CN111265596B - Traditional Chinese medicine composition for preventing and treating middle capsule disease - Google Patents

Abstract

The invention provides a traditional Chinese medicine composition for preventing and treating bursa disease. The traditional Chinese medicine composition comprises the following components in parts by weight: 10-50 parts of isatidis, 10-50 parts of Daqingye and 15-80 parts of propolis. The composition can obviously improve the survival of Chinese honeybee larvae infected with CSBV, can reduce the number of virus copies in the susceptible larvae, improve the growth and development state of the susceptible larvae, and enable the susceptible larvae to metabolize The pupae emerge into adult bees, thereby reducing the fatality rate of infected larvae, preventing the collapse of the infected bee colony, and playing an important role in the recovery and reproduction of the bee colony. The traditional Chinese medicine composition can be used for preparing medicines for preventing and treating Chinese honeycomb larvae and feeding materials, and shows a good market application prospect.

Description

Traditional Chinese medicine composition for preventing and treating middle capsule disease

Technical Field

The invention relates to the field of biological medicine, in particular to a traditional Chinese medicine composition for preventing and treating middle capsule disease.

Background

Bees are important pollination insects in nature, about one third of crops in the world need bee pollination to a certain extent, and some fruits, vegetables and the like completely depend on bee pollination. Bee pollination can significantly improve the yield and quality of crops, thus having important economic value in the aspect of agricultural production. The bee can produce various peak products, such as honey, royal jelly, propolis, bee pollen and the like, the quality of the bee product is closely related to the health state of the bee, the bee product produced by the susceptible bee can bring the potential safety hazard of the bee product, and the medicine for treating bee diseases can also remain in the bee product to cause the safety problem of the bee product.

The Chinese honeybee (Apis cerana) is used as a native bee species in China, has the advantages of strong anti-mite capability, low temperature resistance and being good at collecting sporadic honey powder sources, and plays an important role in the bee breeding industry; meanwhile, the bee germ plasm resource is a unique bee germ plasm resource in China, has a long breeding history, and plays an extremely important role in maintaining the balance of a natural ecosystem (see Bombay, biological characteristic analysis of genes related to the Chinese bee ribosomal protein family and the MAPK signal pathway, 2015). However, Chinese bees are usually harmed by various pathogens, wherein the harm of Chinese sacbrood virus (CSBV) to Chinese bees is the most serious, Chinese bee sacbrood is firstly discovered in the Guangdong in 1972, then rapidly spreads in China and spreads to countries in southeast Asia, and the Chinese bee sacbrood is wide in distribution, high in morbidity and rapid in infection, and once the Chinese bee sacbrood occurs, destructive attack is caused to bee colonies.

The Chinese bee colony is infected by CSBV to seriously affect the larvae and colony vigor of the Chinese bee colony, the infection of virus causes the death of the larvae in the colony, and the larvae can not pupate even when the bee flies survive, so that the number of worker bees newly emerged is insufficient, the Chinese bee colony vigor is weakened, the resistance of the bee colony is reduced, and the Chinese bee colony is more easily attacked by other viruses, thereby showing the phenomenon of multi-virus common infection.

The main propagation paths of the bee viruses are vertical propagation and horizontal propagation, the sacbrood disease virus (SBV) can be propagated in the horizontal direction and the vertical direction in a bee colony of western bees (Apis mellifera), CSBV is taken as a kindred of the SBV, the propagation path is similar to the SBV, the sacbrood virus can be propagated in the bee colony in the modes of food mutual feeding, excrement pollution, mating behavior and gamete reproduction, the damage to the Apis serious to the Apis cerana, and the bee larvae can be killed greatly. The drone can act as a viral vector to transmit the virus to offspring through the reproductive gametes. The non-group-boundary nature of the males may directly determine the genetic diversity of the bee colony, and thus may have a significant impact on the ability of the bee colony to resist disease.

The high incidence of Chinese bee sacbrood is an important factor causing the rapid decrease of the quantity of Chinese bees in China, not only brings great economic loss to Chinese bee breeding industry, but also influences the growth of insect-borne plants depending on Chinese bee pollination and indirectly destroys ecological balance. At present, the disease is mainly prevented and controlled by comprehensive prevention measures of changing the prince-feather of the bee, strictly disinfecting, strengthening management, preventing secondary infection by using antiviral drugs and antibiotics, and the like, the effect is not ideal, the harm and the spread of the virus are difficult to be effectively controlled by common antiviral drugs, the drug residue in bee products is high, the export of the bee products is limited, and the bee products become a great problem which hinders the development of the bee industry in China.

Chinese herbal medicine is a natural product and is widely applied to various diseases such as bacteriostasis, cancer resistance, inflammation resistance, tumor resistance, virus resistance and the like. The research of virus drugs has been generally regarded by the medical community at home and abroad. The hypericum perforatum extract can effectively inhibit porcine reproductive and respiratory syndrome virus in vitro and in vivo. The isatis root water extract has obvious direct killing effect on PRRSV; the astragalus aqueous extract has obvious blocking and inhibiting effects on PRRSV.

The isatis root has a definite curative effect on resisting viruses, and can resist a plurality of types of viruses. The isatis root acidic extract has stronger effect of directly inhibiting influenza viruses. Each part of the isatis root can inhibit herpes simplex virus-I (HSV-I) under different mass concentrations, has the effect of directly inactivating HSV-I, and also has the anti-virus effect on Human Cytomegalovirus (HCMV). The folium isatidis aqueous extract has stronger anti-influenza virus activity, the action mechanism of the folium isatidis aqueous extract is probably related to the enhancement of the immunity of the organism, and the 4(3H) quinazolinone in the folium isatidis has obvious in-vitro anti-H1N 1 type influenza virus activity. Propolis is a purely natural antiviral substance and has a strong effect on a plurality of viruses. Urushisaki and the like research the effect of Brazilian propolis aqueous extract on resisting influenza A virus and prove that caffeoylquinic acid is the substance with the strongest activity on influenza A virus in propolis aqueous extract.

Under the guidance of the theory of traditional Chinese medicine, the radix isatidis, the folium isatidis and the propolis are combined to form a compound, the effect of the compound on the aspect of resisting CSBV is researched, and particularly, the research on preventing and controlling the Chinese sacbrood disease (referred to as Chinese sacbrood disease) is not reported.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine composition for preventing and treating middle capsule disease, which can reduce the fatality rate of Chinese bee (Apis cerana) larvae infected with CSBV.

In order to achieve the purpose of the invention, in a first aspect, the invention provides a traditional Chinese medicine composition (ZN preparation) for preventing and treating middle capsule disease, which comprises the following components in parts by weight: 10-50 parts of isatis root, 10-50 parts of dyers woad leaf and 15-80 parts of propolis.

The composition preferably comprises the following components in parts by weight: 30-40 parts of isatis root, 30-40 parts of dyers woad leaf and 40-50 parts of propolis.

More preferably comprises the following components in parts by weight: 1 part of isatis root, 1 part of dyers woad leaf and 1.5 parts of propolis.

In the present invention, Radix Isatidis (Radix Isatidis) and Folium Isatidis (Folium Isatidis) are obtained from Hongda Korea Biotech Co., Ltd, Beijing. Propolis (proplis), the gum content is 20-60%, preferably 30-50%, more preferably 40-50%, most preferably 50%. Propolis is preferably collected in temperate zone needle broad-leaved mixed forest region, more preferably propolis collected at fixed point in Heilongjiang, Jilin and northeast region of Liaoning.

Experiments show that caffeic acid intervention influences the growth of bee larvae, so that the lower the caffeic acid content in the used propolis, the better. The experimental result shows that the caffeic acid content of the propolis in the east three provinces, particularly the propolis in Heilongjiang and Jilin, is lower than that of the propolis in North China, south China, China and the like. According to seven major vegetation distribution areas in China, the production areas of the Chinese propolis are classified, and the propolis is preferably collected in temperate zone broadleaf mixed forest areas, particularly the propolis collected at fixed points in the northeast of Heilongjiang, Jilin and Liaoning areas.

In a second aspect, the present invention provides a preparation method of the traditional Chinese medicine composition, which comprises: mixing the above materials at a certain proportion, decocting with water, and concentrating or diluting the decoction to concentration of 10-50mg/mL, preferably 22.9 mg/mL. Then, the mixture was autoclaved and stored in a refrigerator at 4 ℃ for further use.

In a third aspect, the invention provides application of the traditional Chinese medicine composition in preparing a medicine for preventing and treating middle capsule diseases. The mesocysticercosis is caused by infection with Chinese bee sacbrood virus (CSBV).

In a fourth aspect, the invention provides application of the traditional Chinese medicine composition in resisting Chinese bee sacbrood virus (including non-treatment purposes).

In a fifth aspect, the invention provides an application of the traditional Chinese medicine composition in inhibiting the virus proliferation (including non-treatment purposes) of Chinese bee sacbrood.

In a sixth aspect, the invention provides application of the traditional Chinese medicine composition in preventing and treating middle capsule diseases. The composition can be used for feeding Chinese bee larva to remarkably reduce CSBV copy number in the body of the infected larva, reduce the fatality rate of the infected larva, improve the growth of the infected larva so that the infected larva can be normally pupated and eclosion into adult bees, and further enhance the physique of the Chinese bee.

In the application, the dosage of the traditional Chinese medicine composition for the bee larvae is more than or equal to 6.4 mg/bee/time. More preferably 6.4 to 30 mg/piece. The time interval of each administration of the ZN preparation is preferably 22-24 h.

Specifically, 6.4 mg/bee/larva/time for 3 days old; bee larva of 4 days old is 9.6 mg/larva; 5 days old bee larva 16 mg/time; bee larva of 6 days old is 25.6 mg/larva.

In the present invention, the dyers woad leaf and the dyers woad root are derived from the same plant, namely Isatis tinctoria Fort (Isatis indigotica Fort.) of the family Cruciferae, the dyers woad leaf is the leaf thereof, and the dyers woad root is the rhizome thereof. The book materia medica just reads clearly states that: the radix isatidis, i.e. indigowoad root, has the same functional taste as indigowoad leaf, can enter the blood system of liver and stomach, and has no excessive effects of clearing heat, detoxifying, avoiding epidemic diseases and killing insects. But leaves are scattered and roots are descending, which are also different from ears. Modern pharmacological research proves that the isatis root has antibacterial and antiviral effects and is a typical representative of antiviral traditional Chinese medicines; it also has effects in improving immunity, destroying leukemia cells, and enhancing phagocytic ability of mononuclear macrophage. The folium Isatidis decoction has antibacterial and antiviral effects, and can inhibit Japanese encephalitis virus, mumps virus, influenza virus, etc. The two herbs are effective in treating wind-heat exterior syndrome and early stage of warm disease because they can clear heat from both the nutrient and blood systems and the defense and qi systems. Propolis is a fragrant colloidal solid prepared by collecting resin (gum) from plant spore or trunk of Apis mellifera (Apis mellifera), mixing with secretion of palate gland and cerifera, and has antibacterial, antiinflammatory, antioxidant, and immunity enhancing effects. The propolis is mainly used for improving immunity and resisting viruses, and the isatis root and the dyers woad leaf are mainly used for resisting viruses and assisting in improving immunity. The three are combined together, the virus is antagonized, the self resistance is improved at the same time, the immunity to the virus is enhanced, the virus meets the requirements that ' vital qi exists in the interior and the pathogen cannot dry ' mentioned in Huangdi's internal meridian, the invasion of exogenous pathogenic factors can be resisted only by improving the self immunity, and the virus is the exogenous pathogenic factor. The technical scheme of the invention is provided for preventing and treating the middle capsule diseases by the principle of 'prevention of diseases' in traditional Chinese medicine, and treating the diseases without the diseases.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the traditional Chinese medicine composition for preventing and treating the middle sac disease provided by the invention has an obvious improvement effect on the survival of Chinese bee larvae infected with the middle sac virus (CSBV), can reduce the virus copy number in the bodies of the infected larvae, improves the growth and development state of the infected larvae, and enables the infected larvae to pupate and emerge into adult bees. Thereby reducing the fatality rate of the infected larvae, preventing the infected bee colony from collapsing, playing an important role in the recovery and the reproduction of the bee colony, and being very suitable for increasing the Chinese bee physique in the Chinese bee breeding production. The traditional Chinese medicine composition can be used for preparing medicines and feeding materials for preventing and treating Chinese bee sacbrood and shows good market application prospect.

Drawings

FIG. 1 is a statistic of mortality of normal Apis cerana larvae and infected larvae with ZN formulation of example 1 of the present invention; wherein, CK: the whole process is fed to the larvae of the syrup. ZN: larvae were fed ZN formulation at 3-6 days of age. CSBV: feeding the syrup with poison at 3 days old, and feeding the larvae with syrup at 4-6 days old. CSBV + ZN: feeding poison preparation at 3 days old, and feeding larva of ZN preparation at 4-6 days old. Data are mean ± SE. N is 3. Using the Least Significant Difference (LSD) test, at the level of P ═ 0.05, the different letters in the columns show statistically significant differences.

FIG. 2 shows the survival of larvae of 4, 5, and 6 day old Apis cerana according to example 1; wherein, CK: the whole process is fed to the larvae of the syrup. ZN: larvae were fed ZN formulation at 3-6 days of age. CSBV: feeding the syrup with poison at 3 days old, and feeding the larvae with syrup at 4-6 days old. CSBV + ZN: feeding poison preparation at 3 days old, and feeding larva of ZN preparation at 4-6 days old. Data are mean ± SE. N is 3.

FIG. 3 shows the growth of the larvae of Apis cerana in example 1 of the present invention; wherein, CK: the whole process is fed to the larvae of the syrup. ZN: the whole process was fed to larvae of ZN formulation. CSBV: feeding the syrup with poison at 3 days old, and feeding the larvae with syrup at 4-6 days old. CSBV + ZN: feeding the poison-containing preparation at 3 days of age, and feeding the larva of ZN preparation with concentration of 0.32mg/mL at 4-6 days of age.

FIG. 4 shows the number of viral copies of larvae treated differently at 4-6 days of age in example 1 of the present invention; wherein, CK: the whole process is fed to the larvae of the syrup. ZN: the whole process was fed to larvae of ZN formulation. CSBV: feeding the syrup with poison at 3 days old, and feeding the larvae with syrup at 4-6 days old. CSBV + ZN: feeding the poison-containing preparation at 3 days of age, and feeding the larva of ZN preparation with concentration of 0.32mg/mL at 4-6 days of age.

FIG. 5 is the statistics of the survival rate of normal larvae by ZN6, ZN7 and ZN8 preparations in example 2 of the invention. Wherein, CK: the whole process is fed to the larvae of the syrup. ZN 6: the larvae are fed with ZN6 preparation at 3-6 days of age. ZN 7: the larvae are fed with ZN7 preparation at 3-6 days of age. ZN 8: the larvae are fed with ZN8 preparation at 3-6 days of age. Data are mean ± SE. N is 3. Using the Least Significant Difference (LSD) test, at the level of P ═ 0.05, the different letters in the columns show statistically significant differences.

FIG. 6 is the statistics of the survival rate of infected larva of ZN6, ZN7 and ZN8 preparations in example 2 of the invention. Wherein, CSBV: feeding the syrup with poison at 3 days old, and feeding the larvae with syrup at 4-6 days old. CSBV + ZN 6: the larvae are fed with the poison preparation at 3 days of age, and fed with ZN6 preparation at 4-6 days of age. CSBV + ZN 7: the larvae are fed with the poison preparation at 3 days of age, and fed with ZN7 preparation at 4-6 days of age. CSBV + ZN 8: the larvae are fed with the poison preparation at 3 days of age, and fed with ZN8 preparation at 4-6 days of age. Data are mean ± SE. N is 3. Using the Least Significant Difference (LSD) test, at the level of P ═ 0.05, the different letters in the columns show statistically significant differences.

Fig. 7 shows the survival of larvae of 4, 5, 6 days old treated differently in example 2 of the invention. Wherein, CK: the whole process is fed to the larvae of the syrup. ZN 8: larvae were fed ZN8 formulation at 3-6 days of age. CSBV: feeding the syrup with poison at 3 days old, and feeding the larvae with syrup at 4-6 days old. CSBV + ZN 8: the larvae are fed with the poison preparation at 3 days of age, and fed with ZN8 preparation at 4-6 days of age. Data are mean ± SE. N is 3.

FIG. 8 is a statistical analysis of the survival rate of normal larvae at different concentrations in example 3 of the present invention. Wherein, CK: feeding the syrup larvae at 3-6 days of age. ZN 8-1: the larvae are fed with ZN8 preparation with concentration of 0.032mg/mL at 3-6 days old. ZN 8-2: larvae were fed with ZN8 formulation at a concentration of 0.32mg/mL at 3-6 days of age. ZN 8-3: larvae were fed with ZN8 preparation at a concentration of 3.2mg/mL at 3-6 days of age. Data are mean ± SE. N is 3. Using the Least Significant Difference (LSD) test, at the level of P ═ 0.05, the different letters in the columns show statistically significant differences.

FIG. 9 is a graph showing the statistics of the survival rate of infected larvae at different concentrations in example 3 of the present invention. Wherein, CSBV: feeding the syrup with poison at 3 days old, and feeding the larvae with syrup at 4-6 days old. CSBV + 0.032: feeding the larvae with the preparation containing toxin at 3 days old, and feeding the larvae with ZN8 preparation with concentration of 0.032mg/mL at 4-6 days old. CSBV + 0.32: feeding the larvae with the toxin-containing preparation at 3 days of age, and feeding the larvae with ZN8 preparation with concentration of 0.32mg/mL at 4-6 days of age. CSBV + 3.2: feeding poison-containing preparation at 3 days of age, and feeding larvae of ZN8 preparation with concentration of 3.2mg/mL at 4-6 days of age. Data are mean ± SE. N is 3. Using the Least Significant Difference (LSD) test, at the level of P ═ 0.05, the different letters in the columns show statistically significant differences.

Fig. 10 shows the larvae dying at 4, 5, and 6 days old for the different treatments of example 3 of the present invention. Wherein, CK: feeding the syrup larvae at 3-6 days of age. 0.032 mg/mL: the larvae are fed with ZN8 preparation with concentration of 0.032mg/mL at 3-6 days old. CSBV: feeding the syrup with poison at 3 days old, and feeding the larvae with syrup at 4-6 days old. CSBV +0.032 mg/mL: the larvae are fed with the poison preparation at 3 days old and the larvae are fed with ZN8 preparation at the concentration of 0.032mg/mL at 4-6 days old. Data are mean ± SE. N is 3.

Figure 11 shows the growth of larvae treated differently in example 3 of the invention. Wherein, A: growth of CK group larvae; b: growth of larvae in group 0.032 mg/mL; c: growth of larvae in the 0.32mg/mL group; d: 3.2mg/mL group larvae growth; e: growth of larvae in the CSBV infected group; f: growth of larvae in the CSBV +0.032mg/mL group; g: growth of CSBV +0.32mg/mL group larvae; h: growth of CSBV +3.2mg/mL group larvae. Bar is 11 mm.

FIG. 12 is a graph showing the effect of feeding different concentrations of caffeic acid on normal larval mortality in example 4 of the present invention. Wherein, 1: larvae fed with syrup at 3-6 days of age (control); 2: feeding larvae with caffeic acid concentration of 0.75 μ g/mL at 3-6 days old; 3: feeding larvae with caffeic acid concentration of 7.5 μ g/mL at 3-6 days old; 4: larvae with caffeic acid concentration of 75 μ g/mL were fed at 3-6 days of age.

FIG. 13 shows the pupation and emergence of healthy larvae and infected larvae with caffeic acid in example 4 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

In the following examples, isatis root and isatis leaf are purchased from the biotechnology limited of Hongda, Beijing, Hongda, and propolis (from Apis mellifera) is obtained from Shandong bee fields (propolis in the areas of deciduous broad-leaved forest in the warm temperate zone and evergreen broad-leaved forest in the subtropic zone, the caffeic acid content of which is higher than that of propolis in the mixed forest area of warm needle broad-leaved trees in the northern bee fields of Heilongjiang, Jilin and Liaoning), and the propolis content is 50%.

Mixing the above materials at a certain proportion, decocting with water, concentrating the water decoction under reduced pressure to dry, weighing, and concocting with double distilled water to obtain 1mL extract containing 22.9 mg. Sterilizing under high pressure, and storing in refrigerator at 4 deg.C.

Example 1 Chinese medicinal composition for preventing and treating middle capsule disease and use thereof

1 materials and methods

1.1 supply sample book

The Apis cerana Fabricius is from Apis cerana Fabricius research institute of China academy of agricultural sciences. In order to obtain 2-instar larvae, after queen bees are closed on the son spleens to lay eggs for 92 hours, the son spleens containing the 2-instar larvae are taken out from the bee colony and put into a 24-well plate, and the son spleens are placed in a constant temperature and humidity incubator with the temperature of 32 +/-1 ℃ and the relative humidity of 75 +/-5% RH for pre-culture.

The test larvae are detected by a reverse transcription-polymerase chain reaction RT-PCR method to be healthy larvae without virus infection to be used as samples of the experiment. RNA extraction RNA was extracted using the RNeasy mini kit (Edley) according to the manufacturer's protocol. Total RNA was eluted in 30. mu.L of elution buffer and used directly for RT-PCR. The method includes a step of removing DNA, thereby ensuring that only RNA is present in the final extract. cDNA was synthesized according to the instructions of the product, and first strand cDNA was immediately synthesized using the extracted RNA using QuantiTec reverse transcription kit (Takara). The amplification reaction was a 50. mu.L system. The cDNA was stored at-20 ℃. The PCR reaction conditions include: denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 15 seconds, 35 cycles. SBV (cysticercosis virus), BQCV (queen bee virus), DWV (parapet virus), IAPV (israel paralysis virus), KBV (kashmir virus), ABPV (acute paralysis virus), CBPV (chronic paralysis virus) were not detected.

1.2 syrup

Syrup preparation and feeding were modified with reference to 2013 standards for feeding apis mellifera (table 1).

TABLE 1 syrup formulation, ratio and dosage to feed

Note: in the table,% represents mass%. See Karl Crailsheim, Robert Brodschneider, Pierrick Aupinel, Dieter Behrens, Elke Genersch, Jutta Vollmann & Ulrike Rissberger-Gall.Standard methods for identifying information retrieval of Apis melifera great. journal of Apicus architral Research, 2013, 52 (1): 1-15.

1.3CSBV

The existence of CSBV was confirmed by RT-PCR from Chinese bee brood in a typical cysticercosis disease form taken from another Chinese bee farm of the institute of bee, Chinese academy of agricultural sciences. To obtain CSBV, 210 larvae infected with CSBV were taken, placed in a sterile triturator and 2100 μ L sterile Phosphate Buffered Saline (PBS) was added and triturated. Grinding, centrifuging at 8000rpm at 4 deg.C for 30min, repeating for 2 times, collecting supernatant, performing RT-PCR detection with cDNA reverse transcribed after RNA extraction as template, and storing the supernatant at-80 deg.C as original venom without other viruses.

Viruses were detected using the absolute quantitative PCR method (see K.M.Hong, H.Najjar, M.Hawley, R.D.Press.quantitative real-time PCR with automatic sample preparation for diagnosis and monitoring of cytological infection in bone matrix laboratory chemistry 2004, 50 (5): 846 856; HU Zhi Gang, CHEN Ke Ping, YAO Qin, GAO Gui Tian, XU Jia-Ping, CHEN Hui-Qi.cloning and Characterification of plasmid PP-BP; a Gene Induced Viral infection in acta III section PCR system 9600, OER 9600, real-time PCR was performed in the PCR system. The real-time fluorescent quantitative PCR reaction system is 15 mu L, comprises 0.3 mu L, SYBR 7.5.5 mu L of each of the forward primer 5'-ccttggagtttgctatttacg-3' and the reverse primer 5'-cctacatccttgggtcag-3', 1 mu L of the template and water which is supplemented to 15 mu L. The qPCR reaction employs a two-step process, the first step comprising: denaturation at 95 deg.C for 3min at constant temperature; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, 40 cycles. The second step is a melting section comprising: denaturation at 95 ℃ for 15 s; annealing at 60 ℃ for 5s and denaturation at 95 ℃ for 15 s. qPCR was performed to determine the concentration of the original venom to be 6.74X 10⁴copies/. mu.L, this original venom was used for subsequent vaccination experiments.

Preparing the toxic syrup: IC based on Pre-test Virus infection₅₀And (3) detecting results, selecting the feed venom according to the proportion of the original venom: syrup (syrup composition see table 1) 1: 3 (volume ratio) was prepared as a mixture of 5. mu.L of virus stock and 15. mu.L of syrup, i.e., CSBV of 1.685. times.10⁴copies/. mu.L of toxic syrup. The toxicant-containing syrup was fed to 3-day-old test larvae. The feeding amount is referred to 1.2.

1.4 mixture ZN (ZN preparation)

The mixture ZN consists of isatis root, dyers woad leaf and propolis, and the mass ratio of the isatis root, the dyers woad leaf and the propolis is 1: 1.5. Decocting with water, concentrating under reduced pressure, filtering, sterilizing, and diluting with syrup of each day age in Table 1 to 0.32 mg/mL.

Preparation of the toxin-containing preparation: mixing the original venom with ZN preparation (syrup composition shown in Table 1) at a volume ratio of 1: 3 to obtain a mixture of 5 μ L virus stock solution and 15 μ L ZN preparation, i.e. CSBV of 1.685 × 10⁴copies/. mu.L of the toxin-containing preparation. The toxicant-containing formulation was fed to 3-day-old test larvae.

1.5ZN preparation for feeding bee larva to resist CSBV

The larvae (3 days old) after pre-culture for 1d were taken out of the incubator, and the larvae with good health status were placed in 48-well plates, randomly divided into control group, infection group, preparation group and anti-virus group, each group containing 24 samples, and repeated three times. Grouping and feeding are shown in table 2.

TABLE 2ZN preparations grouping information for anti-bee virus experiment

The optimal time for infecting bee larvae with CSBV is 2-3 days old, so that CSBV infection and ZN preparation intervention treatment are carried out when the larvae are 3 days old, and the prevention and treatment effects of the ZN preparation on the CSBV infection of bees are analyzed. Feeding the infection group with toxic syrup (preparation method is shown in 1.3), and feeding the intervention group with toxic preparation (preparation method is shown in 1.4); at the age of 4-6 days, the larvae were fed with syrup or formulation, respectively, and only the larvae fed with syrup served as CK control. These foods were placed on the bottom side of the plate to avoid contact with larvae. Feeding was done every 24h and feeding and mortality of larvae were observed and recorded.

Forceps, sterilized with 75% ethanol (prepared with DEPC-treated water) for 5min and rinsed three times with DEPC-treated water, removed the differently treated live larvae, immediately frozen with liquid nitrogen, and then stored in a-80 ℃ freezer for subsequent viral copy number testing.

1.6 Larvae mortality determination method

The death numbers of the larvae in the pre-culture (2 days old) and the virus inoculation day (3 days old) are not counted and analyzed to eliminate the influence of mechanical death of the larvae in the experimental process, the death numbers of the larvae are counted at regular time every day from the next day (4 days old) of virus inoculation, the death larvae are removed, and the steps are repeated until the 6 days old is finished.

The daily larval mortality rate is the number of larvae dead per day/number of larvae alive per day x 100%

The death rate of the larva in the whole process is equal to the death quantity of the larva/the total number of samples multiplied by 100 percent

1.7 registration of larval morphology

The state of the larvae is observed under a body microscope, and photographing records are carried out.

1.8 data interpretation and statistical analysis

Statistical analysis of the data was performed using SPSS 22.0. The effect of ZN preparations on larvae is indicated by "Mean. + -. SE". Changes in viral copy number of CSBV infected larvae were compared using ANOVA and LSD multiple ANOVA methods.

The data for blend ZN after intervention in healthy and virulent larvae are as follows:

(1) mortality of normal and infected larvae by feeding ZN formulation

After the test larvae are fed with syrup at the age of 2 days, respectively feeding syrup and a ZN preparation with the concentration of 0.32mg/mL at the age of 3 to 6 days to investigate the influence of the ZN preparation on the mortality of normal larvae; after the test larvae are pre-cultured at the age of 2 days, the larvae are inoculated with the toxic syrup and the toxic preparation respectively at the age of 3 days, and the larvae are fed with the syrup and the ZN preparation with the concentration of 0.32mg/mL respectively at the age of 4 to 6 days to examine the influence of the ZN preparation on the death rate of the infected larvae, and the results are shown in a figure 1.

The average survival rate of the larvae fed with the syrup in the control group is 94.43%, the average survival rate of the larvae fed with the ZN preparation is 91.67%, and the larvae are not statistically different from the control group, which indicates that the ZN preparation has no influence on the health condition of the larvae; the average survival rate of the susceptible larvae is 43.05%, the survival rate of the susceptible larvae fed with the ZN preparation can reach 90.28%, 47.23% is improved, the statistical difference is extremely obvious (P is less than 0.01), the ZN preparation can effectively improve the survival state of the infected larvae, the survival rate is greatly improved, and the ZN preparation has a prevention and treatment effect on the Chinese bee sacbrood disease.

(2) Mortality rate at different day ages of normal and contaminated larvae by feeding ZN formulation

The larvae of 2 days old were pre-cultured for one day, and the number of larvae of each group was determined to be the same at 3 days old for experimental treatment, and the change in survival number of larvae of 4, 5, and 6 days old was recorded, respectively, and the results are shown in FIG. 2.

For normal larvae, the control group died the most at 5 days of age, with a mortality rate of 2.84%, and relatively few deaths at 4 and 6 days of age, each mortality rate being 1.39%. Compared with the control group, the ZN preparation group has the highest death rate of 4.23 percent at 6 days, 1.39 percent at 4 days and 2.84 percent at 5 days. The ZN preparation group has no significant difference from the control group, and does not influence the normal growth of larvae. For CSBV infected larvae, larvae vaccinated only with the CSBV group had a mortality rate of up to 25% at 4 days of age and then declined gradually, with a mortality rate of 22.70% at 5 days of age and 20.92% at 6 days of age. And the mortality rate of the ZN preparation group at 4 days is only 1.45%, the mortality rate at 5 days is the highest and only reaches 7.07%, which is far lower than the mortality rate at 5 days of the infection group, and the mortality rate at 6 days is reduced to 0. Therefore, ZN preparation greatly improves the survival of infected larvae and greatly reduces the death rate.

(3) Effect of ZN-feeding on the morphology of Normal and infected larvae

The growth of larvae was compared between the CK and ZN, and between the toxicant and + ZN, using a standard diameter of one well of a 48-well plate, and photographed under a microscope, and the results are shown in FIG. 3.

For normal larvae, the larvae fed ZN formulation grew well and developed no differently from the CK group larvae. For infected larvae, infected larvae were smaller than CK group larvae individuals; the larvae fed with ZN preparation containing CSBV grew well, and the size of the larvae was not obviously different from that of CK group.

In the experiment, the morphology of the larva is selected as an index for preventing and treating the Chinese bee sacbrood for the first time, the growth of the infected larva is found to be arrested, the larva grows well after the infected larva is fed with ZN, and certain nutrient substances necessary for promoting the growth and development of the larva are presumed to exist in ZN.

(4) Influence of ZN feeding preparation on CSBV reproduction in Chinese bee larva

After CSBV infects Chinese bee larva, through different treatments, RNA is extracted from larvae of 4 days old, 5 days old and 6 days old, cDNA is synthesized, and quantified to 2 mug, and the virus copy number in the larva is respectively detected (figure 4).

CSBV detection is carried out on apparently healthy 4-6 day old Chinese bee larvae (CK group), the virus copy number of each day old larva is found to be 60-70, which indicates that apparently healthy larvae are also infected by virus and only do not show obvious symptoms, indicates that the virus generally has the phenomenon of invisible infection in bee colonies, and the larvae can live with the virus within a certain concentration, which is identical with the result of Liu shan (2017). After ZN intervention, the virus copy number in the apparent healthy 4-6 day old apis cerana larva bodies is not obviously reduced, and the fact that ZN intervention has no obvious antagonistic effect on apparent healthy larva virus infection is shown.

The virus copy number in the infected larva grows in an explosive manner, and the virus copy number in the larva of 4 days old is as high as 12.1 multiplied by 10⁴This is 1700 times that of CK group, so much virus multiplication leads to the death of infected larvae, with mortality up to 25%, and with growth and development, the virus copy number in 5-day and 6-day-old bodies decreases, but remains at 2.3X10⁴Above, it was shown that the single-day larval mortality rate was still above 20% (fig. 2). After ZN intervention, the virus copy number in the infected larva is obviously reduced, and the 4-day-old virus copy number is reduced to 0.13 multiplied by 10⁴And the copy number of the virus in the larvae of 5 days and 6 days is reduced to about 200, which shows that after ZN is fed, the virulent larvae generate strong antagonistic virus reaction, the infection degree of the virus to the larvae is obviously reduced, and the fatality rate of the virulent larvae is reduced (figure 4).

According to the preference of Chinese sacbrood disease virus to host and tissues thereof, Chinese bee larvae of 2 days old are selected for feeding experiments, and the composition (ZN) has no influence on the characterization change of healthy larvae; the feed additive has an obvious inhibiting effect on pathological changes caused by infection of CSBV larvae, and infected larvae after ZN intervention are well developed and can pupate and emerge into adult bees; ZN can effectively inhibit the proliferation of CSBV in larvae after intervention, greatly reduce the copy number of virus, reduce the fatality rate of infected larvae, prevent infected bee colonies from collapsing and play an important role in the recovery and reproduction of bee colonies.

Example 2 optimization of the dosage ratio of three crude drugs, i.e., radix Isatidis, folium Isatidis and propolis

This example examined the effect of different composition formulation formulations on larvae.

ZN6, ZN7 and ZN8 preparations are all prepared from isatis root, dyers woad leaf and propolis, and the mass ratio of the raw materials of the preparations is as follows: ZN6 at a ratio of 1: 1.8, ZN7 at a ratio of 1: 0.5, ZN8 at a ratio of 1: 1.5, decocting with water, concentrating the decoction under reduced pressure to dryness, adding double distilled water to obtain 1mL preparation containing 22.9mg of extract, autoclaving, and storing in refrigerator at 4 deg.C.

1. Statistics of number of deaths of normal larvae

After the test larvae were fed with syrup at 2-day-old, the survival rates of the larvae were determined for the formulations ZN6, ZN7, ZN8, each at a concentration of 0.32mg/mL, when the larvae were fed with syrup at 3-6-day-old, respectively, and the results are shown in fig. 5.

The average survival rate of the larvae fed with the syrup in the control group is 94.43%, and the average survival rate of the larvae fed with the ZN8 preparation is 91.67%, which is not obviously different from the control group. The average survival rates of the larvae fed with ZN6 and ZN7 preparations are 61.11 percent and 48.61 percent respectively, and are obviously different from the first two groups. It is shown that ZN6 and ZN7 preparations have influence on larva growth, and ZN8 preparations have no influence on larva health condition.

2. Statistics of the number of deaths of infected larvae

After the test larvae were pre-cultured at 2 days, the larvae were inoculated with the toxin-containing syrup and the toxin-containing preparation at 3 days, and the survival rates of the larvae were determined by feeding the larvae with the syrup and the ZN6, ZN7 and ZN8 preparations each having a concentration of 0.32mg/mL at 4 to 6 days, respectively, and the results are shown in FIG. 6.

The average survival rate of the infected larvae is 43.05 percent; after the ZN8 preparation is fed, the average survival rate of the larvae in the intervention group is 90.28 percent, the survival rate is improved by 47.23 percent, and the difference is very obvious (P is less than 0.01); after the ZN6 preparation is fed, the average survival rate of the larvae is 55.56 percent, the survival rate is improved by 12.51 percent, and the treatment effect is poorer than that of ZN8, but the survival rate is obviously different from that of an infected group; the average survival rate of the larvae after the ZN7 preparation is fed is 45.83 percent, the survival rate is only improved by 2.78 percent, and the larvae have no statistical difference from the infected group. ZN8 preparation (the mass ratio of radix isatidis, folium isatidis and propolis is 1: 1.5) has the effect of preventing and treating the Chinese sacbrood, and the formula of the preparation is the preferable formula of the invention.

3. Variation in the number of deaths between larvae of different day ages

The best ZN8 preparation is selected for comparative analysis. The larvae of 2 days old were pre-cultured for one day, and the number of larvae of each group was determined to be the same at 3 days old for experimental treatment, and the change in survival number of larvae of 4, 5, and 6 days old was recorded, respectively, and the results are shown in FIG. 7.

For normal larvae, the control group died the most at 5 days of age, with a mortality rate of 2.84%, and relatively few deaths at 4 and 6 days of age, each mortality rate being 1.39%. Unlike the control group, the ZN8 formulation group had the highest mortality at 6 days of age, 4.23%, and 4 and 5 days of age, 1.39% and 2.84%, respectively. The ZN8 preparation group has no significant difference from the control group, and does not influence the normal growth of larvae.

For infected larvae, larvae vaccinated with CSBV alone had the highest mortality rate of up to 25% at 4 days of age, with subsequent gradual decline in mortality rates, with mortality rates of 22.70% and 20.92% at 5 and 6 days of age, respectively; the mortality rate of the ZN8 preparation group at 4 days is only 1.45%, the mortality rate at 5 days is only 7.07%, the mortality rate is far lower than that of the infection group at 5 days, and the mortality rate at 6 days is reduced to 0, which is very different from that of the CSBV group (P is less than 0.01). The ZN8 feed can improve the survival of infected larva, reduce death rate, and prevent collapse of bee colony caused by CSBV infection.

The screening of the ZN8 preparation group has no influence on the growth of normal larvae and has good curative effect on the growth of infected larvae, so the concentration screening test is carried out on the ZN8 preparation.

EXAMPLE 3 Effect of different concentrations of ZN8 preparation on larval survival

1. Statistics of number of deaths of normal larvae

After the test larvae were fed with syrup at 2 days of age, the survival of larvae was recorded for the group of ZN8 formulations, which were fed with syrup at 3 to 6 days of age, and all at low, medium and high concentrations, and the results are shown in fig. 8.

The average survival rate of the larvae after the control group is fed with the syrup is 94.43%, and the average survival rate of the larvae after the control group is fed with the preparation with the medium concentration (0.32mg/mL) is 91.67%, which is not obviously different from the control group. Medium concentrations are shown to have no effect on larval health. However, the average survival rates of larvae fed with the low concentration (0.032mg/mL) and high concentration (3.2mg/mL) formulations were 77.78% and 81.97%, respectively, which are significantly different from those of the control group (P < 0.05).

2. Statistics of the number of deaths of infected larvae

After 2-day-old pre-culture of the test larvae, 3-day-old larvae were fed with the toxicant-containing syrup and toxicant-containing formulation (containing low, medium and high concentration ZN8 formulations, respectively). The survival rate of larvae when fed with syrup at 4 to 6 days of age, each at a concentration of 0.032mg/mL, 0.32mg/mL, 3.2mg/mL ZN8 formulation was determined and the results are shown in FIG. 9.

The average survival rate of larvae in the infected group is 43.05 percent, the average survival rate of larvae fed with the low-concentration preparation and the high-concentration preparation containing CSBV is 68.05 percent and 75 percent respectively, and is obviously different from that of the infected group (P <0.05), while the average survival rate of larvae fed with the preparation containing CSBV is 93.05 percent and is greatly different from that of the infected group (P < 0.01). It shows that 3 concentrations of ZN8 preparation have control effect on Chinese bee sacbrood, and the effect is best when the concentration is 0.32 mg/mL.

3. Variation in the number of deaths between larvae of different day ages

The larvae of 2 days old were pre-cultured for one day, and the number of larvae in each group was treated by experiment at 3 days old, and the change of death number of larvae of 4, 5, and 6 days old was recorded, respectively, and the results are shown in FIG. 10.

For the normal group, the larva mortality rate of the CK group and the medium concentration group is only 1.39% at the age of 4 days; the mortality rates of the larvae in the CK group and the medium concentration group are both 2.84% at the age of 5 days, the mortality rate of the larvae in the CK group is 2.78% at the age of 6 days, the mortality rate of the larvae in the medium concentration group is 3.17%, and the statistical difference between the larvae in the CK group and the larvae in the medium concentration group is avoided, so that the intervention in the medium concentration group has no adverse effect on the survival of normal larvae.

For the infected group, at 4 days of age, the mortality rate of the larvae in the CSBV group is 25%, and the mortality rate of the larvae in the CSBV + medium concentration group is only 2.78%, so that the mortality rate of the larvae is greatly reduced (P is less than 0.01); the death rate of the larvae of the CSBV group is 22.7 percent at the age of 5 days, and the larvae of the CSBV + medium concentration group do not die; at the age of 6 days, the death rate of the CBSV group larvae is 20.92 percent, and the death rate of the CSBV + medium concentration group larvae is only 7.07 percent, which shows that the intervention of the medium concentration group can obviously reduce the mass death of the larvae caused by the infection of the CSBV, thereby avoiding the collapse of bee colonies caused by the infection of the CSBV.

The concentration of 0.32mg/ml is the optimum concentration for resisting Chinese bee sacbrood.

4. Effect of different concentrations of ZN preparation on larval morphology

The growth of larvae was compared between the CK and ZN, challenge and challenge + ZN preparations, respectively, by microscopic observation and photography, using a single well diameter of a 48-well plate as a standard, and the results are shown in FIG. 11.

For normal larvae, feeding low concentration formulations has an effect on the growth of the larvae; the larvae fed with the medium-concentration preparation and the high-concentration preparation grow well and have no difference with the larvae of the CK group.

For infected larvae, the infected larvae were significantly smaller than CK group larvae individuals; the growth of the larvae fed with the low-concentration preparation containing CSBV is slightly larger than that of the larvae infected with the CSBV, but the development is still unhealthy compared with that of the CK group; the larvae fed with the medium concentration preparation containing CSBV have good growth and development, and have no obvious difference with the larvae of CK group in size; the larva fed with the high-concentration preparation containing CSBV has larger size than that fed with the low-concentration preparation containing toxicity but smaller size than that fed with the medium-concentration preparation containing toxicity, and the development of the larva is influenced to a certain extent.

The screened ZN8 group with the concentration of 0.32mg/mL has no influence on the growth of normal larvae and has good growth promoting effect on infected larvae.

Example 4 Effect of different concentrations of caffeic acid on healthy larva survival

After the test larvae were fed with syrup at 2 days of age, normal larval survival (larvae were fed with syrup at 3 to 6 days of age, caffeic acid concentrations of 0.75mg/mL, 7.5mg/mL, and 75mg/mL, respectively) and larval infection survival (larvae were fed with syrup containing toxicant and toxicant formulations (0.75 mg/mL, 7.5mg/mL, and 75mg/mL, respectively) at 3 days of age, and larvae were fed with syrup, 0.75mg/mL, 7.5mg/mL, and 75mg/mL, respectively, at 4 to 6 days of age, the results are shown in FIG. 12.

For the normal group, the larval mortality rates were 1.39%, 2.84%, 2.92% at 4 to 6 days of age, respectively; the caffeic acid groups with different concentrations cause different degrees of death to 5-day-old larvae, the death rate is 6.94-11.11%, wherein the 0.75 mu g/mL group is very different from the CK group (P is less than 0.01); the death of 6-day-old larvae can be seen on the next day, the mortality rate is 2.78-8.33%, and the 75 mu g/mL group is remarkably different from the CK group (P is less than 0.05).

The average survival rate of the larvae of the CK group with 4-6 days old is 94.43%, while the average survival rate of the larvae fed with caffeic acid with various concentrations is only 81.94-88.89%, and the survival rate is obviously different from that of the CK group (P is less than 0.05), which indicates that the intervention of caffeic acid influences the survival of normal larvae.

Morphological observation revealed that on day 18 of the experiment (i.e., day 22 from the egg stage), the CK group had emerged as a adult bee, but the larvae fed with caffeic acid had retarded growth and development, and the morphology remained in the larval stage and could not pupate, even though the larvae of the caffeic acid group with the highest survival rate of 7.5 μ g/mL, and the experimental results showed that caffeic acid intervention affected the growth and development of normal larvae, and thus caffeic acid was not suitable for feeding apis cerana larvae (fig. 13).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims (12)

1. a Chinese medicine composition for preventing and treating sac disease, is characterized in that, is made up of the composition of following parts by weight: 1 part of Radix Isatidis, 1 part of Daqingye and 1.5 parts of propolis. 2. Chinese medicine composition according to claim 1, is characterized in that, described propolis comes from Italian bee (Apismellifera), and glue content is 20-60%. 3. Chinese medicine composition according to claim 2, is characterized in that, the glue content of described propolis is 30-50%. 4. Chinese medicine composition according to claim 3, is characterized in that, the glue content of described propolis is 40-50%. 5. Chinese medicine composition according to claim 2, is characterized in that, described propolis is collected in temperate coniferous broad-leaved mixed forest area propolis. 6. Chinese medicine composition according to claim 5, is characterized in that, described propolis is collected in the propolis of Heilongjiang, Jilin and northeastern Liaoning region. 7. the preparation method of the described Chinese medicine composition of any one of claim 1-6, it is characterized in that, described method comprises: decoct with water after each composition is mixed in proportion, as required, the decoction is concentrated or diluted to concentration 10 -50mg/mL. 8 . The method according to claim 7 , wherein the method comprises: decocting with water after mixing the components in proportion, and concentrating or diluting the decoction to a concentration of 22.9 mg/mL as required. 9 . 9. The application of the traditional Chinese medicine composition of any one of claims 1-6 in the preparation of a medicine for preventing and treating cystic disease. 10. The application according to claim 9, wherein the mesococcus disease is caused by the infection of mesocystis larvae virus. 11. The application of the Chinese medicinal composition described in any one of claims 1-6 in the non-therapeutic purpose against Chinese bee sac larvae virus. 12. The application of the traditional Chinese medicine composition according to any one of claims 1 to 6 in suppressing the proliferation of Bee Cyst larvae virus for non-therapeutic purposes.

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