CN109939178A - The preparation method of the active compound of targeted inhibition African swine fever virus proliferation - Google Patents
The preparation method of the active compound of targeted inhibition African swine fever virus proliferation Download PDFInfo
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Abstract
The present invention relates to the preparation methods of the active compound of targeted inhibition African swine fever virus proliferation, specifically: each traditional Chinese medicine ingredients including rhizoma polygonati, Prunella vulgaris, radix achyranthis bidentatae, Radix Glycyrrhizae, lucidum spore powder, Schisandra chinensis, great burdock achene, evodia rutaecarpa, Echinacea, psoralea corylifolia, black nightshade and hypericum perforatum are subjected to pulverization process, decoction, filtering, adjustment carbon-nitrogen ratio first, are adjusted rear extracting solution；Extracting solution after adjustment is formulated as culture medium again, and using the stem and leaf of other each Chinese medicinal plants in addition to lucidum spore powder as to culture material, it will be cultivated in above-mentioned culture medium to culture material, then carry out expanding propagation, medicine efficacy screening successively to get active compound of the present invention.Active compound of the present invention is the means acquisition that fermentation is processed by biology, avoids the pollution generated because using chemical method；This active compound to African swine fever virus infection can targeted therapy, drug resistance will not be generated, drug effect is reliable, has no toxic side effect.
Description
Technical field
The invention belongs to application in TCM fields, and in particular to the active compound of targeted inhibition African swine fever virus proliferation
Preparation method.
Background technique
African swine fever is caused by African swine fever virus infection domestic pig and various wild boars (such as red river hog, aper)
A kind of acute, hemorrhagic, deadly infectious disease.World Organization for Animal Health (OIE) is classified as statutory report animal epidemic, the disease
It is also a kind of animal epidemics of China's guard key.It is characterized in that pathogenic process is short, the most acute and acute infection death rate is up to
100%, clinical manifestation is fever (up to 40~42 DEG C), and palpitating speed, expiratory dyspnea, part cough, eye, nose have serosity or viscous
Fluidity purulent secretion, skin cyanosis, the obvious bleeding of lymph node, kidney, gastrointestinal mucosa, African swine fever clinical symptoms and swine fever symptom
It is similar, it can only be made a definite diagnosis by Laboratory Monitoring.
African swine fever virus is the important member that African swine fever section African swine fever virus belongs to, some viral characteristics are similar to iris
Viraceae and Poxviridae.The diameter of virion is 175-215 nanometers, symmetrical in 20 face bodies, there is cyst membrane.Genome is bifilar
Linear DNA, size 170-190kb.In pig body, African swine fever virus can be especially netted in the cytoplasm of several types
It is replicated in endothelial cell and mononuclear macrophage.The virus can be proliferated in turicata, and become main communication media.
At this stage, the vaccine that can effectively prevent African swine fever is not developed in world wide, for African swine fever disease
The drug of poison is also very few, this is for inhibiting very unfavorable for African swine fever virus proliferation.Therefore, us are needed and develops one
Kind is safe and reliable and effectively treats African swine fever virus and does not generate the drug of drug resistance, to cope with the demand in market.
Summary of the invention
In order to solve the above problems existing in the present technology, the present invention provides targeted inhibition African swine fever virus proliferation
The preparation method of active compound.
The technical scheme adopted by the invention is as follows:
The preparation method of the active compound of targeted inhibition African swine fever virus proliferation, includes the following steps:
(1) rhizoma polygonati, Prunella vulgaris, radix achyranthis bidentatae, Radix Glycyrrhizae, Schisandra chinensis, great burdock achene, evodia rutaecarpa, Echinacea, psoralea corylifolia, black nightshade are taken
Successively cleaned, dried with hypericum perforatum, being crushed and lucidum spore powder mixing after obtain crush raw material；
(2) water is added into the crushing raw material, is filtered after decoction, is concentrated, obtains sediment after alcohol precipitation, then will
The sediment is dissolved, and Chinese medicine extract is obtained；
(3) carbon-nitrogen ratio for adjusting the Chinese medicine extract, is adjusted rear extracting solution；
(4) extracting solution after the adjustment is prepared as culture medium；
(5) take in step (1) in addition to lucidum spore powder the stem and leaf of other each Chinese medicinal plants successively carry out disinfection, clean,
It cuts, obtains being cultivated described be put into culture material to obtain cultured products in the culture medium, by institute to culture material
Cultured products are stated after shaking table expands numerous, medicine efficacy screening, obtain the active compound of the targeted inhibition African swine fever virus proliferation.
Further, in step (1), the proportion of each raw material is rhizoma polygonati 10%, Prunella vulgaris 9%, radix achyranthis bidentatae 8%, sweet
Grass 8%, Schisandra chinensis 8%, great burdock achene 8%, evodia rutaecarpa 9%, Echinacea 9%, psoralea corylifolia 7%, black nightshade 8%, hypericum perforatum 8%,
Lucidum spore powder 8%.
Further, in step (1), each raw material is crushed to -250 mesh of 150 mesh.
Further, in step (2), the mass ratio for crushing raw material and water is 1:(8-12).
Further, described to decoct to be cooked by slow fire in step (2), the decocting time is 1.5-2.5h.
Further, in step (2), the concentration is heating concentration, and the heating time is 1-2h.
It further, is that 85%-95% ethanol solution is small to the concentrated liquid alcohol precipitation 2-3 using concentration in step (2)
When, ethyl alcohol is 5-10 times of concentrated liquid quality.
Further, in step (2), sediment is dissolved using reverse osmosis water, the reverse osmosis water is sediment
5-7 times of quality.
Further, in step (3), using ammonium sulfate and glucose by the carbon-nitrogen ratio of the Chinese medicine extract adjust to
(25-29):1。
Further, in step (4), the group of the culture medium is grouped as are as follows: extracting solution 96.248%- after adjustment
96.948%, glucose 1.2%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.15%, yeast extract 0.1%, vitamin B10.01‰、
Vitamin B60.01 ‰, agar 1.3%-2%.
Further, in step (4) or the group of the culture medium is grouped as are as follows: extracting solution 94.3%- after adjustment
94.7%, sucrose 3%, sodium nitrate 0.3%, magnesium sulfate 0.05%, potassium chloride 0.05%, dipotassium hydrogen phosphate 0.1%, tannin
0.1%, peptone 0.1%, agar 1.6%-2%.
Further, in step (5), the weight of the stem and leaf is the 0.5%-1.5% of each Chinese medicinal plant weight.
Further, in step (5), it is the sodium hypochlorite of 8%-12% that the disinfection, which uses concentration,.
Further, in step (5), the time of the disinfection is 8-12min.
Further, in step (5), the stem or leaf are cut to 1cm × 1cm.
Further, in step (5), the cultivation temperature is 30-37 DEG C, and the culture humidity is 40%-50%, described
Incubation time is 5-7 days.
Further, in step (5), the cultured products carry out expansion cultivating system composition when shaking table expands numerous are as follows: adjust
Extracting solution 95.42%, cultured products 1.5%, glucose 2%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.25%, peptone after whole
0.2%, phenylalanine 0.07%, tyrosine 0.06%.
Further, the expansion cultivating system composition in step (5) or when cultured products progress shaking table expands numerous
Are as follows: extracting solution 94.3%, cultured products 2%, sucrose 3%, sodium nitrate 0.3%, magnesium sulfate 0.05%, potassium chloride after adjustment
0.05%, potassium dihydrogen phosphate 0.1%, tannin 0.1%, peptone 0.1%.
Further, in step (5), it is 25-37 DEG C that the shaking table, which expands numerous temperature, and the revolving speed is 130-170rpm,
The numerous number of days of expansion is 5-7 days.
Further, the active compound of the targeted inhibition African swine fever virus proliferation inhibits African swine fever disease in preparation
Application in malicious hyperproliferation agent.
It underdraws below to effect caused by raw material in the present invention.
Rhizoma polygonati, mild-natured, sweet in flavor, returns spleen, lung, kidney channel.Major function: boosting qi and nourishing yin, invigorating the spleen, moistening lung, kidney-nourishing.For taste
Weakness, fatigue and asthenia, dry deficiency of food, deficiency syndrome of the lung cough caused by dryness, asthenia of essence and blood, Heat Diabetes.
Prunella vulgaris, cold in nature, acrid flavour, hardship return liver, gallbladder channel.Major function: relieving inflammation or internal heat, improving eyesight, dissipating bind, detumescence.It is swollen with hot eyes is controlled
Bitterly, eyeball night pain, headache and dizziness, scrofula, goiter, acute mastitis swelling and pain, Thyroid Gland Swell, scrofula, the proliferation of mammary gland, high blood
Pressure.
Radix achyranthis bidentatae, mild-natured, bitter, acid, channel tropism.Major function: the function of nourishing, such as the power of ox.Cure mainly cold-dampness flaccidity syndrome and arthralgia syndrome, four limbs
Muscular constricture, goinyalgia be unable to flex and stretch the extremities, by vim and vigour, that fire of getting mildewed in warm conditions is rotten, long term usage is made light of one's life by commiting suicide is resistance to old, informal dress have tonify the liver and kidney, strong waist and knee, promoting blood circulation,
The effect ensuringd proper downward flow of the blood.
Radix Glycyrrhizae, mild-natured, sweet in flavor, return heart, stomach, spleen, lung channel.Major function: invigorating the spleen and benefiting qi, cough-relieving apophlegmatic, emergency analgesic therapy, adjusting
And pharmacological property.Treat that the weakness of the spleen and the stomach, deficiency of qi in middle-jiao, cough and asthma, skin sore, contraction suddenly has a pain, mitigates that drug is strong, antidote in abdomen
Use is preferably processed in poison, heat-clearing Ying Shengyong, bowl spares.
Lucidum spore powder, mild-natured, sweet in flavor, return heart, lung, liver and kidney channel.Major function: to consumptive disease, cough, asthma, insomnia,
The diseases such as indigestion, malignant tumour have therapeutic effect.
Schisandra chinensis, warm-natured, sour, return lung, kidney, the heart channel of Hang-Shaoyin.Major function: astringing lung-QI nourshing kidney, promote the production of body fluid arrest sweating, arresting seminal emission antidiarrheal, rather
Heart and tranquilizing mind.For chronic cough and dyspnea of deficiency type, injury thirst, spontaneous sweating, emission due to the kidney deficiency, spleen kidney deficiency rushes down, palpitation and insomnia.
Great burdock achene, cold in nature, acrid flavour, hardship attach to the lung and stomach meridians.Major function: diverging wind-heat, promoting eruption of detoxifying, relieving sore-throat.For outer
Feel wind-heat, abscess of throat, measles without adequate eruption, wind-heat dermexanthesis, pathopyretic ulcer, mumps swelling and pain.
Evodia rutaecarpa, property heat, acrid flavour, hardship return liver, spleen, stomach meridian.Major function: eliminating cold to stop pain, soothing the liver lower gas, eliminating dampness drop are inverse.
For jueyin headache, cold hernia stomachache, cold of insufficiency type diarrhea, abdominal distention, vomiting acid regurgitation, foot adverse QI rushing upwards, aphtha aphthae in children.
Echinacea can stimulate the vigor of the immunocytes such as the intracorporal leucocyte of people, have effects that strengthen immunity, also
It can be used for assisting in the treatment of flu, cough and the infection of the upper respiratory tract, for treating skin disease and oidiomycosis, herpes simplex virus
Infection.
Psoralea corylifolia, warm-natured, acrid flavour, hardship return kidney, the spleen channel.Major function: warming kidney and enhancing yang, receiving gas, antidiarrheal.It is lost for impotence
Essence, enuresis frequent micturition, waist and knee crymodynia, kidney deficiency characterized by dyspnea, Diarrhoea, external application curing vitiligo, alopecia areata.
Black nightshade, cold in nature, bitter return liver, stomach meridian.Major function: clearing heat and detoxicating, activating blood circulation and reducing swelling.For cat fever, tooth
Bitterly, chronic bronchitis, acute nephritis, mazoitis etc..
Hypericum perforatum, mild-natured, bitter, puckery, return liver warp.Major function: astringing to arrest bleeding, menstruation regulating promote lactation, are clearing heat and detoxicating, is sharp
It is wet.For spitting blood, spitting blood, discharging fresh blood stool, metrorrhagia and metrostaxis, traumatic hemorrhage, irregular menstruation, newborn woman's agalactia, jaundice, have sore throat,
Red eye, swell pain, urinary tract infections, mouth and nose raw sore, carbuncle and furuncle and pyogenic infections, burn and scald.
The invention has the benefit that
The preparation method of the active compound of targeted inhibition African swine fever virus proliferation of the present invention, specifically: first
Will include rhizoma polygonati, Prunella vulgaris, radix achyranthis bidentatae, Radix Glycyrrhizae, lucidum spore powder, Schisandra chinensis, great burdock achene, evodia rutaecarpa, Echinacea, psoralea corylifolia,
Each traditional Chinese medicine ingredients including black nightshade and hypericum perforatum carry out pulverization process, decoction, filtering, adjustment carbon-nitrogen ratio, mention after being adjusted
Take liquid；Extracting solution after adjustment is formulated as culture medium again, and is made with the stem and leaf of other each Chinese medicinal plants in addition to lucidum spore powder
Will to cultivate in above-mentioned culture medium to culture material to culture material, then expanding propagation, medicine efficacy screening are successively carried out,
Up to active compound of the present invention.Active compound of the present invention is the means acquisition that fermentation is processed by biology, is kept away
The pollution generated because using chemical method is exempted from；This active compound to African swine fever virus infection can targeted therapy, no
Drug resistance can be generated, drug effect is reliable, has no toxic side effect.
The active compound of targeted inhibition African swine fever virus proliferation of the present invention mainly has for African swine fever virus
There is following effect: (1) activating and enhance the immune function of macrophage；(2) inhibit the synthesis of African swine fever virus DNA, in turn
Inhibit the proliferation of virus.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 is the aspect graph after MDBK cell infection VSV virus；
Fig. 2 is normal MDBK cellular morphology figure；
Fig. 3 is 3 gained active compound of embodiment of the present invention dilution 107The aspect graph after MDBK cell is acted on after times；
Fig. 4 is 3 gained active compound of embodiment of the present invention dilution 108The aspect graph of MDBK cell is acted on after times.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, technical solution of the present invention will be carried out below
Detailed description.Obviously, described embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.
Based on the embodiments of the present invention, those of ordinary skill in the art obtained institute without making creative work
There is other embodiment, belongs to the range that the present invention is protected.
Embodiment 1
The present embodiment provides the preparation methods of the active compound of targeted inhibition African swine fever virus proliferation, including walk as follows
It is rapid:
(1) rhizoma polygonati 10g, Prunella vulgaris 9g, radix achyranthis bidentatae 8g, Radix Glycyrrhizae 8g, Schisandra chinensis 8g, great burdock achene 8g, evodia rutaecarpa 9g, purple cone are taken
Chrysanthemum 9g, it psoralea corylifolia 7g, black nightshade 8g, hypericum perforatum 8g, successively cleaned, dried, 150 mesh being crushed to, with 8g lucidum spore powder
It obtains crushing raw material after mixing；
(2) water of 8 times of quality is added into the crushing raw material, is cooked by slow fire 1.5h, then heats 1h and be concentrated, made
Sediment was obtained in 2 hours to the concentrated liquid alcohol precipitation with 85% ethanol solution of 5 times of quality, then reuses 5 times of quality
Reverse osmosis water dissolves the sediment, obtains Chinese medicine extract；
(3) carbon-nitrogen ratio of the Chinese medicine extract is adjusted to 25:1 using ammonium sulfate and glucose, is mentioned after being adjusted
Take liquid；
(4) take extracting solution after the adjustment of suitable weight, glucose, dipotassium hydrogen phosphate, magnesium sulfate, yeast extract, vitamin B1,
Vitamin B6 and agar are uniformly mixed, and obtain culture medium, the culture medium composition are as follows: extracting solution 96.948%, grape after adjustment
Sugared 1.2%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.15%, yeast extract 0.1%, vitamin B10.01 ‰, vitamin
B60.01 ‰, agar 1.3%；
(5) stem and leaf for taking other each Chinese medicinal plant weight 0.5% in addition to lucidum spore powder in step (1), use 8%
Hypochlorite disinfectant 8min is cut to 1cm × 1cm size, is subsequently placed into culture medium and is cultivated after cleaning, 30 DEG C of cultures 5
It, culture humidity is 40%, obtains cultured products；
(6) cultured products carry out expansion cultivating system composition when shaking table expands numerous are as follows: extracting solution after adjustment
95.42%, cultured products 1.5%, glucose 2%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.25%, peptone 0.2%, phenylpropyl alcohol
Above-mentioned each component is carried out shaking table after mixing and expands numerous, shaking speed 130rpm by propylhomoserin 0.07% and tyrosine 0.06%,
25 DEG C are cultivated 5 days, and the active compound of the targeted inhibition African swine fever virus proliferation is obtained after medicine efficacy screening.
Embodiment 2
The present embodiment provides the preparation methods of the active compound of targeted inhibition African swine fever virus proliferation, including walk as follows
It is rapid:
(1) rhizoma polygonati 10g, Prunella vulgaris 9g, radix achyranthis bidentatae 8g, Radix Glycyrrhizae 8g, Schisandra chinensis 8g, great burdock achene 8g, evodia rutaecarpa 9g, purple cone are taken
Chrysanthemum 9g, it psoralea corylifolia 7g, black nightshade 8g, hypericum perforatum 8g, successively cleaned, dried, 250 mesh being crushed to, with 8g lucidum spore powder
It obtains crushing raw material after mixing；
(2) water of 12 times of quality is added into the crushing raw material, is cooked by slow fire 2.5h, then heats 2h and be concentrated,
Sediment was obtained in 3 hours to the concentrated liquid alcohol precipitation using 95% ethanol solution of 10 times of quality, then reuses 7 times of quality
Reverse osmosis water the sediment is dissolved, obtain Chinese medicine extract；
(3) carbon-nitrogen ratio of the Chinese medicine extract is adjusted to 29:1 using ammonium sulfate and glucose, is mentioned after being adjusted
Take liquid；
(4) extracting solution, sucrose, sodium nitrate, magnesium sulfate, potassium chloride, potassium dihydrogen phosphate, pellet after the adjustment of suitable weight are taken
Rather, peptone and agar are uniformly mixed, and obtain culture medium, the culture medium composition are as follows: extracting solution 94.3%, sucrose after adjustment
3%, sodium nitrate 0.3%, magnesium sulfate 0.05%, potassium chloride 0.05%, dipotassium hydrogen phosphate 0.1%, tannin 0.1%, peptone
0.1% and agar 2%；
(5) stem and leaf for taking other each Chinese medicinal plant weight 1.5% in addition to lucidum spore powder in step (1), use 12%
Hypochlorite disinfectant 12min, 1cm × 1cm size is cut to after cleaning, is subsequently placed into the culture medium and is cultivated, 37
DEG C culture 7 days, culture humidity be 50%, obtain cultured products；
(6) cultured products carry out expansion cultivating system composition when shaking table expands numerous are as follows: extracting solution 94.3% after adjustment,
Cultured products 2%, sucrose 3%, sodium nitrate 0.3%, magnesium sulfate 0.05%, potassium chloride 0.05%, potassium dihydrogen phosphate 0.1%, pellet
Peaceful 0.1% and peptone 0.1%, above-mentioned each component is carried out to shaking table after mixing and expands numerous, shaking speed 170rpm, 37 DEG C
Culture 7 days obtains the active compound of the targeted inhibition African swine fever virus proliferation after medicine efficacy screening.
Embodiment 3
The present embodiment provides the preparation methods of the active compound of targeted inhibition African swine fever virus proliferation, including walk as follows
It is rapid:
(1) rhizoma polygonati 10g, Prunella vulgaris 9g, radix achyranthis bidentatae 8g, Radix Glycyrrhizae 8g, Schisandra chinensis 8g, great burdock achene 8g, evodia rutaecarpa 9g, purple cone are taken
Chrysanthemum 9g, it psoralea corylifolia 7g, black nightshade 8g, hypericum perforatum 8g, successively cleaned, dried, 200 mesh being crushed to, with 8g lucidum spore powder
It obtains crushing raw material after mixing；
(2) water of 10 times of quality is added into the crushing raw material, is cooked by slow fire 2h, then heats 1.5h and be concentrated,
Sediment was obtained in 2.5 hours to the concentrated liquid alcohol precipitation using 90% ethanol solution of 8 times of quality, then reuses 6 times of matter
The reverse osmosis water of amount dissolves the sediment, obtains Chinese medicine extract；
(3) carbon-nitrogen ratio of the Chinese medicine extract is adjusted to 27:1 using ammonium sulfate and glucose, is mentioned after being adjusted
Take liquid；
(4) extracting solution, glucose, dipotassium hydrogen phosphate, magnesium sulfate, yeast extract, vitamin B after the adjustment of suitable weight are taken1、
Vitamin B6It is uniformly mixed with agar, obtains culture medium, the culture medium composition are as follows: extracting solution 96.548%, grape after adjustment
Sugared 1.2%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.15%, yeast extract 0.1%, vitamin B10.01 ‰, vitamin
B60.01 ‰, agar 1.7%；
(5) stem and leaf for taking other each Chinese medicinal plant weight 1% in addition to lucidum spore powder in step (1), use 10%
Hypochlorite disinfectant 10min is cut to 1cm × 1cm size after cleaning, be subsequently placed into the culture medium and cultivated, and 34 DEG C
Culture 6 days, culture humidity are 45%, obtain cultured products；
(6) cultured products carry out expansion cultivating system composition when shaking table expands numerous are as follows: extracting solution after adjustment
95.42%, cultured products 1.5%, glucose 2%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.25%, peptone 0.2%, phenylpropyl alcohol
Above-mentioned each component is carried out shaking table after mixing and expands numerous, shaking speed 150rpm by propylhomoserin 0.07% and tyrosine 0.06%,
31 DEG C are cultivated 6 days, and the active compound of the targeted inhibition African swine fever virus proliferation is obtained after medicine efficacy screening.
Experimental example
Cell toxicity test and effect are carried out to the active compound of 3 gained targeted inhibition African swine fever virus of embodiment proliferation
Fruit test.
One, test objective
It is tested by cell bio-activity, can detection 3 gained active compound sample of the embodiment of the present invention improve cell
Immunity and toxicity to cell.
Two, test method
1. the recovery and culture of bovine kidney cells (MDBK)；
Viral 2. (VSV) malicious valence measurement；
3. sample dilutes: taking in active compound sample 30ul to No. 1 pipe of 3 gained of the embodiment of the present invention, add dilution
270ul is to get 10 times of dilute samples；It takes 30ul sample into No. 2 pipes from No. 1 pipe, adds dilution 270ul dilute to get 100 times
Release sample；It takes 30ul sample into No. 3 pipes from No. 2 pipes, adds dilution 270ul to get 1000 times of dilute samples, with such
It pushes away to get 10,100,1000,104、105、106、107、108、109、1010Times dilute sample；
4. sample accesses MDBK cell: taking each extension rate sample 100ul, be sequentially ingressed into 96 from small to large by extension rate
In the G row of orifice plate, H behavior repetitive operation observes cell by the metamorphosis after drug effect；
5. inoculation VSV virus: being respectively connected to 100ul virus (VSV) into each hole of G, H row, observe cell death feelings afterwards for 24 hours
Condition.
Three, result is observed
Observe this product dilution 10,100,1000,104、105、106、107、108、109、1010The cell of dilute sample again is dead
Situation is died, as a result such as table 1:
1 experimental observation result of table
Note: "-" indicates 70% or more cell death；The cell death of "+" expression 40-70%；" ++ " indicates 10-40%
Cell death；" +++ " indicates 10% cell death below.
Fig. 1-Fig. 4 is respectively virus function in cell, blank control, only 107Times dilute sample acts on cell, only 108Times
Dilute sample acts on the cellular morphology figure after cell, it can be seen that after virus infected cell, cellular morphology changes, carefully
Born of the same parents' death rate increases significantly, and 107Times dilute sample and 108Times dilute sample acts on cell, does not have toxicity, cell to cell
Form is good.
Four, conclusion (of pressure testing)
By related data in table 1 it follows that
1. 3 gained active compound sample of embodiment of the present invention dilution 106There is protective effect to MDBK cell after times, has
Inhibit the function of VSV virus infected cell, but effect is unobvious；
2. 3 gained active compound sample of embodiment of the present invention dilution 104There is good protection to make in MDBK cell after times
With having the function of inhibiting VSV virus infected cell, effect is obvious.
This test is carried out using VSV virus substitution African swine fever virus, 3 gained activity of the embodiment of the present invention can be deduced
Composition can protect cell, from the infection of African swine fever virus.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain
Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (10)
1. the preparation method of the active compound of targeted inhibition African swine fever virus proliferation, which comprises the steps of:
(1) rhizoma polygonati, Prunella vulgaris, radix achyranthis bidentatae, Radix Glycyrrhizae, Schisandra chinensis, great burdock achene, evodia rutaecarpa, Echinacea, psoralea corylifolia, black nightshade are taken and is passed through
Leaf Fructus Forsythiae successively cleaned, dried, being crushed and lucidum spore powder mixing after obtain crush raw material；
(2) water is added into the crushing raw material, is filtered, is concentrated after decoction, obtains sediment after alcohol precipitation, it then will be described
Sediment is dissolved, and Chinese medicine extract is obtained；
(3) carbon-nitrogen ratio for adjusting the Chinese medicine extract, is adjusted rear extracting solution；
(4) extracting solution after the adjustment is prepared as culture medium；
(5) take in step (1) that the stem and leaf of other each Chinese medicinal plants are successively carried out disinfection, clean, cut in addition to lucidum spore powder,
It obtains being cultivated described be put into culture material to obtain cultured products in the culture medium, by the training to culture material
Product is supported after shaking table expands numerous, medicine efficacy screening, obtains the active compound of the targeted inhibition African swine fever virus proliferation.
2. the preparation method of the active compound of targeted inhibition African swine fever virus proliferation according to claim 1, special
Sign is, in step (1), the proportion of each raw material is rhizoma polygonati 10%, Prunella vulgaris 9%, radix achyranthis bidentatae 8%, Radix Glycyrrhizae 8%, the five tastes
Sub 8%, great burdock achene 8%, evodia rutaecarpa 9%, Echinacea 9%, psoralea corylifolia 7%, black nightshade 8%, hypericum perforatum 8%, lucidum spore powder
8%.
3. the preparation method of the active compound of targeted inhibition African swine fever virus proliferation according to claim 1, special
Sign is, in step (1), each raw material is crushed to -250 mesh of 150 mesh.
4. the preparation method of the active compound of targeted inhibition African swine fever virus proliferation according to claim 1, special
Sign is, in step (2), the mass ratio for crushing raw material and water is 1:(8-12)；
Described to decoct to be cooked by slow fire, the decocting time is 1.5-2.5h；
The concentration is heating concentration, and the heating time is 1-2h；
It is 85%-95% ethanol solution to the concentrated liquid alcohol precipitation 2-3 hours using concentration, ethyl alcohol is concentrated liquid quality
5-10 times；
Sediment is dissolved using reverse osmosis water, the reverse osmosis water is 5-7 times of sediment quality.
5. the preparation method of the active compound of targeted inhibition African swine fever virus proliferation according to claim 1, special
Sign is, in step (3), is adjusted the carbon-nitrogen ratio of the Chinese medicine extract to (25-29) using ammonium sulfate and glucose: 1.
6. the preparation method of the active compound of targeted inhibition African swine fever virus proliferation according to claim 1, special
Sign is that in step (4), the group of the culture medium is grouped as are as follows: extracting solution 96.248%-96.948%, glucose after adjustment
1.2%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.15%, yeast extract 0.1%, vitamin B10.01 ‰, vitamin B60.01‰、
Agar 1.3%-2%；
Alternatively, the group of the culture medium is grouped as are as follows: extracting solution 94.3%-94.7%, sucrose 3%, sodium nitrate after adjustment
0.3%, magnesium sulfate 0.05%, potassium chloride 0.05%, dipotassium hydrogen phosphate 0.1%, tannin 0.1%, peptone 0.1%, agar
1.6%-2%.
7. the preparation method of the active compound of targeted inhibition African swine fever virus proliferation according to claim 1, special
Sign is, in step (5), the weight of the stem and leaf is the 0.5%-1.5% of each Chinese medicinal plant weight；
It is the sodium hypochlorite of 8%-12% that the disinfection, which uses concentration, and the time of the disinfection is 8-12min.
8. the preparation method of the active compound of targeted inhibition African swine fever virus proliferation according to claim 1, special
Sign is, in step (5), the stem or leaf are cut to 1cm × 1cm；
The cultivation temperature is 30-37 DEG C, and the culture humidity is 40%-50%, and the incubation time is 5-7 days.
9. the preparation method of the active compound of targeted inhibition African swine fever virus proliferation according to claim 1, special
Sign is, in step (5), the cultured products carry out expansion cultivating system composition when shaking table expands numerous are as follows: extracting solution after adjustment
95.42%, cultured products 1.5%, glucose 2%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.25%, peptone 0.2%, phenylpropyl alcohol
Propylhomoserin 0.07%, tyrosine 0.06%；
Alternatively, the cultured products carry out expansion cultivating system composition when shaking table expands numerous are as follows: extracting solution 94.3%, training after adjustment
Support product 2%, sucrose 3%, sodium nitrate 0.3%, magnesium sulfate 0.05%, potassium chloride 0.05%, potassium dihydrogen phosphate 0.1%, tannin
0.1%, peptone 0.1%；
It is 25-37 DEG C that the shaking table, which expands numerous temperature, and the revolving speed is 130-170rpm, and the numerous number of days of expansion is 5-7 days.
10. the active compound of targeted inhibition African swine fever virus proliferation inhibits African pig in preparation according to claim 1
Application in pestivirus hyperproliferation agent.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1960743A (en) * | 2003-04-16 | 2007-05-09 | 薰衣草作物项目有限公司 | Herbal composition |
| CN105902989A (en) * | 2016-06-22 | 2016-08-31 | 蒙焕文 | Medicine for preventing and treating hog cholera |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1960743A (en) * | 2003-04-16 | 2007-05-09 | 薰衣草作物项目有限公司 | Herbal composition |
| CN105902989A (en) * | 2016-06-22 | 2016-08-31 | 蒙焕文 | Medicine for preventing and treating hog cholera |
Non-Patent Citations (2)
| Title |
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| COSTARD S，等: "Epidemiology of African swine fever virus", 《VIRUS RESERCH》 * |
| 刘冰: "中国非洲猪瘟最先在母猪群和育肥猪群发生的原因探讨 ", 《饲料与畜牧》 * |
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Application publication date: 20190628 |