阅读说明：本技术 芡实叶子的乙醇提取物的制备方法及其应用 (Preparation method and application of ethanol extract of gorgon fruit leaves ) 是由 叶满红 周斌 于 2020-07-20 设计创作，主要内容包括：芡实叶子的乙醇提取物的制备方法及其应用,涉及蜜蜂的蜜蜂美洲幼虫病防治技术领域。将阴干的芡实叶子研磨成粉末后采用乙醇水溶液浸泡,经抽滤,取滤液旋转蒸发,取得芡实叶子的乙醇提取物。将该提取物溶解于二甲基亚砜中,用于防治蜜蜂美洲幼虫病。该提取物的使用不存在停药期,在蜂群的整个生产及繁殖阶段均可安全使用,有助于维护蜂群的健康,促进蜂群整体生产力的提高。(A preparation method and application of an ethanol extract of gordon euryale leaves relate to the technical field of prevention and treatment of bee American larva diseases of bees. Grinding the dried gordon euryale seeds in the shade into powder, soaking the powder in an ethanol water solution, performing suction filtration, taking filtrate, and performing rotary evaporation to obtain an ethanol extract of the gordon euryale seeds. The extract is dissolved in dimethyl sulfoxide for preventing and treating American larva of bee. The extract has no drug withdrawal period, and can be safely used in the whole production and reproduction stage of bee colony, which is helpful for maintaining health of bee colony and promoting improvement of integral productivity of bee colony.)

1. The preparation method of the ethanol extract of the gorgon fruit leaves is characterized by comprising the following steps: grinding the dried gordon euryale seeds in the shade into powder, soaking the powder in an ethanol water solution, performing suction filtration, taking filtrate, and performing rotary evaporation to obtain an ethanol extract of the gordon euryale seeds.

2. The method of claim 1, wherein: the water content of the ethanol extract of the gorgon fruit leaves is 10-20 wt.%.

3. The production method according to claim 1 or 2, characterized in that: the volume percentage of the ethanol in the ethanol water solution is 75-85%.

4. The use of the ethanol extract of euryale ferox leaves obtained by the preparation method according to claim 1, is characterized in that: and (3) dissolving the ethanol extract of the gorgon euryale leaves in dimethyl sulfoxide, and using the ethanol extract to prevent and treat the American larva of bees.

Technical Field

The invention relates to the technical field of prevention and treatment of American larva diseases of bees.

Background

The bees are important economic insects, the bees are important components of a pollination medium and are important components forming global biodiversity, and the bee pollination medium has pollination activity on crops, fruits and wild plants, so that the bee pollination medium becomes one of the most important pollination insects in the world, has great significance for agricultural production and plays an important role in maintaining the biodiversity. Meanwhile, bee products such as honey, bee pollen, royal jelly, propolis and the like produced by the bees are also important health-care food and chemical raw materials.

American larva disease (AFB), also called American foul brood and American larva putrid disease for short, is a malignant bee larva infectious disease and occurs all around the world. American larva disease is related to the infection of larval bacillus, the occurrence of the disease is greatly related to the resistance of larva, and the disease is rapidly spread among the larva of a bee colony by feeding the larva of the bee colony with infected bees. The modern treatment method is to burn the bee colony in situ after the disease is diagnosed, so as to prevent the disease from spreading. The etiological agent of AFB is bacillus larvae (paenibacillus larvae), which belongs to a gram-positive bacterium. The sensitivity of bee larvae to bacillus larvae decreases with increasing age of the day, producing hundreds of millions of spores with infective ability in each infected larva, which spores, due to their extreme resistance to heat and chemicals, survive for as long as 35 years, causing scales, hives, products and equipment of larval debris to be a potential source of contamination after AFB infection.

At present, a lot of antibiotics are used for treating AFB, such as sulfonamides, oxytetracycline hydrochloride, tetracycline, penicillin, streptomycin, erythromycin, kanamycin, erythromycin, tylosin and the like, and the medicaments are usually mixed with syrup or honey water and sprayed after being blended. The dosage of syrup and honey water is 150mL per spleen, and the spraying is carried out for 1 time at intervals of 4-5 days. However, continuous use of antibiotics has the potential to contaminate bee products. For this purpose, the dosage is controlled to be appropriate, especially to avoid the production season, and to prevent and treat the antibiotic residue in the bee product. Moreover, the early spring or late autumn is selected as much as possible, and the treatment effect is better. However, in general, antibiotics can remain in bee products, seriously affect the quality of the bee products and pose a threat to human health. In addition, the antibiotics can also enhance the drug resistance of bee pathogens, cause corresponding variation of the pathogens and further cause the bee colony to be more serious.

Therefore, the AFB is prevented and controlled by using the antibiotics, the residual antibiotics in bee products exceed the standard due to excessive use or improper use time of the antibiotics, the antibiotics can only inhibit clinical symptoms and cannot play a role in spores, larvae and adult bees contacting the antibiotics are obviously shortened in service life, and more seriously, the generation of the drug resistance is the main reason for the fact that the antibiotics cannot effectively control the AFB, huge economic loss is caused to bee-keepers in the world, and meanwhile, the food safety problem of people is directly influenced.

In order to reduce the use of antibiotics in bee-keeping production, people adopt plants to prevent and treat bee diseases. For example, the pure Chinese medicinal preparation of Shanxi Shaoxing fish bee pharmaceutical company, Dumanjun, the Chinese herbal plant powder of Gansu Tianshui Huo Tao bee pharmaceutical company, Jianfeng anti-mite incense powder, and the Chinese herbal preparation of Shanxi Weipeng pharmaceutical company, Fengkang, etc. However, the current products for treating bee diseases cannot better meet the development needs of the green bee industry from the aspects of variety and curative effect. And the biological prevention and control function is also achieved through antagonistic bacteria, for example, the cultured P.larvae is successfully used for carrying out biological prevention and control on AFB caused by the larval bacillus for the first time; the original dew technology developed in Japan has obvious prevention effect on bee diseases, especially on crawl bee disease, big belly disease, larva disease, bee dysentery and the like, but has narrow action spectrum, slow drug effect, short lasting effect and is easily influenced by external conditions.

At present, the population quantity of bees worldwide decreases year by year, and among a plurality of factors causing sharp reduction of bee populations, pathogens generate drug resistance to antibiotics, which is a main reason for failure in prevention and control of bee colony diseases. With the long-term use of antibiotic drugs, the drug-resistant germs of bee colonies continuously appear, the problem of drug resistance is obvious day by day, and a vicious circle is formed because the drug effect is gradually reduced and the dosage of antibiotics is continuously increased.

Disclosure of Invention

The invention aims to provide a natural ethanol extract of gorgon euryale leaves capable of replacing plant sources of antibiotics and used for preventing and treating American larva diseases of bees.

The preparation method of the ethanol extract of the gorgon fruit leaves comprises the following steps: grinding the dried gordon euryale seeds in the shade into powder, soaking the powder in an ethanol water solution, performing suction filtration, taking filtrate, and performing rotary evaporation to obtain an ethanol extract of the gordon euryale seeds.

The water content of the ethanol extract of the gorgon fruit leaves is 10-20 wt.%.

The volume percentage of the ethanol in the ethanol water solution is 75-85%.

The invention has simple process, and experiments prove that the bacillus cereus inhibitor has an inhibiting effect on the bacillus cereus.

The invention also aims to provide the application of the ethanol extract of the gorgon fruit leaves obtained by the preparation method.

And (3) dissolving the ethanol extract of the gorgon euryale leaves in dimethyl sulfoxide, and using the ethanol extract to prevent and treat the American larva of bees.

Experiments prove that different ethanol extracts of gorgon euryale leaves have the inhibiting effect on the bacillus larvae, which lays a foundation for developing natural medicines and feeds for preventing and controlling AFB, and simultaneously finds out a novel and natural plant source preparation capable of replacing antibiotics for green bee-keeping production.

More preferably, the ethanol extract of the gorgon fruit leaves does not have a drug withdrawal period, can be safely used in the whole production and reproduction stage of the bee colony, is beneficial to maintaining the health of the bee colony and promotes the improvement of the whole productivity of the bee colony. The plant source antibiotic substitute preparation can reduce the use of antibiotics, reduce the antibiotic residue in bee products, and improve the yield and quality of bee products.

Drawings

FIG. 1 is an agarose gel electrophoresis of the colony PCR products.

FIG. 2 is a diagram of the bacteriostatic effect of the extract of euryale ferox leaves.

Detailed Description

1. The material and the method are as follows:

1.1 Experimental materials

1.1.1 Collection of Experimental samples

Collecting adult and larva of bee in bee field, placing adult bee with clinical symptoms and larva with clinical symptoms in aseptic finger-shaped tube, and placing on ice. Meanwhile, adult bees and larvae from the same hive and comb, which appear to be healthy, were harvested and returned and placed in a refrigerated storage at-70 ℃.

1.1.2 preparation of extract of Gorgon fruit leaves

Grinding collected gordon euryale leaves dried in the shade, weighing 20g of powder respectively, soaking the powder in 75-85% ethanol water solution according to the mass ratio of 1:20 for 2 hours, carrying out suction filtration, further soaking the powder subjected to suction filtration for 24 hours, carrying out suction filtration again, repeating the steps for 3 and 4 times, combining filtrates obtained by suction filtration, and carrying out reduced pressure concentration by using a rotary evaporator to obtain the gordon euryale leaf ethanol extract.

The water content of the gorgon fruit leaf ethanol extract obtained by detection is 10-20 wt.%.

1.1.3 preparation of Experimental reagents

Preparing Luria-Bertani liquid culture medium: mixing 1g of yeast extract, 0.5g of tryptone and 0.5g of sodium chloride, adjusting the pH value to 7.2 by adopting a buffer solution, adding a proper amount of double distilled water to fix the volume to 100mL, sterilizing at high pressure, and cooling to obtain the Luria-Bertani liquid culture medium.

Preparing the culture powder of the bacillus larvae: mixing 25% of royal jelly, 50% of drone pupa powder, 15% of protein powder and 10% of queen bee larva powder, totaling to 100%, and freeze-drying to obtain the culture powder of the bee larva bacillus.

Packaging the Bacillus larvae culture powder, sealing, and storing at-20-20 deg.C.

1.1.4 Experimental instruments

A water making machine produced by Nanjing Yopu environmental protection equipment Limited; mettletelodedo electronic balance; a SHEL LAB water bath; 5834R model refrigerated centrifuge; a Telstar LyoQuest lyophilizer; GHX9160B-1 incubator; an ultra-low temperature refrigerator; SW-CJ-2HD superclean bench.

1.2. Experimental methods

1.2.1 treatment of bee samples

The larvae from the diseased bee colony were homogenized with PBS (ratio of larvae to PBS 10 g: 100ml) to obtain a suspension, which was then heated at 80 deg.C for 10min or 95-96 deg.C for 3-5 min.

Adult bees in a nidus of a disease are sufficiently washed with PBS, and the obtained samples are divided into three parts, and each part is respectively treated by three treatments: without heat treatment; heat treating at 80 deg.C for 10 min; heat treatment at 95 deg.C for 3 min.

Adult bees in the affected bee field were thoroughly washed with PBS, and the intestines were taken out and homogenized with PBS (the ratio of the intestines to PBS was 10 g: 100ml) for 30 sec. The homogenate was filtered through filter paper, centrifuged, and the pellet was resuspended in PBS.

1.2.2 isolation and culture of bacteria

The Bacillus honeybee larva culture solution is obtained by dissolving the Bacillus honeybee larva culture powder in PBS (the mixing ratio of the culture powder to the PBS is 10 g: 100ml), and sterilizing with a disposable filter.

Mixing the culture solution of the bacillus honeybee and a liquid Luria-Bertarfi culture medium according to the volume ratio of 20% to obtain the culture solution.

And (3) respectively smearing the culture solutions on a sterile culture tray, respectively smearing the samples treated by 1.2.1 on each culture medium, and culturing at 37 ℃ for 1-3 days.

As a result, it was found that: bacteria in each sterile culture dish grow vigorously and colonies aggregate into clusters.

Description of the drawings: the above culture powder does contain the substances required for the growth of the larval bacillus, and can be used for laboratory culture of the larval bacillus after being added to a common LB culture medium as a nutrient component.

Single colonies were picked from each medium and transferred to the same medium for amplification culture.

Typically, a single colony of Bacillus larvae is small, regular, mostly rough, flat or convex, white to beige in color.

1.2.3 extraction and amplification of bacterial DNA

Extracting DNA of each single colony after the amplification culture by the following method: single colonies growing on each medium were picked with sterilized toothpicks, suspended in 50. mu.L of distilled water, heated to 95 ℃ for 15min, centrifuged for 5min, and 1-5. mu.L of supernatant was used as a template for PCR.

PCR (50. mu.L) reaction system comprising: 1-5. mu.L of template DNA; 50pmol/L Forward (AFB-F) and reverse primer (AF)B-R); comprises 10nmol/L dNTPs and 2mM MgCl₂1-2.5U of Mix of Taq polymerase; ddH₂O。

Amplification conditions: pre-denaturation at 95 ℃ for 1min, denaturation at 93 ℃ for 1min, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1.5min, and extension at 72 ℃ for 5min after 30 cycles.

1.2.4 colony PCR identification

And (3) performing colony PCR identification on the bacterial monoclonals growing on the culture medium: the PCR product was detected by 0.8% agarose gel electrophoresis, and the band of interest appeared around the position of about 1100bp, as shown in FIG. 1.

In FIG. 1, lane M is DL2000 DNA marker, lane 2 is a negative control, and lanes 1,3-8 are amplification products of colony PCR.

The agarose gel electrophoresis picture of the colony PCR product illustrates: the samples treated at 1.2.1 all carried AFB-carrying Bacillus larvae.

1.2.5 determination of bacteriostatic effect of euryale ferox leaf extract

Dissolving the ethanol extract of the gordon euryale leaves by using dimethyl sulfoxide to prepare a solution with the concentration of 1g/mL, and then performing gradient dilution by using the dimethyl sulfoxide to prepare 5 ethanol extracts of the gordon euryale leaves with the concentration gradients of 200mg/mL, 100mg/mL, 50mg/mL, 25mg/mL and 12.5mg/mL respectively.

Respectively smearing the culture solutions on an aseptic culture disc, respectively coating a flat plate with the sample treated by 1.2.1, after the bacterial liquid is dried, pricking a plurality of small holes on the coated flat plate after each bacterial liquid is dried by using a sterilized gun head, respectively dripping the ethanol extracts of the euryale ferox leaves with the 5 concentration gradients in the small holes, and simultaneously leaving the ethanol extracts of the euryale ferox leaves which are not dripped as a control group.

Then, each of the above plates was placed in an incubator at 37 ℃ for 3 days.

And (4) observing results: and measuring the diameter of each inhibition zone by a cross method for 3 times, and taking the average value as the final diameter of the inhibition zone.

2 results of the experiment

The diameter of the transparent inhibition zone is enlarged in the small hole on the culture dish which is dripped with ethanol extracts of different gordon euryale seeds leaves, as shown in figure 2.

And the small hole on the culture plate without dripping the ethanol extract of the gorgon fruit leaves is not changed.

Therefore, the ethanol extract of the gorgon euryale leaves shows certain bacteriostatic activity to the larval bacillus and also shows certain bacteriostatic effect even at lower concentration.

The following table is a comparison table of antibacterial effects corresponding to different concentrations of ethanol extracts of gorgon fruit leaves.

As can be seen from the above table, the higher the concentration of the ethanol extract of the gorgon fruit leaves is, the better the bacteriostatic effect is.

And (4) conclusion: the ethanol extract of semen euryales can be used for preventing and treating AFB.