CN110638871B

CN110638871B - Anti-bee paralysis virus extract and preparation method thereof

Info

Publication number: CN110638871B
Application number: CN201910966087.0A
Authority: CN
Inventors: 侯春生; 邓帅; 杨卅; 刁青云; 王新玲
Original assignee: Institute of Apicultural Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Apicultural Research of Chinese Academy of Agricultural Sciences
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2021-07-13
Anticipated expiration: 2039-10-12
Also published as: CN110638871A

Abstract

The invention discloses a composition for resisting bee paralytic virus, and a preparation and application thereof. The composition for resisting the bee paralytic virus is characterized by comprising the following components in percentage by mass: 50-65% of cinnamon, 25-35% of Balaurel and 5-25% of ginseng flower. The composition for resisting the bee paralysis virus is suitable for preventing and treating the bee paralysis and improving the overall antiviral ability of bee colonies.

Description

Anti-bee paralysis virus extract and preparation method thereof

Technical Field

The invention relates to an anti-bee paralysis virus extract and a preparation method thereof.

Background

Bees are social insects, and like other livestock and poultry animals, bees are also attacked by various diseases such as bacteria, fungi, viruses, parasites and the like, and among the various diseases of the bees, bee virus diseases are the most special and the most complex disease causing mechanisms. In addition, the globalization of bees and bee product trade leads to the rapid spread of bee diseases and enemies in different areas of the world, and seriously threatens the health of the bees. The virus disease is the most serious disease which is prevalent so far, and is also one of the important factors threatening the health of the bee colony. Although some chemicals are available, the efficacy is not significant and the virus is not completely eliminated, resulting in a latent infection of the virus in the bee colony and a new pandemic in the spring of the next year.

The mechanism for preventing and treating the bee paralytic virus globally available at present is mainly aimed at inhibiting or blocking RNA polymerase or helicase dependent on virus RNA. The rapid propagation of the virus can be reduced at that time, but the virus has the biggest characteristic of extremely rapid variation, and is easy to escape from host or drug resistance so as to change the sequence of the original gene, so that the virus disease cannot be effectively prevented and treated, and the risk of polluting bee products exists.

The natural product is considered to be the most widely used raw material for drug development or disease prevention and control all the time, and has the advantages of no toxicity, no harm, no toxic or side effect, easy degradation, no residue and capability of exerting the function of natural product functional molecules to the maximum extent.

The bee virus disease, especially one of the diseases with the biggest damage of paralysis disease to bees, is not solved completely by the insect virus field, once bees are infected, the bees are easy to remain latent, and great infectious sources and damage are caused.

Traditionally, viral diseases have not been treated as truly as other bacterial and parasitic diseases. However, with the rapid development of molecular biotechnology in this field, new substances with antiviral properties are continuously discovered. The existing medicine has the defects of large toxic and side effects, long-term use, inaccurate compatibility, temporary solution, virus eradication, failure in inhibiting the diffusion of the paralytic virus of bees, low immunity of bee colonies and finally death of the bee colonies.

Disclosure of Invention

The invention is based on modern biotechnology, and long-term experimental verification, is prepared by adopting a new process, and proves that the plant extract has great potential in treating the intractable bee disease caused by virus infection such as bee acute paralysis, israel bee paralysis, bee chronic paralysis and the like by preventing and treating a large number of bee colonies caused by bee paralysis virus.

Based on the above, the invention aims to provide the anti-bee paralysis virus composition which has proper drug compatibility, maintains the original natural component effects through a modern extraction process, has the effects of resisting virus, enhancing immunity, improving bee colony health and the like, is suitable for preventing and treating israeli bee paralysis, bee acute paralysis and bee chronic paralysis, as well as an extracting solution and a preparation thereof, and a preparation method thereof.

The composition for resisting the bee paralytic virus is composed of the following components in percentage by mass: 50-65% of salvia miltiorrhiza and cassia bark leaf, 25-35% of august leaf and 5-25% of ginseng flower.

As a preferable scheme of the invention, the composition for resisting the bee paralytic virus comprises 55% of cortex cinnamomi, 30% of fructus cinnamomi, and 15% of ginseng flower by mass.

The invention also provides an anti-bee paralysis virus extract.

The anti-bee paralytic virus extract is prepared by utilizing the composition, and the preparation method of the anti-bee paralytic virus extract comprises the following steps: extracting saponin from the ginseng flower, and then mixing the saponin with the salvia miltiorrhiza and cinnamomum japonicum leaf powder to obtain the bee paralysis virus resistant extract. Wherein, the method for extracting saponin by using ginseng flower is to extract by using 60-75% ethanol solution as solvent. The powder of the red sage root and cassia twig is obtained by superfine grinding, and the particle size is preferably 80-120 meshes.

In one embodiment of the present invention, the preparation method of the anti-bee paralytic virus extract comprises the following steps:

1) leaching flos Ginseng with 60-75 vol% ethanol solution as solvent, collecting the percolate, recovering ethanol, and concentrating at 55 deg.C to obtain flos Ginseng extractive solution with relative density of 1.25-1.50; the main component of the ginseng flower extract is saponin.

2) Pulverizing cortex Cinnamomi and Augusticica leaves by 80-120 mesh, and sieving;

3) adding the powder of the salvia miltiorrhiza and cinnamomum bayenii leaves in the step 2) and the saponin extracting solution obtained in the step 1) into water, uniformly mixing, filtering (80-120 meshes), and concentrating the filtrate at 55 ℃ to obtain a concentrated solution with the relative density of 1.25-1.50, namely the bee paralysis virus resisting extract.

The above extract can also be made into capsule, paste or spray.

The application of the composition for resisting the bee paralysis virus or the extract for resisting the bee paralysis virus in preventing and treating the bee paralysis disease and the application in improving the integral resistance of bee colonies to the bee paralysis virus also belong to the protection scope of the invention.

The composition for resisting the bee paralysis virus has proper compatibility of medicines, maintains the original natural component effects through a modern extraction process, has the effects of resisting viruses, enhancing immunity, improving bee colony health and the like, is suitable for resisting the bee paralysis virus for preventing and treating the israeli bee paralysis, the bee acute paralysis and the bee chronic paralysis, and has excellent effect.

Drawings

FIG. 1 shows the antiviral effect of the composition against poliovirus of example 3.

Fig. 2 is a comparison of the test results of dead bees on the fifth and sixth days with the blank control group.

Lanes

5 and 6 are blank control bee samples on fifth and sixth days, respectively;

lanes

1, 7 are day five and six virus-infected cohort bee-like samples;

lanes

4, 8 are day six virus-infected cohort bee-like samples;

lanes

2 and 3 are control bee samples at day five and six virus plus antiviral extract doses of 100 μ g/mL;

lanes

9 and 10 are bee samples from day five and six virus groups at a dose of 10 μ g/mL antiviral extract; lane 11 is a fifth day virus plus antiviral extract dose of 50. mu.g/mL swarms.

Detailed Description

The methods in the following examples are conventional methods unless otherwise specified.

The materials used in the examples of the invention are as follows:

leaf of red-rooted cinnamon: purchased from online mall of Alibara, weights are on a dry basis.

And (3) Bayue cinnamon leaf: purchased from online mall of Alibara, weights are on a dry basis.

Ginseng flower: purchased from online mall of Alibara, weights are on a dry basis.

Example 1 preparation of composition against bee paralytic virus and extract thereof

In the embodiment, the composition for resisting the bee paralytic viruses comprises 50% of salvia miltiorrhiza and cassia twig, 25% of octopus chinensis and 25% of ginseng flower by mass.

The preparation method of the extract of the composition for resisting the bee paralytic virus comprises the following steps:

(1) weighing 5 g of salvia miltiorrhiza and cassia twig, 2.5 g of augustum leaf and 2.5 g of ginseng flower according to the formula, selecting clean materials, cleaning and drying for later use. Leaching flos Ginseng with 60 vol% ethanol solution as solvent, collecting the leaching solution, recovering ethanol, and concentrating at 55 deg.C to obtain flos Ginseng extractive solution with relative density of 1.25-1.50; the main component of the ginseng flower extract is saponin.

(2) Pulverizing cortex Cinnamomi and Augusticica leaves by 80-120 mesh, and sieving;

(3) adding the powder of the cinnamomum wilsonii leaves and the cinnamomum wilsonii leaves in the step 2) and the saponin extracting solution obtained in the step 1) into water, uniformly mixing, filtering to remove impurities, and concentrating the filtrate at 55 ℃ to obtain a concentrated solution with the relative density of 1.25-1.50, namely the bee paralysis virus resisting extract.

(4) And (4) uniformly mixing the extracting solution obtained in the step (3), preparing a mixed extracting solution, and finally preparing capsules, paste or spray.

Example 2 preparation of composition against bee paralytic virus and extract thereof

In the embodiment, the composition for resisting the bee paralytic viruses comprises 60 mass percent of salvia miltiorrhiza and cassia twig, 25 mass percent of octopus chinensis and 15 mass percent of ginseng flower.

The preparation method of the bee paralysis virus resisting extract comprises the following steps:

(1) weighing 6 g of salvia miltiorrhiza and cassia twig, 2.5 g of augustum leaf and 1.5 g of ginseng flower according to the formula, selecting clean materials, cleaning and drying for later use. Leaching flos Ginseng with 75 vol% ethanol solution as solvent, collecting the leaching solution, recovering ethanol, and concentrating at 55 deg.C to obtain flos Ginseng extractive solution with relative density of 1.25-1.50; the main component of the ginseng flower extract is saponin.

(3) adding the powder of the cinnamomum wilsonii leaves and the cinnamomum wilsonii leaves in the step (2) and the ginseng flower extracting solution obtained in the step 1) into water, uniformly mixing, filtering to remove impurities, and concentrating the filtrate at 55 ℃ to obtain a concentrated solution with the relative density of 1.25-1.50, namely the bee paralysis virus resisting extract.

(4) And (4) uniformly mixing the extracting solution obtained in the step (3), preparing a mixed extracting solution, and finally preparing capsules, paste or spray. Example 3 preparation of composition against bee paralytic virus and extract thereof

In the embodiment, the composition for resisting the bee paralytic viruses comprises 55% of cinnamomum subavenium, 30% of cinnamomum octovalvis and 15% of ginseng flower by mass.

(1) weighing 5.5 g of salvia miltiorrhiza and cassia twig, 3.0 g of augustum leaf and 1.5 g of ginseng flower according to the formula, selecting clean materials, cleaning and drying for later use. Leaching flos Ginseng with 75 vol% ethanol solution as solvent, collecting the leaching solution, recovering ethanol, and concentrating at 55 deg.C to obtain flos Ginseng extractive solution with relative density of 1.25-1.50; the main component of the ginseng flower extract is saponin.

Example 4 application of composition against poliovirus in preventing and treating poliomyelitis disease in bees the anti-poliovirus extracts prepared in examples 1-3 were subjected to the following experiments:

first, safety experiment

(1) The anti-paralytic virus extract prepared in examples 1-3 was added to 50% sucrose solution at concentrations of 0.1. mu.g/mL, 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL and 500. mu.g/mL to feed healthy bees, 30 bees per group, 3 groups per dose, continuously for 10 days.

(2) The other two groups were fed with PBS buffer solution as negative control, and 50% sucrose solution as blank control. The number of dead individuals and mortality of bees in each group were recorded daily for all groups.

The results show that no individual death is found in any group, and the antiviral drug has no influence on the health of bee colonies.

Second, antiviral detection

(1) According to the above experimental data, the anti-paralytic virus extract prepared in examples 1-3 was added in an amount of 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL as the experimental dose plusThe viruses of the above experimental and blank groups were obtained by ultracentrifugation and contained about 10 per microliter of virus + D100 (extract concentration 100. mu.g/mL, IAPV + D100 in FIG. 1), virus + D50 (extract concentration 50. mu.g/mL, IAPV + D50 in FIG. 1), virus + D10 (extract concentration 10. mu.g/mL, IAPV + D10 in FIG. 1), and three groups of virus-containing 50% sucrose solution as virus group (IAPV), and virus-free 50% sucrose solution as blank control (ck) were fed to bees as three groups of experimental and blank groups of virus-containing 50% sucrose solution as virus-containing solution (IAPV), respectively¹²Copy number was added to 50% sucrose solution for feeding. In addition, bee virus I, which is known to have been previously shown to have inhibitory effect on bee paralytic virus, was used as a drug control group. Mortality and number of dead individuals were recorded for each group of bees every day for all groups. The results are shown in FIG. 1.

(2) A226-bp fragment was amplified by fluorescent quantitative PCR detection of paralytic virus using a pre-primer 5'-CCAGCCGTGAAACATGTTCTTACC-3' and a reverse primer 5'-ACATAGTTGCACGCCAATACGAGAAC-3' by hybridization of a Western blot to a dead bee using fluorescent quantitative PCR. The housekeeping genes used beta-actin 5'-TTGTATGCCAACACTGTCCTTT-3' and actin 5'-TGGCGCGATGATCTTAATTT-3' as forward and reverse primers, respectively. The reaction conditions were 95 degrees for 3 seconds, 60 degrees for 30 seconds, 72 degrees for 20 seconds, and 40 cycles. And calculating the obtained data by adopting an absolute quantitative method. Protein hybridization was detected by grinding bees into PMSF containing 0.2% DTT and 1uM, centrifuging the supernatant for 10 minutes at 12500 rpm at 15 deg.C, then adding 200ul chloroform and then 10 minutes at 12500 rpm. The resulting protein was boiled for 3 minutes, applied to SDS-PAGE, run at 110V for about 1 hour, and then imaged with an ECL detection kit against an antibody against the paralytic capsid protein VP3, and the results were stored. The fluorescence quantitative results are shown in table 1, the results of the protein hybridization detection part are shown in fig. 2, and the results show that the bee infection virus titer of the virus and drug treatment group is obviously lower than that of the virus treatment group; and some virus and drug treated bees can not detect the expressed protein after 5 or 7 days. Indicating that the virus inhibitor has better virus inhibition effect.

TABLE 1 results of fluorescence quantitative measurement of viral titre

Example 5 use of an anti-polio virus composition to improve the overall resistance of a colony to polio virus.

According to the above experimental data, 10. mu.g/mL, 50. mu.g/mL, and 100. mu.g/mL were selected as experimental doses, and the bee was fed with a sucrose solution containing the anti-paralytic virus extract prepared in example 3 at final concentrations of 10. mu.g/mL, 50. mu.g/mL, and 100. mu.g/mL (virus + D100, virus + D50, and virus + D10 in tables 2-4) as experimental groups, and three groups, one group was fed with PBS buffer alone as a negative control, and the other group was fed with normal sugar water alone as a blank control. In addition, bee virus I, which is known to have been previously shown to have inhibitory effect on bee paralytic virus, was used as a drug control group. Each group had 3 bees, each about 7 frames. The treatment groups were fed once every 7 days for a total of 2 feeds and observed for 20 days. All groups were recorded daily for the number of pre-hive dead individuals and bee mortality for each group. The bees are killed before cleaning the beehive every morning, and the number of bees with paralysis symptoms is recorded.

The inhibition effect of the antiviral drugs is calculated by adopting a fluorescent quantitative PCR method on dead bees, and the results are shown in Table 2, and the bee infection virus titer of the virus and drug treatment group is obviously lower than that of the virus treatment group. Meanwhile, the comparison of bees showing paralysis symptoms with healthy is determined, and the results are shown in tables 3 and 4, and the difference of viability shows that the bee venom has better virus inhibition effect. Fluorescent quantitative PCR detection of paralytic virus the pre-primer 5'-CCAGCCGTGAAACATGTTCTTACC-3' and the reverse primer 5'-ACATAGTTGCACGCCAATACGAGAAC-3' were used to amplify a 226-bp fragment. The housekeeping genes used beta-actin 5'-TTGTATGCCAACACTGTCCTTT-3' and actin 5'-TGGCGCGATGATCTTAATTT-3' as forward and reverse primers, respectively. The reaction conditions were 95 degrees for 3 seconds, 60 degrees for 30 seconds, 72 degrees for 20 seconds, and 40 cycles. And calculating the obtained data by adopting an absolute quantitative method.

TABLE 2 fluorescence quantitation results

TABLE 3 survival Rate

TABLE 4 comparison of the number of bees with symptoms of paralysis

Claims

1. The composition for resisting the bee paralytic virus is characterized by comprising the following components in percentage by mass: 50-65% of salvia miltiorrhiza and cassia bark leaf, 25-35% of august leaf and 5-25% of ginseng flower.

2. The composition against poliovirus of honey bees according to claim 1, wherein: the traditional Chinese medicine composition comprises 55% of salvia miltiorrhiza and cassia twig leaves, 30% of augusticum acutiloba leaves and 15% of ginseng flowers by mass percentage.

3. A preparation method of an anti-bee paralytic virus extract, which is prepared by the composition of claim 1 or 2, and comprises the following steps: extracting saponin from the ginseng flower, and then mixing the saponin with the salvia miltiorrhiza and cinnamomum japonicum leaf powder to obtain the bee paralysis virus resistant extract.

4. The method for preparing the anti-paralytic virus bee extract according to claim 3, comprising the steps of:

1) leaching flos Ginseng with 60-75% ethanol solution as solvent, collecting the solution, recovering ethanol, and concentrating at 55 deg.C to obtain saponin extract with relative density of 1.25-1.50;

3) adding the powder of the cinnamomum wilsonii leaves and the cinnamomum wilsonii leaves in the step 2) and the saponin extracting solution obtained in the step 1) into water, uniformly mixing, filtering to remove impurities, and concentrating the filtrate at 55 ℃ to obtain a concentrated solution with the relative density of 1.25-1.50, namely the bee paralysis virus resisting extract.

5. An anti-poliovirus extract prepared by the method of claim 3 or 4.

6. Use of the composition against poliovirus of claim 1 or 2 for the preparation of a medicament for the prevention and treatment of poliomyelitis disease in bees.

7. Use of the composition against poliovirus of honey bees according to claim 1 or 2 for the manufacture of a medicament for improving the overall resistance of a bee colony against poliovirus.

8. The composition against poliovirus of claim 1 or 2, the use of the extract against poliovirus of claim 5 for preparing a preparation against poliovirus.

9. The use of the anti-paralytic virus extract of claim 5 in the manufacture of a medicament for the prevention and treatment of bee paralysis.

10. Use of the anti-paralytic virus bee extract of claim 5 in the manufacture of a medicament for improving the overall resistance of a colony to paralytic virus bees.