阅读说明：本技术 一种治疗蜜蜂真菌病的制剂组方及其制备方法、应用方法 (Preparation formula for treating bee mycosis and preparation method and application method thereof ) 是由 张魁 侯丽娜 李大威 高见红 于 2021-10-15 设计创作，主要内容包括：本发明公开了一种治疗蜜蜂真菌病的制剂组方及其制备方法、应用方法,属于中药制剂技术领域,包括如下步骤：(1)低温真空干燥处理；(2)原料称取；(3)初步浸提；(4)电晕-火焰交替处理；(5)二次浸提；(6)成品制备。本申请将茯苓、苦参、金银花等按照合适的比例进行搭配,与现代生物、物理化学提取技术结合起来,以促进各原料中活性成分充分的溶出,并形成独特的配伍,最终制备的制剂绿色安全,对蜜蜂真菌病具有很好的治疗效果。(The invention discloses a preparation formula for treating bee mycosis, a preparation method and an application method thereof, belonging to the technical field of traditional Chinese medicine preparations and comprising the following steps: (1) carrying out low-temperature vacuum drying treatment; (2) weighing raw materials; (3) primary leaching; (4) performing corona-flame alternate treatment; (5) secondary leaching; (6) and (5) preparing a finished product. According to the application, the poria cocos, the radix sophorae flavescentis, the honeysuckle and the like are matched according to a proper proportion, and are combined with modern biological and physical-chemical extraction technologies, so that active ingredients in the raw materials are fully dissolved out, unique compatibility is formed, and the finally prepared preparation is green and safe and has a good treatment effect on bee mycosis.)

1. The preparation of the preparation for treating bee mycosis is characterized by comprising the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven for vacuum drying treatment, and respectively taking out;

(2) weighing raw materials:

weighing 20-30 parts by weight of poria cocos, 13-17 parts by weight of radix sophorae flavescentis, 10-14 parts by weight of honeysuckle, 7-9 parts by weight of folium isatidis, 3-4 parts by weight of safflower carthamus, 2-3 parts by weight of ligusticum wallichii, 1-3 parts by weight of folium artemisiae argyi, 3-5 parts by weight of coptis chinensis, 5-10 parts by weight of golden cypress, 7-9 parts by weight of rheum officinale and 7-13 parts by weight of liquorice after low-temperature vacuum drying treatment in step (1) for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary extraction, and collecting a first extracting solution and filter residues for later use;

(4) corona-flame alternate treatment:

carrying out corona-flame alternate treatment on the filter residue collected in the step (3), and taking out for later use after the completion;

(5) secondary leaching:

placing the filter residue treated in the step (4) in a Soxhlet extraction device for secondary leaching to obtain a second extracting solution for later use;

(6) and (3) preparing a finished product:

and (4) uniformly mixing the first extracting solution obtained in the step (3) and the second extracting solution obtained in the step (5) and then concentrating.

2. The preparation of claim 1, wherein the vacuum drying treatment in step (1) is performed under a vacuum degree of 1.3 to 1.7Pa, at a temperature of 30 to 50 ℃, and drying to a moisture content of 1 to 1.4%.

3. The preparation of claim 1, wherein the extraction agent used in the first leaching in step (3) is diethyl ether, and the number of refluxing times is 20-30 times.

4. The preparation of the agent for treating bee mycosis according to claim 1, wherein the specific procedure of corona-flame alternate treatment in step (4) is: corona treatment is carried out for 30-50 s, then flame treatment is carried out for 3-5 min, then corona treatment is carried out for 20-30 s, and finally flame treatment is carried out for 1-2 min.

5. The preparation of claim 4, wherein the corona treatment voltage is 10-12 kV, and the flame treatment is flame exposure.

6. The preparation of claim 1, wherein the extractant used in the second leaching in step (5) is absolute ethanol, and the number of refluxing times is 10-20.

7. The preparation of claim 1, wherein the concentration in step (6) is performed by a rotary evaporator.

8. A formulation for the treatment of bee mycosis, characterized in that it is prepared according to the method of any one of claims 1 to 7.

9. An application method of a preparation for treating bee mycoses is characterized in that the preparation for treating the bee mycoses is particularly applied to the treatment of the bee mycoses.

Technical Field

The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a preparation formula for treating bee mycosis, and a preparation method and an application method thereof.

Background

The bee mycosis mainly comprises "chalkbrood disease" and fungal creeping bee disease, wherein the "chalkbrood disease" is also called "calcareous" disease and is caused by ascosphaera apis, and the fungi of the genera ascomycete, ascomycete subclass, saccharales, saccharaceae and saccharaceae. The chalkbrood disease mainly harms bee larvae, and is a common and stubborn fungal infectious disease in the actual bee-keeping process.

The current methods for preventing and treating bee mycosis mainly comprise: (1) thoroughly disinfecting the original beehives; (2) the bee species with no medical history and strong disease resistance are exchanged; (3) excessive heat preservation and excessive moisture are avoided in the spring breeding period; (4) the honeycomb must be thoroughly sterilized; (5) drug treatment; the drug therapy is the most effective therapy method, but the research on the preparation for preventing and treating bee mycosis is very limited nowadays, and although a large amount of traditional Chinese medicine preparations are used, the treatment effect is general, and the great waste of traditional Chinese medicine raw materials is caused.

Disclosure of Invention

The invention aims to solve the existing problems and provides a preparation formula for treating bee mycosis, a preparation method and an application method thereof.

The invention is realized by the following technical scheme:

1. designed preparation formula

The preparation formula is as follows: poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix. Wherein:

poria, neutral in nature, sweet and bland in flavor, is commonly used for inducing diuresis to eliminate dampness, invigorating spleen and tranquilizing;

lightyellow sophora root, cold in nature and bitter in taste, is commonly used for clearing heat and drying dampness, killing parasites and relieving itching and inducing diuresis;

honeysuckle, sweet in nature and cold in nature, is commonly used for clearing heat, removing toxicity and dispelling wind and heat;

folium Isatidis, cold in nature and bitter in taste, is commonly used for clearing heat and removing toxicity, cooling blood and relieving sore throat;

safflower, warm in nature and pungent in flavor, is commonly used for activating blood and dredging meridians, removing blood stasis and relieving pain;

chuan Xiong is warm in nature and pungent in flavor, and is commonly used for activating blood and qi, dispelling wind and alleviating pain;

folium Artemisiae Argyi, warm in nature, pungent and bitter in flavor, is commonly used for warming meridians to stop bleeding, dispelling cold to alleviate pain;

coptis root, rhizoma Coptidis, being cold in nature and bitter in taste, is commonly used for clearing heat and drying dampness, purging fire and removing toxicity;

phellodendron bark, cortex Phellodendri, with cold nature and bitter taste, is commonly used for clearing heat and drying dampness, purging fire and removing toxicity, and relieving deficiency heat;

rhubarb, being cold in nature and bitter in taste, is commonly used for purging and removing accumulation, clearing heat and purging fire, detoxifying and stopping bleeding, activating blood and dissolving stasis;

licorice root, radix Glycyrrhizae is neutral in nature and sweet in taste, and is commonly used for tonifying qi and strengthening middle energizer, eliminating phlegm and relieving cough, removing toxicity, relieving spasm and pain, and moderating drug properties.

2. Determination of preparation method

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven for vacuum drying treatment, and respectively taking out;

(2) weighing raw materials:

weighing 20-30 parts by weight of poria cocos, 13-17 parts by weight of radix sophorae flavescentis, 10-14 parts by weight of honeysuckle, 7-9 parts by weight of folium isatidis, 3-4 parts by weight of safflower carthamus, 2-3 parts by weight of ligusticum wallichii, 1-3 parts by weight of folium artemisiae argyi, 3-5 parts by weight of coptis chinensis, 5-10 parts by weight of golden cypress, 7-9 parts by weight of rheum officinale and 7-13 parts by weight of liquorice after low-temperature vacuum drying treatment in step (1) for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary extraction, and collecting a first extracting solution and filter residues for later use;

(4) corona-flame alternate treatment:

carrying out corona-flame alternate treatment on the filter residue collected in the step (3), and taking out for later use after the completion;

(5) secondary leaching:

placing the filter residue treated in the step (4) in a Soxhlet extraction device for secondary leaching to obtain a second extracting solution for later use;

(6) and (3) preparing a finished product:

and (4) uniformly mixing the first extracting solution obtained in the step (3) and the second extracting solution obtained in the step (5) and then concentrating.

Further, the vacuum degree is controlled to be 1.3-1.7 Pa during the vacuum drying treatment in the step (1), the temperature is controlled to be 30-50 ℃, and the drying is carried out until the water content is 1-1.4%.

By adopting the technical scheme, the raw materials are respectively and independently placed in the vacuum drying box for vacuum drying treatment and are dried under the condition of vacuum low temperature, so that the loss or inactivation of active ingredients, particularly volatile oil ingredients, in the raw materials is prevented, the cell permeability of the raw materials can be improved, and a foundation is laid for subsequent leaching.

Further, the extractant used in the primary leaching in the step (3) is diethyl ether, and the reflux times are 20-30 times.

By adopting the technical scheme, the dried raw materials are mixed and crushed, and the repeated reflux extraction is carried out by using diethyl ether as an extracting agent by using a Soxhlet extraction device, so that the extraction rate is effectively improved.

Further, the specific procedure of the corona-flame alternate treatment in the step (4) is as follows: corona treatment is carried out for 30-50 s, then flame treatment is carried out for 3-5 min, then corona treatment is carried out for 20-30 s, and finally flame treatment is carried out for 1-2 min.

Further, the working voltage during corona treatment is 10-12 kV, and flame treatment is flame outer flame treatment.

Further, the extractant used in the secondary leaching in the step (5) is absolute ethyl alcohol, and the reflux times are 10-20 times.

Further, the concentration in the step (6) is performed by using a rotary evaporator.

By adopting the technical scheme, the filter residue collected by primary leaching in the step (3) is subjected to corona-flame alternate treatment, the filter residue can be dried through the discharge and flame oxidation effects, the surface wettability of the filter residue is improved, countless micro-concave dense holes are formed on the surface of the filter residue, the leaching rate is improved, and in addition, the generation of chemical substances which are helpful for treating bee mycosis can be promoted. The extractant adopted in the secondary extraction is absolute ethyl alcohol, so that the problems of single extractant and limited extraction ingredients are avoided, the full dissolution of effective ingredients is promoted, and the utilization rate of the raw materials is improved.

A formulation for the treatment of bee mycosis, prepared according to the method of any one of claims 1 to 7.

3. Determining an application method

The application of a preparation for treating bee mycoses is to treat the bee mycoses, and the preparation is diluted by different times and mixed with syrup with the same amount to perform the steps of 1: 1, feeding after mixing, observing the effect and determining the application method.

Compared with the prior art, the invention has the following advantages:

according to the application, the poria cocos, the radix sophorae flavescentis, the honeysuckle and the like are matched according to a proper proportion, and are combined with modern biological and physical-chemical extraction technologies, so that active ingredients in the raw materials are fully dissolved out, unique compatibility is formed, and the finally prepared preparation is green and safe and has a good treatment effect on bee mycosis.

Detailed Description

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing poria cocos, sophora flavescens, honeysuckle, folium isatidis, safflower, ligusticum wallichii, folium artemisiae argyi, coptis chinensis, phellodendron, rheum officinale and liquorice in a vacuum drying oven for vacuum drying treatment, and taking out the materials for later use respectively, wherein the vacuum degree is controlled to be 1.3-1.7 Pa, the temperature is controlled to be 30-50 ℃, and the materials are dried until the water content is 1-1.4%;

(2) weighing raw materials:

weighing 20-30 parts by weight of poria cocos, 13-17 parts by weight of radix sophorae flavescentis, 10-14 parts by weight of honeysuckle, 7-9 parts by weight of folium isatidis, 3-4 parts by weight of safflower carthamus, 2-3 parts by weight of ligusticum wallichii, 1-3 parts by weight of folium artemisiae argyi, 3-5 parts by weight of coptis chinensis, 5-10 parts by weight of golden cypress, 7-9 parts by weight of rheum officinale and 7-13 parts by weight of liquorice after low-temperature vacuum drying treatment in step (1) for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary leaching, wherein the used extraction agent is diethyl ether, the reflux frequency is 20-30 times, and collecting a first extracting solution and filter residues for later use;

(4) corona-flame alternate treatment:

performing corona-flame alternate treatment on the filter residue collected in the step (3), firstly performing corona treatment for 30-50 s, then performing flame treatment for 3-5 min, then performing corona treatment for 20-30 s, and finally performing flame treatment for 1-2 min, and taking out for later use after the corona treatment is completed, wherein the working voltage during the corona treatment is 10-12 kV, and the flame treatment is flame outer flame treatment;

(5) secondary leaching:

placing the filter residue treated in the step (4) in a Soxhlet extraction device for secondary extraction, wherein the used extraction agent is absolute ethyl alcohol, the reflux frequency is 10-20 times, and a second extracting solution is obtained for later use;

(6) and (3) preparing a finished product:

and (3) uniformly mixing the first extracting solution obtained in the step (3) and the second extracting solution obtained in the step (5), and then placing the mixture into a rotary evaporator for concentration.

For further explanation of the present invention, reference will now be made to the following specific examples.

Example 1

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven for vacuum drying, respectively taking out, controlling vacuum degree at 1.3Pa and temperature at 30 deg.C, and drying until water content is 1%;

(2) weighing raw materials:

weighing 20 parts of poria cocos, 13 parts of radix sophorae flavescentis, 10 parts of honeysuckle, 7 parts of folium isatidis, 3 parts of safflower carthamus, 2 parts of ligusticum wallichii, 1 part of folium artemisiae argyi, 3 parts of coptis chinensis, 5 parts of golden cypress, 7 parts of rheum officinale and 7 parts of liquorice which are subjected to low-temperature vacuum drying treatment in the step (1) in corresponding parts by weight for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary leaching, wherein the used extraction agent is diethyl ether, the reflux frequency is 20 times, and collecting a first extracting solution and filter residues for later use;

(4) corona-flame alternate treatment:

performing corona-flame alternate treatment on the filter residue collected in the step (3), firstly performing corona treatment for 30s, then performing flame treatment for 3min, then performing corona treatment for 20s, and finally performing flame treatment for 1min, and taking out the filter residue for standby after the corona treatment is completed, wherein the working voltage during the corona treatment is 10kV, and the flame treatment is flame outer flame treatment;

(5) secondary leaching:

placing the filter residue treated in the step (4) in a Soxhlet extraction device for secondary extraction, wherein the used extraction agent is absolute ethyl alcohol, the reflux frequency is 10 times, and a second extracting solution is obtained for later use;

(6) and (3) preparing a finished product:

and (3) uniformly mixing the first extracting solution obtained in the step (3) and the second extracting solution obtained in the step (5), and then placing the mixture into a rotary evaporator for concentration.

Example 2

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven, vacuum drying, and drying at 40 deg.C under vacuum degree of 1.5Pa to water content of 1.2%;

(2) weighing raw materials:

weighing 25 parts of poria cocos, 15 parts of radix sophorae flavescentis, 12 parts of honeysuckle, 8 parts of folium isatidis, 3.5 parts of safflower, 2.5 parts of ligusticum wallichii, 2 parts of folium artemisiae argyi, 4 parts of coptis chinensis, 7.5 parts of cortex phellodendri, 8 parts of rheum officinale and 10 parts of liquorice which are subjected to low-temperature vacuum drying treatment in the step (1) in corresponding parts by weight for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary leaching, wherein the used extraction agent is diethyl ether, the reflux frequency is 25 times, and collecting a first extracting solution and filter residues for later use;

(4) corona-flame alternate treatment:

performing corona-flame alternate treatment on the filter residue collected in the step (3), wherein the corona treatment is performed for 40s, then the flame treatment is performed for 4min, then the corona treatment is performed for 25s, finally the flame treatment is performed for 1.5min, and then the filter residue is taken out for standby after the flame treatment is completed, wherein the working voltage during the corona treatment is 11kV, and the flame treatment is flame outer flame treatment;

(5) secondary leaching:

placing the filter residue treated in the step (4) in a Soxhlet extraction device for secondary extraction, wherein the used extraction agent is absolute ethyl alcohol, the reflux frequency is 15 times, and a second extracting solution is obtained for later use;

(6) and (3) preparing a finished product:

and (3) uniformly mixing the first extracting solution obtained in the step (3) and the second extracting solution obtained in the step (5), and then placing the mixture into a rotary evaporator for concentration.

Example 3

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven, vacuum drying, respectively taking out, controlling vacuum degree at 1.7Pa and temperature at 50 deg.C, and drying to water content of 1.4%;

(2) weighing raw materials:

weighing 30 parts of poria cocos, 17 parts of radix sophorae flavescentis, 14 parts of honeysuckle, 9 parts of folium isatidis, 4 parts of safflower carthamus, 3 parts of ligusticum wallichii, 3 parts of folium artemisiae argyi, 5 parts of coptis chinensis, 10 parts of golden cypress, 9 parts of rheum officinale and 13 parts of liquorice which are subjected to low-temperature vacuum drying treatment in the step (1) in corresponding parts by weight for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary leaching, wherein the used extraction agent is diethyl ether, the reflux frequency is 30 times, and collecting a first extracting solution and filter residues for later use;

(4) corona-flame alternate treatment:

performing corona-flame alternate treatment on the filter residue collected in the step (3), firstly performing corona treatment for 50s, then performing flame treatment for 5min, then performing corona treatment for 30s, and finally performing flame treatment for 2min, and taking out the filter residue for standby after the corona treatment is completed, wherein the working voltage during the corona treatment is 12kV, and the flame treatment is flame outer flame treatment;

(5) secondary leaching:

placing the filter residue treated in the step (4) in a Soxhlet extraction device for secondary extraction, wherein the used extraction agent is absolute ethyl alcohol, the reflux frequency is 20 times, and a second extracting solution is obtained for later use;

(6) and (3) preparing a finished product:

and (3) uniformly mixing the first extracting solution obtained in the step (3) and the second extracting solution obtained in the step (5), and then placing the mixture into a rotary evaporator for concentration.

Example 4

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven, vacuum drying, and drying at 40 deg.C under vacuum degree of 1.5Pa to water content of 1.2%;

(2) weighing raw materials:

weighing 25 parts of poria cocos, 15 parts of radix sophorae flavescentis, 12 parts of honeysuckle, 8 parts of folium isatidis, 3.5 parts of safflower, 2.5 parts of ligusticum wallichii, 2 parts of folium artemisiae argyi, 4 parts of coptis chinensis, 7.5 parts of cortex phellodendri, 8 parts of rheum officinale and 10 parts of liquorice which are subjected to low-temperature vacuum drying treatment in the step (1) in corresponding parts by weight for later use;

(3) corona-flame alternate treatment:

mixing and crushing the raw materials weighed in the step (2), and then carrying out corona-flame alternative treatment, wherein the corona treatment is firstly carried out for 40s, then the flame treatment is carried out for 4min, then the corona treatment is carried out for 25s, finally the flame treatment is carried out for 1.5min, and then the raw materials are taken out for standby after the completion of the flame treatment, wherein the working voltage during the corona treatment is 11kV, and the flame treatment is flame outer flame treatment;

(4) leaching

Placing the raw materials treated in the step (3) in a Soxhlet extraction device for extraction, wherein the used extractant is absolute ethyl alcohol, the reflux times are 15 times, and an extracting solution is obtained for later use;

(5) and (3) preparing a finished product:

and (4) uniformly mixing the extracting solution obtained in the step (4), and then placing the mixture into a rotary evaporator for concentration.

Example 5

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven, vacuum drying, and drying at 40 deg.C under vacuum degree of 1.5Pa to water content of 1.2%;

(2) weighing raw materials:

weighing 25 parts of poria cocos, 15 parts of radix sophorae flavescentis, 12 parts of honeysuckle, 8 parts of folium isatidis, 3.5 parts of safflower, 2.5 parts of ligusticum wallichii, 2 parts of folium artemisiae argyi, 4 parts of coptis chinensis, 7.5 parts of cortex phellodendri, 8 parts of rheum officinale and 10 parts of liquorice which are subjected to low-temperature vacuum drying treatment in the step (1) in corresponding parts by weight for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary leaching, wherein the used extraction agent is diethyl ether, the reflux frequency is 25 times, and collecting a first extracting solution and filter residues for later use;

(4) corona treatment:

carrying out corona treatment on the filter residue collected in the step (3) for 40s, and taking out for later use after the corona treatment is finished, wherein the flame treatment is flame outer flame treatment;

(5) secondary leaching:

placing the filter residue treated in the step (4) in a Soxhlet extraction device for secondary extraction, wherein the used extraction agent is absolute ethyl alcohol, the reflux frequency is 15 times, and a second extracting solution is obtained for later use;

(6) and (3) preparing a finished product:

and (3) uniformly mixing the first extracting solution obtained in the step (3) and the second extracting solution obtained in the step (5), and then placing the mixture into a rotary evaporator for concentration.

Example 6

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven, vacuum drying, and drying at 40 deg.C under vacuum degree of 1.5Pa to water content of 1.2%;

(2) weighing raw materials:

weighing 25 parts of poria cocos, 15 parts of radix sophorae flavescentis, 12 parts of honeysuckle, 8 parts of folium isatidis, 3.5 parts of safflower, 2.5 parts of ligusticum wallichii, 2 parts of folium artemisiae argyi, 4 parts of coptis chinensis, 7.5 parts of cortex phellodendri, 8 parts of rheum officinale and 10 parts of liquorice which are subjected to low-temperature vacuum drying treatment in the step (1) in corresponding parts by weight for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary leaching, wherein the used extraction agent is diethyl ether, the reflux frequency is 25 times, and collecting a first extracting solution and filter residues for later use;

(4) flame treatment:

performing flame treatment on the filter residue collected in the step (3), wherein the flame treatment is flame outer flame treatment for 4 min;

(5) secondary leaching:

placing the filter residue treated in the step (4) in a Soxhlet extraction device for secondary extraction, wherein the used extraction agent is absolute ethyl alcohol, the reflux frequency is 15 times, and a second extracting solution is obtained for later use;

(6) and (3) preparing a finished product:

and (3) uniformly mixing the first extracting solution obtained in the step (3) and the second extracting solution obtained in the step (5), and then placing the mixture into a rotary evaporator for concentration.

Example 7

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven, vacuum drying, and drying at 40 deg.C under vacuum degree of 1.5Pa to water content of 1.2%;

(2) weighing raw materials:

weighing 25 parts of poria cocos, 15 parts of radix sophorae flavescentis, 12 parts of honeysuckle, 8 parts of folium isatidis, 3.5 parts of safflower, 2.5 parts of ligusticum wallichii, 2 parts of folium artemisiae argyi, 4 parts of coptis chinensis, 7.5 parts of cortex phellodendri, 8 parts of rheum officinale and 10 parts of liquorice which are subjected to low-temperature vacuum drying treatment in the step (1) in corresponding parts by weight for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary leaching, wherein the used extraction agent is diethyl ether, the reflux frequency is 25 times, and collecting a first extracting solution and filter residues for later use;

(4) secondary leaching

Placing the filter residue obtained in the step (3) in a Soxhlet extraction device for secondary extraction, wherein the used extraction agent is absolute ethyl alcohol, the reflux frequency is 15 times, and a second extracting solution is obtained for later use;

(5) and (3) preparing a finished product:

and (4) uniformly mixing the first extracting solution obtained in the step (3) and the second extracting solution obtained in the step (4), and then placing the mixture into a rotary evaporator for concentration.

Example 8

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven, vacuum drying, and drying at 40 deg.C under vacuum degree of 1.5Pa to water content of 1.2%;

(2) weighing raw materials:

weighing 25 parts of poria cocos, 15 parts of radix sophorae flavescentis, 12 parts of honeysuckle, 8 parts of folium isatidis, 3.5 parts of safflower, 2.5 parts of ligusticum wallichii, 2 parts of folium artemisiae argyi, 4 parts of coptis chinensis, 7.5 parts of cortex phellodendri, 8 parts of rheum officinale and 10 parts of liquorice which are subjected to low-temperature vacuum drying treatment in the step (1) in corresponding parts by weight for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary leaching, wherein the used extraction agent is diethyl ether, the reflux frequency is 25 times, and collecting a first extracting solution and filter residues for later use;

(4) and (3) preparing a finished product:

and (4) placing the first extracting solution obtained in the step (3) into a rotary evaporator for concentration.

Example 9

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven, vacuum drying, and drying at 40 deg.C under vacuum degree of 1.5Pa to water content of 1.2%;

(2) weighing raw materials:

weighing 25 parts of poria cocos, 15 parts of radix sophorae flavescentis, 12 parts of honeysuckle, 8 parts of folium isatidis, 3.5 parts of safflower, 2.5 parts of ligusticum wallichii, 2 parts of folium artemisiae argyi, 4 parts of coptis chinensis, 7.5 parts of cortex phellodendri, 8 parts of rheum officinale and 10 parts of liquorice which are subjected to low-temperature vacuum drying treatment in the step (1) in corresponding parts by weight for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary extraction, wherein the used extraction agent is absolute ethyl alcohol, the reflux frequency is 25 times, and collecting a first extraction solution and filter residues for later use;

(4) corona-flame alternate treatment:

performing corona-flame alternate treatment on the filter residue collected in the step (3), wherein the corona treatment is performed for 40s, then the flame treatment is performed for 4min, then the corona treatment is performed for 25s, finally the flame treatment is performed for 1.5min, and then the filter residue is taken out for standby after the flame treatment is completed, wherein the working voltage during the corona treatment is 11kV, and the flame treatment is flame outer flame treatment;

(5) secondary leaching:

placing the filter residue treated in the step (4) in a Soxhlet extraction device for secondary extraction, wherein the used extraction agent is absolute ethyl alcohol, the reflux frequency is 15 times, and a second extracting solution is obtained for later use;

(6) and (3) preparing a finished product:

and (3) uniformly mixing the first extracting solution obtained in the step (3) and the second extracting solution obtained in the step (5), and then placing the mixture into a rotary evaporator for concentration.

Example 10

The preparation of a preparation for treating bee mycosis comprises the following steps:

(1) low-temperature vacuum drying treatment:

respectively placing Poria, radix Sophorae Flavescentis, flos Lonicerae, folium Isatidis, Carthami flos, rhizoma Ligustici Chuanxiong, folium Artemisiae Argyi, Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, and Glycyrrhrizae radix in a vacuum drying oven, vacuum drying, and drying at 40 deg.C under vacuum degree of 1.5Pa to water content of 1.2%;

(2) weighing raw materials:

weighing 25 parts of poria cocos, 15 parts of radix sophorae flavescentis, 12 parts of honeysuckle, 8 parts of folium isatidis, 3.5 parts of safflower, 2.5 parts of ligusticum wallichii, 2 parts of folium artemisiae argyi, 4 parts of coptis chinensis, 7.5 parts of cortex phellodendri, 8 parts of rheum officinale and 10 parts of liquorice which are subjected to low-temperature vacuum drying treatment in the step (1) in corresponding parts by weight for later use;

(3) primary leaching:

mixing and crushing the raw materials weighed in the step (2), placing the mixture in a Soxhlet extraction device for primary leaching, wherein the used extraction agent is diethyl ether, the reflux frequency is 25 times, and collecting a first extracting solution and filter residues for later use;

(4) corona-flame alternate treatment:

performing corona-flame alternate treatment on the filter residue collected in the step (3), wherein the corona treatment is performed for 40s, then the flame treatment is performed for 4min, then the corona treatment is performed for 25s, finally the flame treatment is performed for 1.5min, and then the filter residue is taken out for standby after the flame treatment is completed, wherein the working voltage during the corona treatment is 11kV, and the flame treatment is flame outer flame treatment;

(5) secondary leaching:

placing the filter residue treated in the step (4) in a Soxhlet extraction device for secondary extraction, wherein the used extractant is diethyl ether, the reflux times are 15 times, and a second extracting solution is obtained for later use;

(6) and (3) preparing a finished product:

and (3) uniformly mixing the first extracting solution obtained in the step (3) and the second extracting solution obtained in the step (5), and then placing the mixture into a rotary evaporator for concentration.

In order to compare the technical effects of the application, the preparation is prepared by the methods of the above example 2 and examples 4 to 10 respectively, and then the functional animal test is carried out, specifically:

1. safety toxicology evaluation test

1.1 sample:

the formulation prepared by the method of example 2. During the test, the test object is prepared by using distilled water as a solvent and is refrigerated for storage.

1.2 Experimental animals:

20 KM mice, 20 SD rats, and half male and female mice, which are derived from the Experimental animal center of Yanbian university.

1.3 acute oral toxicity test:

one dosage group of 15000mg/kg.BW is set, and the stomach is drenched once by mouth according to 20 mL/kg.BW. Animals are fasted for 16h before gavage, drinking water is not limited, and the animals are observed for 7 days after the first contamination.

By adopting a maximum tolerated dose method, the rats and mice are respectively randomly divided into two groups, 4 groups in total, and each group has 10 animals with the same sex. The test substance 75g is weighed, added with distilled water to 100mL and mixed for standby. The test substance was orally administered to both rats and mice once. The animals were observed for signs of intoxication and mortality within one week and were sacrificed for gross dissection after the test was completed and weighed. And judging the toxicity grading of the test object according to the LD50 value.

Table 1 example 2 results of acute oral toxicity test of formulation on rats and mice

Acute oral toxicity test results: after the test subjects, no abnormality was observed in the general performance and behavior of the rats and mice, no death of the animals was observed during the observation period (table 1), and no abnormality was observed with gross dissection. The oral liquid has acute oral MTD value of over 15000mg/kg.BW for rat and mouse, and belongs to nontoxic grade according to acute toxicity grade.

That is, the formulations prepared by the methods of the present application are not harmful to animals.

2. Test of therapeutic Effect

2.1 test methods: in 8 months of 2021, 3 beehives of 80 beehives of a bee field in Yangji city, Jilin province are taken for testing, 8 groups of a control group and test groups 1-8 are divided, 10 beehives of each group are provided, and each beehive has 9000 plus 10000 bees. The preparations correspondingly prepared in the embodiments 2 and 4-10 are respectively used for treatment, namely, the natural growth and decay after fungal infection are observed. The usage and dosage are as follows: adding distilled water into 1 part of the preparation to 1000ml, mixing, adding 1000ml syrup (white sugar and water are decocted according to a ratio of 100: 60), mixing, feeding 200ml per box of bee colony, artificially infecting chalkbrood according to a conventional method until local infection is obviously formed in 5 days, and then carrying out preparation treatment. The dosage of the drug for the uninfected part is 10mL, the drug is taken twice a day, the drug is continuously taken for 2 weeks, and the skin lesions are observed to observe the natural growth and disappearance of the fungus after the infection;

2.2 evaluation criteria of clinical efficacy: the standard of cure is as follows: the hyphae of the skin lesion is faded, no newly infected larva exists, no clinical symptoms exist, the fungus microscopic examination is continuous and secondary negative, and no relapse occurs after the medicine is stopped for one week; the effective standard is as follows: the hyphae disappear, or no newly infected larva exists, or no clinical symptoms exist, and the hyphae possibly recur after stopping taking the medicine for one week; invalidation criteria: the hyphae of the skin lesions are not faded, newly infected larvae exist, clinical symptoms exist, and the skin lesions recur after the medicine is stopped for one week. Or the fungus is positive by microscopic examination, or the skin damage appears in the original place after one week after the clinical recovery is stopped, and the fungus is positive by microscopic examination.

Specific experimental comparative data are shown in Table 2 below

TABLE 2 therapeutic effect of formulations on bee chalkbrood disease

	Cure (case)	Effective (case)	Invalid (case)
				Example 2	9	1	0
Example 4	3	7	0
				Example 5	7	3	0
Example 6	8	2	0
				Example 7	4	6	0
Example 8	3	7	0
				Example 9	7	3	0
Example 10	7	3	0

As can be seen from table 2 above, in the application, poria cocos, sophora flavescens, honeysuckle and the like are matched according to a proper proportion, and are combined with modern biological and physicochemical extraction technologies, so that the finally prepared preparation has a good treatment effect on bee mycosis.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the present invention is not limited to the illustrated embodiments, and all the modifications and equivalents of the embodiments may be made without departing from the spirit of the present invention.