In vivo anti-H1N 1 Performance test (see FIGS. 2-9)
Establishment of H1N1 infected mice model
Randomly dividing mice into normal group, model group, high, medium, low, dose group and oseltamivir phosphate group (32 mg/kg) of ethyl acetate extraction part of Callicarpa nudiflora, feeding 10 mice each for 3 days, anesthetizing (anesthetic is isoflurane) by using a small animal anesthesia machine, taking H1N1 virus stock solution, diluting 100 times, and dropping 20 μl of rhinovirus (LD 50 of virus is 10) -3 /mL, i.e. diluting the virus solution 10 3 Half of the mice can die by 20 μl inoculation, the experimental mice have an infectious viral load of 10LD 50), and 0.2mL is administered by gastric lavage 2h after infection.
In vivo anti-H1N 1 effect study
After 6 days of continuous gastric lavage, the mice are killed by neck removal, lung tissues of the mice are taken, the lung index (lung weight/body weight multiplied by 100%) is calculated, two lungs close to one side of the heart are taken and fixed by paraformaldehyde, and after dehydration, tissue transparency, wax dipping, embedding, slicing and drying, HE staining is carried out, and the pathological changes of the lungs of the mice are observed. The H1N1 viral load of the lung tissue of the mice was measured, and the level of inflammatory factors in the peripheral blood of the mice was measured. The beautyberry ethyl acetate extract can obviously reduce the lung index increase caused by H1N1 infection, increase the spleen index of mice, reduce the H1N1 virus load of lung tissues of mice and reduce the level of inflammatory factors in peripheral blood.
According to the experimental results, after the H1N1 virus invades the body, inflammatory factors such as IL-1 beta, TNF-alpha, IFN-gamma and the like are induced to cause a series of inflammatory injuries, and lung tissue inflammatory infiltration leads to increase of lung index. The ethyl acetate extract of the callicarpa nudiflora can obviously inhibit the increase of inflammatory factors, reduce the lung index and relieve the inflammatory injury caused by H1N1 infection. The q-PCR method is used for detecting the proliferation condition of H1N1 in the lung of a mouse, and the ethyl acetate extract of callicarpa nudiflora can obviously reduce the virus load of the lung tissue H1N1 of the mouse, so that the ethyl acetate extract can inhibit the proliferation of H1N1 in the lung of the mouse. The antibody produced by the organism under the stimulation of influenza virus mainly comprises IgG, the ethyl acetate extract of the callicarpa nudiflora can obviously increase the IgG level of mouse serum and the spleen index of the mouse, which indicates that the ethyl acetate extract of the callicarpa nudiflora can enhance the immune response of the mouse. Thus, it was suggested that the ethyl acetate extract of Callicarpa nudiflora can exert an anti-H1N 1 effect from various routes such as direct inhibition of viruses, anti-inflammation and immunity enhancement.
The invention provides an extraction method of beautyberry extract and application of the extract in resisting H1N1 virus, beautyberry medicinal materials are taken, crushed, repeatedly extracted in ethanol and evaporated and concentrated, finally extracted in petroleum ether and ethyl acetate respectively, evaporated and concentrated, and volatilized in water bath to obtain beautyberry ethyl acetate extract; the ethyl acetate extract of the callicarpa nudiflora can effectively relieve lung tissue injury caused by H1N1 infection, inhibit excessive inflammatory reaction, enhance immunity, obviously reduce the viral load of lung tissues, inhibit the proliferation of viruses in the lung, and can play a role in resisting H1N1 from multiple ways of directly inhibiting viruses, resisting inflammation, enhancing immunity and the like. In summary, the beneficial effects of the invention are as follows: can effectively relieve lung tissue injury caused by H1N1 infection, inhibit excessive inflammatory reaction, enhance immunity, remarkably reduce virus load of lung tissue, and inhibit proliferation of virus in lung.
Of course, the present invention is capable of other various embodiments and its several details are capable of modification and variation in light of the present invention, as will be apparent to those skilled in the art, without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. The application of the beautyberry ethyl acetate extract in preparing the anti-H1N 1 virus medicine is characterized in that the extraction method of the beautyberry ethyl acetate extract comprises the following steps:
step one extraction of Callicarpa nudiflora
Pulverizing and sieving beautyberry medicinal materials, and adding 94-96% ethanol, wherein the mass ratio of beautyberry medicinal material powder to ethanol is 1: 7-9, soaking, reflux-extracting and filtering to obtain a first filtrate and a first filter residue; 70-80% ethanol is added into the first filter residue, and the mass ratio of the first filter residue to the ethanol is 1: 7-9, reflux-extracting and filtering to obtain a second filtrate and a second filter residue; adding water into the second filter residue, wherein the mass ratio of the second filter residue to the water is 1: 7-9, reflux-extracting and filtering to obtain a third filtrate;
combining the first filtrate, the second filtrate and the third filtrate, and evaporating and concentrating to obtain concentrated solution;
step two, extraction of the effective components
Petroleum ether is added into the concentrated solution, and the volume ratio of the petroleum ether to the concentrated solution is 1: 0.8-1.2, extracting with petroleum ether, separating, and retaining a water phase;
adding ethyl acetate into the petroleum ether extracted water phase, wherein the volume ratio of the ethyl acetate to the petroleum ether extracted water phase is 1: 0.8-1.2, extracting and separating by using ethyl acetate, retaining an ethyl acetate extraction layer to obtain an ethyl acetate extract, evaporating the ethyl acetate extract, volatilizing in a water bath, and obtaining the callicarpa nudiflora ethyl acetate extract.
2. The use of the ethyl acetate extract of beautyberry of claim 1, wherein the size of the screen of the crushing and sieving is 30-50 mesh.
3. The use of ethyl acetate extract of beautyberry of claim 1, wherein the apparatus used for the evaporative concentration is a rotary evaporator.
4. The use of the ethyl acetate extract of beautyberry of claim 1, wherein the number of extraction times of petroleum ether and ethyl acetate is 3.
5. The use of the ethyl acetate extract of beautyberry of claim 1, wherein the soaking time is 30-35 minutes.
6. The use of the ethyl acetate extract of beautyberry of claim 1, wherein the time of the reflux extraction is 110 to 130 minutes.
7. The use of ethyl acetate extract of beautyberry of claim 1, wherein the optimal mass ratio of beautyberry powder to ethanol, the optimal mass ratio of the first filter residue to ethanol, and the optimal mass ratio of the second filter residue to water are all 1:8.
8. the use of the beautyberry ethyl acetate extract as claimed in claim 1, wherein the optimum volume ratio of petroleum ether to concentrate and the optimum volume ratio of ethyl acetate to petroleum ether extracted aqueous phase are each 1:1.
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Citations (3)
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|---|---|---|---|---|
| CN106266262A (en) * | 2015-06-05 | 2017-01-04 | 九芝堂股份有限公司 | A kind of Callicarpa nudiflora extract with antiinflammatory action |
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| CN108686045A (en) * | 2018-07-03 | 2018-10-23 | 江西普正制药有限公司 | A kind of callicarpa nudiflora composition and its application for treating pharyngitis |
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|---|---|---|---|---|
| CN106266262A (en) * | 2015-06-05 | 2017-01-04 | 九芝堂股份有限公司 | A kind of Callicarpa nudiflora extract with antiinflammatory action |
| CN106924448A (en) * | 2017-01-10 | 2017-07-07 | 湖南华纳大药厂天然药物有限公司 | Treat pharmaceutical composition of anemopyretic cold and preparation method thereof |
| CN108686045A (en) * | 2018-07-03 | 2018-10-23 | 江西普正制药有限公司 | A kind of callicarpa nudiflora composition and its application for treating pharyngitis |
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| Progress in Traditional Chinese Medicine Against Respiratory Viruses: A Review;Bao-Hong Li，等;Front Pharmacol.;第12卷;第743623篇第1-17页 * |
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