CN112691138B - Preparation method of mulberry resource extract with in-vitro inhibitory activity on African swine fever virus - Google Patents
Preparation method of mulberry resource extract with in-vitro inhibitory activity on African swine fever virus Download PDFInfo
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- A61K36/18—Magnoliophyta (angiosperms)
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Abstract
The invention discloses a preparation method of a mulberry resource extract with the activity of inhibiting African swine fever virus in vitro, which comprises the following steps: s1, taking fresh mulberry leaves and mulberry twigs, removing impurities, cleaning, drying and crushing; s2, mixing the crushed mulberry leaves and mulberry twigs according to a proportion, and adding an enzyme solution for enzymolysis; s3, leaching the mixed powder of the mulberry leaves and the mulberry branches subjected to enzymolysis for 1 time by using acidic hot water and alkaline hot water respectively, filtering an extracting solution by using an ultrafiltration membrane, carrying out alcohol precipitation and centrifugation on a filtrate, taking a supernatant, and carrying out reduced pressure concentration to obtain a concentrated solution I; s4, extracting the residues in the S3 by using three-stage ethanol, combining extracting solutions, and concentrating under reduced pressure to obtain a concentrated solution II; and S5, combining the concentrated solutions I and II, performing combined elution, and performing reduced pressure concentration and drying to obtain the compound.
Description
Technical Field
The invention relates to a preparation method of a mulberry resource extract with the in-vitro activity of inhibiting African swine fever virus.
Background
The mulberry leaves are dry leaves of Morus alba L of Morus, which are produced in most areas of China and have abundant resources. The mulberry leaves are excellent traditional Chinese medicinal materials from ancient times except for silkworm breeding, and have long clinical medicinal history recorded in various medical classics in China, such as Shen nong Ben Cao Jing and Ben Cao gang mu, and modern pharmacological research shows that the mulberry leaves have the effects of reducing blood sugar, resisting oxidation, resisting aging, reducing blood fat and the like. Mature fruit of mulberry belonging to the family Moraceae, as mentioned in Ben Cao Jing Shu: mulberry, fructus Mori, sweet and cold in flavor, tonifying blood and removing heat, is a medicine for cooling blood, enriching blood and nourishing yin. The record of the compendium of materia Medica: the juice is pounded to drink, the alcoholism is relieved, the wine is brewed and taken, the water and the gas are promoted, and the swelling is reduced. The mulberry contains multiple active ingredients due to the special growth environment of the mulberry, and has the effects of resisting oxidation, protecting nerves, resisting cerebral ischemia, reducing blood sugar, reducing blood fat, reducing blood pressure, resisting cancer and the like.
African Swine fever is an acute, hemorrhagic, virulent infectious disease caused by African Swine virus (ASFV) infecting domestic pigs and various wild pigs (such as African wild pigs, European wild pigs, etc.). The world animal health organization classifies the animal epidemic disease as a legal report animal epidemic disease, and the disease is also a kind of animal epidemic situation which is mainly prevented in China. Since the first African swine fever epidemic situation is diagnosed in 8 months and 3 days in 2018, viruses are rapidly spread to most regions in China, and huge economic losses are caused. At present, the 3D fine structure of African swine fever virus is decoded, but no specific vaccine or antiviral drug aiming at ASFV can effectively control the spread of the virus in time when epidemic outbreak occurs.
At present, aiming at African swine fever, people mainly take the following measures to deal with the African swine fever:
1. and killing sick pigs infected with African swine fever virus. Once the sick pigs infected with the African swine fever virus are confirmed, the pigs are killed immediately, and a series of work such as strict blocking and isolation of the activity places of the sick pigs, disinfection, restriction of movement of the sick pigs, marketing of meat products and the like is performed immediately. The sick pigs eat the residual forage or drink water and are burnt or buried deeply. Although the treatment method can inhibit the spread of the swine fever to a certain extent, the cost is huge, and serious economic loss can be caused to pig farmers.
2. Improve the immunity of the swinery so as to achieve the effect of prevention. At present, no effective drug capable of directly treating African swine fever is reported. The patent CN 103705771B discloses a medicine for preventing and treating swine fever, which is prepared by proportioning spina gleditsiae, reed rhizome, astragalus polysaccharide and other raw materials according to a certain mass part, and the medicine is used in advance for pigs without being infected with swine fever, can improve the immunity of the pigs, has good prevention effect on the swine fever, and has good treatment effect on the pigs with symptoms of the swine fever. However, the swine fever targeted by the drugs in the above patents is caused by Classical Swine Fever Virus (CSFV). Classical Swine Fever Virus (CSFV), a small RNA virus, belongs to the Flaviviridae family (Flavivivirisae) pestivirus genus (pestivirus). While the African Swine Fever Virus (ASFV) is a double-stranded nucleoplasmic large DNA virus, the only species of the African swine fever virus family (Asfarviridae), infectious and highly pathogenic. The African Swine Fever Virus (ASFV) and the swine fever virus (CSFV) have obvious difference in virus structure, pathogenicity, infectivity, lethality rate and the like.
At present, medicines which can directly play a good role in preventing and treating African Swine Fever Virus (ASFV), in particular traditional Chinese medicinal materials, are deeply dug.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a preparation method of a natural mulberry resource extract with the activity of inhibiting African Swine Fever Virus (ASFV) in vitro, and the method is simple to operate, high in safety, low in cost and easy to popularize.
The invention adopts the following technical scheme to realize the purpose of the invention:
a preparation method of mulberry resource extract with in vitro inhibitory activity on African swine fever virus comprises the following steps:
s1, taking fresh mulberry leaves and mulberry twigs, removing impurities, cleaning, drying and crushing;
s2, mixing the crushed mulberry leaves and mulberry twigs in proportion, adding a proper amount of enzyme solution, and performing enzymolysis for 11-13 hours;
s3, leaching the mixed powder of the mulberry leaves and the mulberry branches subjected to enzymolysis in the step S2 for 1 time by using acidic hot water, then leaching for 1 time by using alkaline hot water, combining extracting solutions, and reserving residues for later use; filtering the extracting solution by an ultrafiltration membrane, collecting filtrate, carrying out alcohol precipitation and centrifugation on the filtrate, taking supernatant, and carrying out reduced pressure concentration to obtain a concentrated solution I;
s4, extracting the residues in the step S3 by three-stage ethanol, combining extracting solutions, and concentrating under reduced pressure to obtain a concentrated solution II;
s5, combining the concentrated solution I and the concentrated solution II, and adjusting the pH value to be weakly acidic; and (3) carrying out combined elution on the combined solution sequentially through nonpolar, low-polarity and medium-polarity macroporous resins, combining the eluates, and carrying out reduced pressure concentration and drying to obtain the mulberry resource extract with the activity of inhibiting the African swine fever virus in vitro.
In step S2, the mass ratio of mulberry leaves to mulberry twigs is 4: 1-6: 1, preferably 5: 1.
wherein, in step S2, the enzyme type is selected from one or more of cellulase, hemicellulase or pectinase; the enzymolysis conditions are as follows: the enzymolysis temperature is 40-60 ℃, and preferably 50 ℃; the pH value is 4.5-6.5, and the preferable pH value is 5.0; in the step S2, the active substances in the cells can be leached out more easily by adding enzyme to degrade and breaking cell walls.
In step S3, the acidic hot water leaching conditions are: the ratio of material to liquid is 1: 6-1: 15, preferably 1: 10; the pH value is 1.0-3.0, and the preferable pH value is 2.0; the temperature of the hot water is 60-100 ℃, and preferably 80 ℃; the extraction time is 60-120 min.
In step S3, the alkaline hot water leaching conditions are: the ratio of material to liquid is 1: 6-1: 15, preferably 1: 10; the pH value is 10.0-12.0, and the preferable pH value is 11.0; the temperature of the hot water is 60-80 ℃, and the preferred temperature is 70 ℃; the extraction time is 60-120 min.
Wherein, in step S3, the ultrafiltration membrane is selected from one of cellulose acetate membrane, polysulfone membrane and polyamide membrane; the ultrafiltration membrane has the intercepted relative molecular mass of 20000-50000 daltons.
In the step S3, in the alcohol precipitation process, the final volume concentration of the ethanol is 60-70%, and the alcohol precipitation time is 24-36 h.
In the step S3, macromolecular proteoglycan and the like can be removed by acid and alkali simulated digestion extraction, i.e., semi-bionic extraction, combined with membrane filtration and alcohol precipitation at the later stage, thereby obtaining a mulberry leaf active extract and a mulberry twig active extract.
In step S4, the three-stage ethanol extraction is performed by sequentially using 30%, 50%, and 70% ethanol by volume, where the ratio of material to liquid is 1: 8, the extraction temperature is 55-65 ℃, the extraction time is 55-65 min, and the extraction times are 1-2.
In the step S4, the residues are subjected to fractional alcohol extraction to obtain purer mulberry leaf active extracts and mulberry twig active extracts.
Wherein, in step S5, the nonpolar macroporous resin is selected from one of D-101, XAD-1 and HP-20; the weak-polarity macroporous resin is selected from one of AB-8, DS-401 and HZ-841; the medium-polarity macroporous resin is selected from one of XAD-6, DM-301 and HZ-806.
In step S5, the pH value of the combined solution is 4 to 6, preferably 5; the eluent is ethanol eluent, and the volume concentration of the ethanol is 70-90%; the drying mode is selected from one of freeze drying, spray drying or vacuum drying.
The invention also discloses a mulberry resource extract with the activity of inhibiting the African swine fever virus in vitro, which is obtained by the preparation method.
Compared with the prior art, the invention has the following beneficial effects:
1) according to the invention, mulberry leaves and mulberry twigs in a specific compound ratio are subjected to specific sequential steps of enzyme method assistance, semi-bionic extraction, tertiary alcohol extraction and combined elution purification and specific process parameters, and the prepared mulberry resource extract containing the mulberry leaf active extract and the mulberry twigs active extract has excellent in-vitro African swine fever virus inhibition activity.
2) The preparation method disclosed by the invention is simple in preparation process, high in safety, low in production cost and good in industrial application prospect.
Detailed Description
The present invention is further illustrated by the following specific examples, which are, however, not intended to limit the scope of the invention.
The raw materials used in the examples of the present invention and the comparative examples are commercially available products.
The in vitro inhibition of African swine fever virus activity of mulberry resource extract was tested by the following cell assay:
1. experimental methods
1) Viruses and cells: chinese African swine fever virus isolate (ASFV) was isolated and deposited by the military veterinary institute of military medical academy of sciences. Aseptically collecting healthy pig lung, injecting sterilized PBS into the lung, irrigating for several times, collecting lavage fluid, centrifuging to collect pig alveolar macrophage (B-PAM), and collecting content of 5 × 106The 96-well plate was inoculated at a concentration of 100. mu.L/well per mL. The plate is paved on the next day,and (6) carrying out testing.
2) And (3) virus titer determination: after ASFV virus is activated and proliferated in cells, the ASFV virus is diluted to 10 times of gradient-10Separately infecting B-PAM cells cultured in a 96-well plate to a monolayer, setting 8 multiple wells at each concentration, and culturing at 37 deg.C and 5% CO2The medium was cultured for 4 days under the conditions, and cytopathic effect (CPE) was observed and recorded under an inverted microscope every day, and the degree of CPE was judged according to the following criteria: "-" indicates that the cells grow normally and no lesions appear; "+" indicates that cytopathic effect is about 0-25% of the monolayer cells in the well; "+ +" indicates that the cell disease is 25-50%; "+ + + +" indicates that the cell disease is 50-75%; the cytopathic effect is 75-100%. Calculating the infection dose of half cell by Reed-Muench method (TCID)50 )。
3) Cytotoxicity experiments: inoculating B-PAM cells to a 96-well plate, culturing to a single layer, discarding culture solution, respectively adding mulberry leaf and mulberry twig active extracts diluted into different concentrations by a maintenance solution double dilution method, setting 4 multiple holes for each concentration, setting a cell control group, continuously culturing, observing cell change every day, and measuring the maximum non-toxic concentration (MNCC) of a mulberry leaf and mulberry twig extract sample after 72 h.
4) In vitro anti-ASFV activity assay: test group, active extract samples of mulberry leaves and mulberry twigs with different concentrations and concentration of 100 XTCID in the nontoxic concentration range50After 50 mu L of each virus suspension is mixed evenly, the mixture is added into a monolayer B-PAM cell of a 96-well plate, 4 multiple wells are arranged for each concentration, and a cell control group and a virus control group are arranged at the same time. Placing at 37 ℃ and 5% CO2The culture was continued in the incubator and the cytopathic condition was observed and recorded daily. When the CPE of the virus control group is "+ + + + +" and the CPE of the cell control group is "-", the concentration of the sample with CPE of "+ +" is taken as the half Inhibitory Concentration (IC) of the sample to the virus, wherein the IC is 50 percent of inhibition concentration of the sample50）。
2. The experimental results are as follows: as shown in table 1.
Example 1
A preparation method of mulberry resource extract with in vitro inhibitory activity on African swine fever virus comprises the following steps:
s1, taking fresh mulberry leaves and mulberry twigs, removing impurities, cleaning, drying and crushing;
s2, mixing the crushed mulberry leaves and mulberry twigs according to the mass ratio of 5: 1, mixing, adding a proper amount of cellulase liquid, carrying out enzymolysis for 12 hours at the temperature of 50 ℃ and the pH value of 5.0;
s3, leaching the mixed powder of the mulberry leaves and the mulberry twigs subjected to enzymolysis in the step S2 for 1 time by using acidic hot water, wherein the leaching conditions of the acidic hot water are as follows: the ratio of material to liquid is 1: 10; the pH value is 2.0; the temperature of the hot water is 80 ℃; the extraction time is 90 min; leaching for 1 time by using alkaline hot water, wherein the alkaline hot water leaching conditions are as follows: the ratio of material to liquid is 1: 10; the pH value is 11.0; the temperature of the hot water is 70 ℃; the extraction time is 90 min; mixing extractive solutions, and collecting residue; filtering the extracting solution by a cellulose acetate membrane ultrafiltration membrane, wherein the relative molecular mass intercepted by the ultrafiltration membrane is 30000 daltons, collecting filtrate, carrying out alcohol precipitation on the filtrate (in the alcohol precipitation process, the final volume concentration of ethanol is 65%, and the alcohol precipitation time is 30 hours), centrifuging, taking supernate, and concentrating under reduced pressure to obtain concentrated solution I;
s4, subjecting the residue in the step S3 to three-stage ethanol extraction, wherein the three-stage ethanol extraction is implemented by sequentially using 30%, 50% and 70% ethanol in volume concentration, and the material-liquid ratio is 1: 8, extracting at 60 ℃ for 60min for 1 time, combining the extracting solutions, and concentrating under reduced pressure to obtain a concentrated solution II;
s5, combining the concentrated solution I and the concentrated solution II, and adjusting the pH value to 5; and (3) carrying out combined elution on the combined solution sequentially through D-101, AB-8 and XAD-6 macroporous resin, wherein the eluent is an ethanol eluent, the volume concentration of the ethanol is 80%, combining the eluates, and carrying out reduced pressure concentration and freeze drying to obtain the mulberry resource extract with the activity of inhibiting the African swine fever virus in vitro.
The prepared mulberry resource extract has performance index IC for inhibiting activity of African swine fever virus in vitro50(ug/mL) is shown in Table 1.
Example 2
A preparation method of mulberry resource extract with in vitro inhibitory activity on African swine fever virus comprises the following steps:
s1, taking fresh mulberry leaves and mulberry twigs, removing impurities, cleaning, drying and crushing;
s2, mixing the crushed mulberry leaves and mulberry twigs according to the mass ratio of 4: 1, mixing, adding a proper amount of hemicellulase solution, carrying out enzymolysis for 11 hours at the temperature of 40 ℃ and the pH value of 6.5;
s3, leaching the mixed powder of the mulberry leaves and the mulberry twigs subjected to enzymolysis in the step S2 for 1 time by using acidic hot water, wherein the leaching conditions of the acidic hot water are as follows: the ratio of material to liquid is 1: 15; the pH value is 1.0; the temperature of the hot water is 60 ℃; the extraction time is 120 min; leaching for 1 time by using alkaline hot water, wherein the alkaline hot water leaching conditions are as follows: the ratio of material to liquid is 1: 6; the pH value is 10.0; the temperature of the hot water is 60 ℃; the extraction time is 120 min; mixing extractive solutions, and collecting residue; filtering the extracting solution by a polysulfone membrane ultrafiltration membrane, wherein the relative molecular mass intercepted by the ultrafiltration membrane is 20000 daltons, collecting filtrate, carrying out alcohol precipitation on the filtrate (in the alcohol precipitation process, the final volume concentration of the ethanol is 60%, and the alcohol precipitation time is 36 hours), centrifuging, taking supernatant, and concentrating under reduced pressure to obtain concentrated solution I;
s4, subjecting the residue in the step S3 to three-stage ethanol extraction, wherein the three-stage ethanol extraction is implemented by sequentially using 30%, 50% and 70% ethanol in volume concentration, and the material-liquid ratio is 1: 8, extracting at 60 ℃ for 60min for 1 time, combining the extracting solutions, and concentrating under reduced pressure to obtain a concentrated solution II;
s5, combining the concentrated solution I and the concentrated solution II, and adjusting the pH value to 6; and (3) carrying out combined elution on the combined solution sequentially through XAD-1, DS-401 and DM-301 macroporous resin, wherein the eluent is an ethanol eluent, the volume concentration of the ethanol is 70%, combining the eluates, and carrying out reduced pressure concentration and spray drying to obtain the mulberry resource extract with the activity of inhibiting the African swine fever virus in vitro.
Performance index IC of mulberry resource extract prepared by the method and having in-vitro African swine fever virus inhibition activity50(ug/mL) is shown in Table 1.
Example 3
A preparation method of mulberry resource extract with in vitro inhibitory activity on African swine fever virus comprises the following steps:
s1, taking fresh mulberry leaves and mulberry twigs, removing impurities, cleaning, drying and crushing;
s2, mixing the crushed mulberry leaves and mulberry twigs according to the mass ratio of 6: 1, mixing, adding a proper amount of pectinase liquid, carrying out enzymolysis for 11 hours at the temperature of 60 ℃ and the pH value of 4.5;
s3, leaching the mixed powder of the mulberry leaves and the mulberry twigs subjected to enzymolysis in the step S2 for 1 time by using acidic hot water, wherein the leaching conditions of the acidic hot water are as follows: the ratio of material to liquid is 1: 6; the pH value is 3.0; the temperature of the hot water is 100 ℃; the extraction time is 60 min; leaching for 1 time by using alkaline hot water, wherein the alkaline hot water leaching conditions are as follows: the ratio of material to liquid is 1: 15; the pH value is 12.0; the temperature of the hot water is 80 ℃; the extraction time is 60 min; mixing extractive solutions, and collecting residue; filtering the extracting solution by a polyamide membrane ultrafiltration membrane, wherein the relative molecular mass intercepted by the ultrafiltration membrane is 50000 daltons, collecting filtrate, carrying out alcohol precipitation on the filtrate (in the alcohol precipitation process, the final volume concentration of ethanol is 70%, and the alcohol precipitation time is 24 hours), centrifuging, taking supernate, and concentrating under reduced pressure to obtain concentrated solution I;
s4, subjecting the residue in the step S3 to three-stage ethanol extraction, wherein the three-stage ethanol extraction is implemented by sequentially using 30%, 50% and 70% ethanol in volume concentration, and the material-liquid ratio is 1: 8, extracting at 60 ℃ for 60min for 1 time, combining the extracting solutions, and concentrating under reduced pressure to obtain a concentrated solution II;
s5, combining the concentrated solution I and the concentrated solution II, and adjusting the pH value to 4; and (3) the combined solution is sequentially subjected to HP-20, HZ-841 and HZ-806 macroporous resin for joint elution, the eluent is ethanol eluent, the volume concentration of the ethanol is 90%, the eluent is combined, and the mulberry resource extract with the activity of inhibiting the African swine fever virus in vitro is obtained by decompression concentration and vacuum drying.
Performance index IC of mulberry resource extract prepared by the method and having in-vitro African swine fever virus inhibition activity50(ug/mL) is shown in Table 1.
Comparative example 1:
a method for preparing mulberry resource extract with in vitro inhibitory activity on African swine fever virus, without the enzyme method auxiliary step of step S2); the rest is the same as example 1.
Performance index IC of mulberry resource extract prepared by the method and having in-vitro African swine fever virus inhibition activity50(ug/mL) is shown in Table 1.
Comparative example 2:
a method for preparing mulberry resource extract with in vitro inhibitory activity on African swine fever virus replaces the semi-bionic extraction step of step S3) with conventional hot water extraction: namely the ratio of material to liquid is 1: 10; the pH value is natural; the temperature of the hot water is 100 ℃; the extraction time is 90 min; the rest is the same as example 1.
Performance index IC of mulberry resource extract prepared by the method and having in-vitro African swine fever virus inhibition activity50(ug/mL) is shown in Table 1.
Comparative example 3
A preparation method of mulberry resource extract for inhibiting the activity of African swine fever virus in vitro, which replaces the tertiary alcohol extraction step of the step S4) with the conventional alcohol extraction: namely leaching by using 70% ethanol, wherein the material-liquid ratio is 1: 10, the extraction temperature is 60 ℃, the extraction time is 60min, and the extraction times are 3 times; the rest is the same as example 1.
Performance index IC of mulberry resource extract prepared by the method and having in-vitro African swine fever virus inhibition activity50(ug/mL) is shown in Table 1.
Comparative example 4
A preparation method of mulberry resource extract with in vitro inhibitory activity against African swine fever virus comprises replacing the combined elution and purification step of step S5) with elution with D-101 macroporous resin, wherein the eluate is ethanol eluate with volume concentration of 70%; the rest is the same as example 1.
Performance index IC of mulberry resource extract prepared by the method and having in-vitro African swine fever virus inhibition activity50(ug/mL) is shown in Table 1.
Comparative example 5
A preparation method of a mulberry resource extract with the in vitro activity of inhibiting African swine fever virus comprises the step S2), wherein the mass ratio of mulberry leaves to mulberry twigs is 2: 1; the rest is the same as example 1.
Performance index IC of mulberry resource extract prepared by the method and having in-vitro African swine fever virus inhibition activity50(ug/mL) is shown in Table 1.
Comparative example 6
A preparation method of a mulberry resource extract with the in vitro activity of inhibiting African swine fever virus comprises the step S2), wherein the mass ratio of mulberry leaves to mulberry twigs is 8: 1; the rest is the same as example 1.
Performance index IC of mulberry resource extract prepared by the method and having in-vitro African swine fever virus inhibition activity50(ug/mL) is shown in Table 1.
TABLE 1
| Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 | Comparative example 6 | |
| IC50（ug/mL） | 3.9 | 7.8 | 7.8 | 62.5 | 125.0 | 125.0 | 500.0 | 62.5 | 31.2 |
Claims (7)
1. A preparation method of mulberry resource extract with in vitro inhibitory activity on African swine fever virus comprises the following steps:
s1, taking fresh mulberry leaves and mulberry twigs, removing impurities, cleaning, drying and crushing;
s2, mixing the crushed mulberry leaves and mulberry twigs in proportion, adding a proper amount of enzyme solution, and performing enzymolysis for 11-13 hours;
s3, leaching the mixed powder of the mulberry leaves and the mulberry branches subjected to enzymolysis in the step S2 for 1 time by using acidic hot water, then leaching for 1 time by using alkaline hot water, combining extracting solutions, and reserving residues for later use; filtering the extracting solution by an ultrafiltration membrane, collecting filtrate, carrying out alcohol precipitation and centrifugation on the filtrate, taking supernatant, and carrying out reduced pressure concentration to obtain a concentrated solution I;
s4, extracting the residues in the step S3 by three-stage ethanol, combining extracting solutions, and concentrating under reduced pressure to obtain a concentrated solution II;
s5, combining the concentrated solution I and the concentrated solution II, and adjusting the pH value to be weakly acidic; the combined solution is sequentially subjected to combined elution through nonpolar, low-polarity and medium-polarity macroporous resins, the eluents are combined, and the mulberry resource extract with the activity of inhibiting the African swine fever virus in vitro is obtained after decompression, concentration and drying;
in the step S2, the mass ratio of the mulberry leaves to the mulberry twigs is 4: 1-6: 1;
in step S2, the enzyme type is selected from one or more of cellulase, hemicellulase or pectinase; the enzymolysis conditions are as follows: the enzymolysis temperature is 40-60 ℃; the pH value is 4.5-6.5;
in step S3, the acidic hot water leaching conditions are: the ratio of material to liquid is 1: 6-1: 15; the pH value is 1.0-3.0; the temperature of the hot water is 60-100 ℃; the extraction time is 60-120 min;
in step S3, the alkaline hot water leaching conditions are: the ratio of material to liquid is 1: 6-1: 15; the pH value is 10.0-12.0; the temperature of the hot water is 60-80 ℃; the extraction time is 60-120 min;
in step S3, the ultrafiltration membrane is selected from one of a cellulose acetate membrane, a polysulfone membrane and a polyamide membrane; the intercepted relative molecular mass of the ultrafiltration membrane is 20000-50000 daltons; in the alcohol precipitation process, the final volume concentration of the ethanol is 60-70%, and the alcohol precipitation time is 24-36 h;
in step S4, the three-stage ethanol extraction is performed by sequentially using 30%, 50%, and 70% ethanol by volume, where the ratio of material to liquid is 1: 8, extracting at the temperature of 55-65 ℃ for 55-65 min for 1-2 times;
in step S5, the nonpolar macroporous resin is selected from one of D-101, XAD-1 and HP-20; the weak-polarity macroporous resin is selected from one of AB-8, DS-401 and HZ-841; the medium-polarity macroporous resin is selected from one of XAD-6, DM-301 and HZ-806;
in step S5, the pH value of the combined solution is pH = 4-6; the eluent is ethanol eluent, and the volume concentration of the ethanol is 70-90%; the drying mode is selected from one of freeze drying, spray drying or vacuum drying.
2. The method for preparing a mulberry resource extract having an activity of inhibiting African swine fever virus in vitro according to claim 1, wherein in step S2, the mass ratio of mulberry leaves to mulberry twigs is 5: 1.
3. the method for preparing the mulberry resource extract with the activity of inhibiting the African swine fever virus according to claim 1, wherein in step S2, the enzymatic hydrolysis conditions are as follows: the enzymolysis temperature is 50 ℃; the pH was 5.0.
4. The method for preparing the mulberry resource extract having the activity of inhibiting African swine fever virus according to claim 1, wherein in step S3, the acidic hot water leaching conditions are as follows: the ratio of material to liquid is 1: 10; the pH value is 2.0; the hot water temperature was 80 ℃.
5. The method for preparing the mulberry resource extract with the activity of inhibiting the African swine fever virus according to claim 1, wherein in step S3, the alkaline hot water leaching conditions are as follows: the ratio of material to liquid is 1: 10; the pH value is 11.0; the hot water temperature was 70 ℃.
6. The method for preparing the mulberry resource extract having the activity of inhibiting African swine fever virus in vitro according to claim 1, wherein in step S5, the pH value of the combined solution is pH = 5.
7. A mulberry resource extract having an in vitro inhibitory activity against African swine fever virus obtained by the method according to any one of claims 1 to 6.
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