CN111617116A - Preparation method and application of ethanolic extract of rhizoma leaves - Google Patents

Abstract

The invention relates to a preparation method and application of ethanolic extract from leaves of Rhizoma rhizoma, and relates to the technical field of honeybee American larvae control. The dried rhizoma leaves are ground into powder, soaked in ethanol aqueous solution, filtered by suction, and the filtrate is taken by rotary evaporation to obtain the ethanolic extract of rhizoma leaves. The extract was dissolved in dimethyl sulfoxide and used to control honeybee American larvae. The use of the extract does not have a withdrawal period, and can be safely used in the entire production and reproduction stages of the bee colony, which helps maintain the health of the bee colony and promotes the improvement of the overall productivity of the bee colony.

Description

Preparation method and application of ethanolic extract from leaves of Rhizoma Rhizoma

technical field

The invention relates to the technical field of honeybee American larval disease control.

Background technique

Bees are important economic insects, and bees are an important part of pollinators and constitute an important component of global biodiversity. Their pollination activities on crops, fruits and wild plants make them the most important worldwide. One of the pollinators is not only of great significance to agricultural production, but also plays an important role in maintaining biodiversity. At the same time, honey, bee pollen, royal jelly, propolis and other bee products produced by bees are also important health food and chemical raw materials.

American Foulbrood (AFB, also known as American larvae foul disease, American larvae rot disease), referred to as American rot disease, is a vicious bee larval infectious disease that occurs worldwide. American larval disease is related to Bacillus larvae infection. The occurrence of the disease is closely related to the resistance of the larvae. It is mainly fed by the infected bees to the larvae of the colony, which makes the disease spread rapidly among the larvae of the colony. . Usually the modern disposal method is to incinerate the bee colony after diagnosis of the disease to prevent the disease from spreading. The causative agent of AFB is Paenibacillus larvae, a Gram-positive bacterium. The susceptibility of honeybee larvae to Bacillus larvae decreases with age, producing hundreds of millions of infectious spores in each infected larvae due to their extreme resistance to heat and chemicals , which can survive for up to 35 years, leading to AFB infection. Scales, beehives, products and equipment of larval remains are all potential sources of contamination.

There are many antibiotics currently used to treat AFB, such as sulfonamides, oxytetracycline hydrochloride, tetracycline, penicillin, streptomycin, erythromycin, kanamycin, erythromycin, tylosin, etc. Syrup or honey water, spray after blending. The dosage of syrup and honey water is 100-150mL per spleen, and sprayed once every 4-5d. However, continuous use of antibiotics has the potential to contaminate bee products. To this end, the dosage of the drug should be properly controlled, especially the production season should be avoided to prevent the residue of antibiotics in bee products. Moreover, try to choose early spring or late autumn, the treatment effect will be better. However, in general, antibiotics will remain in bee products, seriously affecting the quality of bee products and posing a threat to human health. In addition, antibiotics can also increase the resistance of bee pathogens, causing the pathogens to mutate accordingly, making the bee colony more sick.

It can be seen that the use of antibiotics to prevent and control AFB is often caused by excessive use or improper use of antibiotics, which can easily lead to excessive antibiotic residues in bee products. Antibiotics can only suppress clinical symptoms and cannot act on spores. The life span of larvae and adult bees is significantly shortened, and more seriously, the emergence of drug resistance is the main reason that they cannot effectively control AFB, causing huge economic losses to beekeepers all over the world. At the same time, it also directly affects people's food safety issues.

In order to reduce the use of antibiotics in beekeeping, people use plants to control bee diseases. For example, the pure Chinese medicine preparation of Shanxi Zhenxing Yubee Pharmaceutical Co., Ltd.-Dox mites once clean, the Chinese herbal plant powder of Gansu Tianshui Huitao Bee Industry Co., Ltd.-Jianbee anti-mite fragrance powder, the Chinese herbal medicine preparation of Shanxi Weipeng Pharmaceutical Co., Ltd.-Fengkang Wait. However, at present, these products for treating bee diseases cannot well meet the development needs of green bee industry in terms of type and curative effect. There are also biological control effects through antagonistic bacteria. For example, Evans and Armstrong successfully used the cultured P. larvae for the first time to carry out biological control of AFB caused by Bacillus larvae; In particular, it has obvious preventive effect on crawling bee disease, pot stomach disease, larvae disease and bee dysentery, but its action spectrum is narrow, the drug effect is slow, the lasting effect is short, and it is easily affected by external conditions.

At present, the number of bee populations around the world is decreasing year by year. Among the many factors that cause the sharp decline of bee populations, the resistance of pathogens to antibiotics is the main reason for the failure of bee colony disease prevention and control. With the long-term use of antibiotics, bee colony-resistant bacteria continue to emerge, and the problem of drug resistance becomes increasingly prominent. Because the efficacy of drugs gradually decreases, the dosage of antibiotics continues to increase, thus forming a vicious circle.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a natural, plant-derived ethanol extract of Rhizoma chinensis that can replace antibiotics for preventing and treating Bee American larvae.

The preparation method of the leaf ethanol extract of Rhizoma Rhizoma of the present invention is as follows: the dried Rhizoma Rhizoma leaves are ground into powder, soaked in ethanol aqueous solution, filtered by suction, and the filtrate is obtained by rotary evaporation to obtain the leaf ethanol extract of Rhizoma Rhizoma.

The water content of the ethanolic extract of the leaves of Rhizoma Rhizoma is 5-10 wt.%.

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

The process of the invention is simple, and it is proved by experiments that it has an inhibitory effect on the honeybee larvae Bacillus.

Another object of the present invention is to propose the application of the ethanolic extract of the leaves of Rhizoma rhizoma obtained by the above preparation method.

The ethanolic extract of the leaves of the rhizoma rhizoma is dissolved in dimethyl sulfoxide, and is used for preventing and treating honeybee American larvae.

It has been confirmed by experiments that the ethanolic extracts of different rhizoma leaves have inhibitory effects on Bacillus larvae, which lays the foundation for the development of natural medicines and feeds for the prevention and treatment of AFB, and also provides new and natural products for green beekeeping. Plant-derived preparations that can replace antibiotics.

What's more superior is that there is no withdrawal period for the use of the ethanolic extract of the leaves of the rhizome, and it can be safely used in the entire production and reproduction stages of the bee colony, which helps to maintain the health of the bee colony and promote the improvement of the overall productivity of the bee colony. Plant-derived antibiotic substitution formulations can reduce the use of antibiotics, reduce antibiotic residues in bee products, and improve the yield and quality of bee products.

Description of drawings

Figure 1 is an agarose gel electrophoresis image of colony PCR products.

Figure 2 is a graph showing the antibacterial effect of the extract of Rhizoma rhizoma leaves.

Detailed ways

1. Materials and methods:

1.1 Experimental materials

1.1.1 Collection of experimental samples

Adult honeybees and larvae were collected from the local bee farms where the bees were relocated. In the bee colony with clinical symptoms, adult bees with clinical symptoms and larvae with clinical symptoms were taken into sterile finger-shaped tubes. on ice. At the same time, adult bees and larvae from healthy-looking hives and comb spleens from the same beehive were collected and brought back, and stored at -70°C for cryopreservation.

1.1.2 Preparation of the ethanolic extract of the leaves of Rhizoma

Grind the collected dried rhizoma leaves, weigh 20 g of powder respectively, soak them in an aqueous ethanol solution with a volume percentage of 75 to 85% for 2 hours, and then filter the powders after immersion in a mass ratio of 1:20. After 24 hours, suction filtration is repeated, repeated 3 or 4 times, the filtrates obtained by suction filtration are combined, and the filtrate obtained by suction filtration is concentrated to a viscous state under reduced pressure using a rotary evaporator to obtain the ethanolic extract of the leaves of Rhizoma rhizoma.

The water content of the ethanolic extract of the leaves of Rhizoma rhizoma obtained through testing is 5-10 wt.%.

1.1.3 Configuration of experimental reagents

Preparation of Luria-Bertani liquid medium: Mix 1 g of yeast extract, 0.5 g of tryptone and 0.5 g of sodium chloride, adjust the pH value to 7.2 with buffer, add an appropriate amount of double-distilled water to make up to 100 mL, and autoclave Bacteria, cool down, and obtain Luria-Bertani liquid medium.

Preparation of Bacillus honeybee larvae culture powder: Mix 25% royal jelly, 50% drone pupa powder, 15% protein powder and 10% queen bee larvae powder to make a total of 100%, freeze-dry to obtain Bacillus bee larvae culture powder.

The culture powder of Bacillus bee larvae was packaged and sealed and stored at -20-20°C.

1.1.4 Experimental Instruments

Nanjing Youpu Environmental Protection Equipment Co., Ltd. water maker; METTLERTOLEDO electronic balance; SHEL LAB water bath; 5834R refrigerated centrifuge; Telstar LyoQuest freeze dryer; GHX9160B-1 incubator; ultra-low temperature refrigerator; SW-CJ-2HD ultra-clean workbench .

1.2. Experimental method

1.2.1 Processing of honeybee samples

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

The adult bees in the infected hive were washed with PBS, and the obtained samples were divided into three parts, and each part was treated with three kinds of treatments: no heat treatment; heat treatment at 80 °C for 10 min; heat treatment at 95 °C for 3 min.

The adult bees in the diseased hive were thoroughly washed with PBS, and the intestines were taken out and homogenized with PBS (the mixing ratio of intestines and PBS was 10 g: 100 ml) for 30 sec. The homogenate was then filtered through filter paper, centrifuged, and the pellet was resuspended in PBS.

1.2.2 Isolation and culture of bacteria

The culture powder of Bacillus bee larvae was dissolved in PBS (the mixing ratio of culture powder and PBS was 10g:100ml) and sterilized with a disposable filter to obtain the culture solution of Bacillus bee.

The culture solution of Bacillus apis and the liquid Luria-Bertarfi medium were mixed in a proportion of 20% by volume to obtain a culture solution.

The above culture solutions were smeared on sterile culture plates, and then the samples treated in 1.2.1 were smeared on each culture medium, and cultured at 37°C for 1 to 3 days.

The results showed that the bacteria in each sterile culture plate grew vigorously, and the colonies aggregated into clusters.

Description: The above culture powder does contain substances required for the growth of Bacillus larvae, and it can be used for laboratory culture of Bacillus larvae after adding it as a nutrient component to ordinary LB medium.

Pick a single colony on each medium and transfer it into the same medium as the corresponding medium for expansion.

Usually larval Bacillus single colonies are small, regular, mostly rough, flat or raised, white to beige in color.

1.2.3 Extraction and amplification of bacterial DNA

The DNA of each single colony after expanded culture was extracted as follows: pick a single colony grown on each medium with a sterilized toothpick, suspend it in 50 μL of distilled water, heat it to 95 °C for 15 min, and centrifuge for 5 min. Take 1-5 μL of the supernatant to use as a template for PCR.

PCR (50μL) reaction system, including: 1-5μL template DNA; 50pmol/L forward (AFB-F) and reverse primers (AFB-R); including 10nmol/L dNTPs, 2mM MgCl ₂ , 1-2.5U Mix of Taq polymerase; ddH ₂ O.

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

1.2.4 Colony PCR identification

Colony PCR identification was performed on the bacterial clones grown on the medium: PCR products were detected by 0.8% agarose gel electrophoresis, and the target band appeared near the position of about 1100 bp, as shown in Figure 1 .

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

From the agarose gel electrophoresis of the colony PCR products, it is indicated that the samples processed in 1.2.1 above all carry Bacillus larvae of AFB.

1.2.5 Determination of the antibacterial effect of the ethanolic extract of the leaves

Take the ethanolic extract of the leaves of the rhizome and dissolve it in dimethyl sulfoxide to make a solution of 1 g/mL, and then carry out gradient dilution with dimethyl sulfoxide to make the concentration of 200mg/mL, 100mg/mL, 50mg/mL, 25mg/mL and 12.5mg/mL 5 different concentration gradients of the ethanolic extract of Rhododendron leaves.

Apply the above culture solution on the sterile culture plate respectively, and then use the samples treated in 1.2.1 to coat the plate respectively. After the bacteria solution is dry, use a sterilized pipette tip to coat the dried bacteria solution. Poke a few small holes on the plate, and then drop the ethanol extract of the leaves of Rhizoma Rhizoma with the above 5 concentration gradients respectively in the small holes, while leaving the ethanol extract of Rhizoma Rhizoma leaves without dripping as a control group.

Then each of the above culture plates was placed in a 37°C incubator for 3 days.

Observation results: The diameter of the inhibition zone was measured by the cross method, and each inhibition zone was measured 3 times, and the average value was taken as the final inhibition zone diameter.

2. Experimental results:

The diameter of the transparent bacteriostatic zone expanded in the small holes on the culture plate to which different concentrations of the ethanol extract of the leaves of the rhizoma were added dropwise, as shown in Figure 2.

The small holes on the culture plate without the dropwise addition of the ethanolic extract of Rhododendron leaves did not change.

It can be seen that the ethanolic extracts of the leaves of Rhizoma Rhizoma have a certain bacteriostatic activity against Bacillus larvae, even at a lower concentration.

The following table is a comparison table of the antibacterial effect corresponding to the concentration of the ethanolic extract of different rhizoma leaves.

It can be seen from the above table that the higher the concentration of the ethanolic extract of the leaves, the better the bacteriostatic effect.

CONCLUSION: The ethanolic extract of the leaves of Rhizoma Rhizoma can be used to control AFB.

Claims (4)

1. the preparation method of the leaf ethanolic extract of Rhizoma Rhizoma, it is characterized in that: adopt ethanol aqueous solution to soak after the Rhizoma rhizoma leaf dried in the shade is ground into powder, through suction filtration, get the filtrate rotary evaporation, obtain the leaf ethanolic extract of Rhizoma Rhizoma. 2 . The preparation method according to claim 1 , wherein the water content of the ethanolic extract of the leaves of the Rhizoma Rhizoma is 5-10 wt.%. 3 . 3. The preparation method according to claim 1 or 2, wherein the volume percentage of ethanol in the ethanol aqueous solution is 75-85%. 4. adopt the application of the leaf ethanolic extract of the rhizoma rhizoma that the preparation method as claimed in claim 1 obtains, it is characterized in that: the leaf ethanolic extract of described rhizoma rhizoma is dissolved in dimethyl sulfoxide, for preventing and treating honey bee larval disease.

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