Application of radix angelicae dahuricae extract in preparation of aquatic pathogenic bacteria resisting medicine or feed additive

Abstract

The invention discloses an application of a radix angelicae dahuricae extract in preparation of aquatic pathogenic bacteria resisting drugs or feed additives. The radix angelicae dahuricae extract has antibacterial activity of inhibiting aquatic pathogenic bacteria, and active substances of the radix angelicae dahuricae extract comprise isocoralline, alloimperatorin, beta-sitosterol and stigmasterol. The radix angelicae dahuricae extract has an obvious antibacterial effect on aquatic pathogenic bacteria aeromonas veronii, vibrio harveyi and vibrio parahaemolyticus, the antibacterial concentration is in the range of 50-100 mg / mL, and the radix angelicae dahuricae extract does not generate drug resistance, is safe and non-toxic, so that the radix angelicae dahuricae extract can be used for preparing the aquatic pathogenic bacteria resisting drugs or feed additives, and can be used for preparing the aquatic pathogenic bacteria resisting drugs or feed additives. Therefore, the composition is used for preventing or treating aquatic animal infection caused by aquatic pathogenic bacteria.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of angelica dahurica extract in preparing anti-aquatic pathogenic bacteria medicine or feed additive. Background Art

[0002] The main pathogens of aquatic animals are mainly bacteria, viruses and parasites. Among them, the pathogens of bacterial diseases mainly include some Aeromonas viridis, Vibrio harveyi and Vibrio parahaemolyticus. These pathogens pose a serious threat to a variety of fish organisms, especially to fish with high breeding density, such as tilapia, largemouth bass, sea bass, Macrobrachium rosenbergii and other aquatic products, which seriously restrict the healthy development of aquaculture. At present, antibacterial drugs used in aquaculture rely on medical or veterinary antibiotics, and there are few types of antibacterial raw materials specifically used for aquatic products. Although antibiotics take effect quickly, they are not easy to degrade and remain in aquatic animals for a long time, causing environmental pollution, thereby threatening human health. Excessive use of antibiotics will also cause the production of multi-drug resistant strains and drug-resistant genes, which will cause great difficulties in disease prevention and control. Therefore, it is urgent to develop a safe and effective antibacterial drug for the treatment of pathogenic bacteria infections in aquatic animals.

[0003] Chinese herbal medicines have the advantages of abundant resources, easy access, low cost, minimal toxicity and side effects, and less residue, which makes them have potential therapeutic efficacy and broad application prospects in the development of prevention and treatment of aquatic pathogens. As a well-known Chinese medicine for both medicine and food, Angelica dahurica is widely used in clinical practice for its significant medicinal value and edible safety. With its advantages of low price and abundant resources, it can be used as an ideal substitute for antibacterial drugs and antibiotics. The natural Chinese medicine Angelica dahurica, which is both medicine and food, plays an important role in the prevention and treatment of animal diseases. A large number of studies have shown that Angelica dahurica extracts contain rich plant active ingredients, including coumarins, volatile oils, alkaloids, and phenolic compounds. It has significant antibacterial effects and can enhance the body's immunity, showing a wide range of pharmacological effects. However, the antibacterial activity of Angelica dahurica extracts against aquatic pathogens is still unclear. Therefore, the identification and evaluation of the active ingredients of Angelica dahurica extracts against aquatic pathogens is of great significance for the development of green and environmentally friendly therapeutic drugs. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an application of an Angelica dahurica extract in the preparation of an anti-aquatic pathogen drug or a feed additive, so as to make up for the above-mentioned deficiencies of the prior art.

[0005] In order to achieve the above object, the present invention provides the following technical solutions.

[0006] The invention provides application of angelica dahurica extract in preparing medicine for resisting aquatic pathogens or feed additive.

[0007] Furthermore, the angelica dahurica extract has antibacterial activity against aquatic pathogens.

[0008] Furthermore, the aquatic pathogens include Aeromonas vermifuge, Vibrio harveyi and Vibrio parahaemolyticus.

[0009] Furthermore, the active substances in the Angelica dahurica extract include isocortin, alloimperatorin, β-sitosterol and stigmasterol.

[0010] Furthermore, the Angelica dahurica extract is used in preventing or treating vibriosis, hemorrhagic septicemia and intestinal inflammation caused by aquatic pathogens.

[0011] Furthermore, the angelica dahurica extract is extracted and separated from the natural Chinese medicine angelica dahurica.

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] 1) The angelica dahurica extract provided by the present invention has a significant inhibitory effect on aquatic pathogens Aeromonas veronii, Vibrio harveyi and Vibrio parahaemolyticus.

[0014] 2) The Angelica dahurica extract provided by the present invention is easy to obtain, low in cost, has minimal toxic and side effects, little residue, and no adverse reactions, and is suitable for preparing drugs or feed additives against aquatic pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A schematic diagram of a culture plate for Aeromonas vermifuge, Vibrio harveyi and Vibrio parahaemolyticus used in the present invention;

[0016] Figure 2 The diagram is a schematic diagram of the minimum inhibitory concentration of Aeromonas vernix and the antibacterial efficiency results of 96-well plate according to the present invention;

[0017] Figure 3 It is a schematic diagram of the minimum inhibitory concentration of Vibrio parahaemolyticus in a 96-well plate and the results of the antibacterial efficiency according to the present invention;

[0018] Figure 4 It is a schematic diagram of the minimum inhibitory concentration of Vibrio harveyi in a 96-well plate and the antibacterial efficiency results according to the present invention.

[0019] Figure 5 This is a schematic diagram of the results of the effective active ingredients detected in the Angelica dahurica extract;

[0021] Figure 6 The figure is a schematic diagram for predicting the antibacterial activity analysis of the natural active product of the Angelica dahurica extract of the present invention. DETAILED DESCRIPTION

[0022] In order to further illustrate the technical methods and antibacterial effects adopted by the present invention to achieve the predetermined purpose, the following is a further description of the embodiments of the present invention in conjunction with the accompanying drawings and examples, but it should not be understood as a limitation of the present invention. The technical solutions described in the present invention, unless otherwise specified, are conventional techniques in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0023] Example 1 Determination of the antibacterial activity of Angelica dahurica extract against aquatic pathogens

[0024] 1. Preparation of Angelica dahurica Extract

[0025] The natural Chinese medicinal herb Angelica dahurica is dried and crushed at room temperature to obtain a crude powder of Angelica dahurica, which is sieved and placed in a beaker and soaked in 65% ethanol overnight. The powder is concentrated and recovered by an evaporator and rotary evaporator, and reflux extraction is performed twice, each time for 3 hours. The extracts are filtered and separated, and the extracts are combined and concentrated to 250 mL by vacuum distillation to obtain a 1:4 concentrated extract (1 mL of concentrated extract is equivalent to 4.0 g of original medicinal material). After being evenly mixed, the extracts are spray-dried to obtain a Chinese medicinal extract of Angelica dahurica.

[0026] 2. Bacteria Activation

[0027] Weigh tryptone (1.0012 g), yeast extract (0.5003 g), sodium chloride (1.0016 g), and agar (1.5004 g) on ​​a balance and pour them into a 250 mL conical flask. Use a measuring cylinder to measure 100 mL of ultrapure water and pour it into the conical flask. Stir to completely dissolve it. Adjust the pH to 8.1 with sodium hydroxide. After sealing with a sealing film, put the conical flask into a sterilizer and sterilize it with wet heat at 121°C for 20 min.

[0028] After sterilization, take out the triangular flask containing the culture medium, place it in the clean bench, light the alcohol lamp, and when the temperature cools to about 45°C, pour the culture medium evenly into 6 sterile flat culture dishes. After the culture medium is completely solidified, take 20 μL of Aeromonas viridis, Vibrio harveyi and Vibrio parahaemolyticus liquid, and spread them on the aforementioned culture medium plates, 2 plates for each strain. After the coating is completed, mark the flat culture plates, place the plates in a microbial incubator, and culture at 30°C for 24 hours.

[0029] 3. Preparation of Bacterial Suspension

[0030] Weigh peptone (2.0012g), yeast extract (1.0016g), and sodium chloride (2.0021g) on ​​a balance and dissolve them in 200mL of ultrapure water. After they are completely dissolved, adjust the pH to 8.05 with sodium hydroxide, and dispense the liquid culture medium into 50mL triangular flasks. Each bottle is dispensed with about 20mL of liquid culture medium, and a total of 6 bottles are dispensed. The remaining liquid culture medium is stored in 250mL triangular flasks for later use. Seal all triangular flasks with sealing film and place them in an autoclave for wet heat sterilization at 121°C for 20min. After sterilization, take out the culture medium and place it in an ultra-clean workbench. Light the alcohol lamp. After the culture medium has completely cooled, select a complete single colony from the flat culture dish and place it in the dispensed 50mL triangular flask. Mark it, and then place it in a shaker at 30°C and 170rpm for culture. When the concentration of the bacterial suspension is 10 7 -10 8 cfu / mL, take it out for later use.

[0031] 4. Setting the concentration of Angelica dahurica extract

[0032] Weigh 0.5006g of Angelica dahurica extract on a balance, add 2mL of dimethyl sulfoxide (DMSO) to dissolve, and set the concentration of the Angelica dahurica extract mother solution to 250mg / mL. After complete dissolution, dilute with the sterile liquid culture medium prepared in step 3 to obtain an initial concentration of 100mg / mL, and then dilute twice with the sterile liquid culture medium to obtain a concentration gradient of: 100mg / mL, 50mg / mL, 25mg / mL, 12.5mg / mL, 6.25mg / mL, 3.125mg / mL, 1.563mg / mL, 0.78mg / mL, 0.39mg / mL, 0.195mg / mL, 0.098mg / mL, 0.049mg / mL.

[0033] 5.96-well plate antibacterial experiment

[0034] Table 1 96-well plate layout in antibacterial experiment

[0035]

[0036]

[0037] The distribution of the wells of the 96-well plate in Table 1 is shown in the table. The following description is made for the additives in each row and column of the distribution wells shown in Table 1:

[0038] (1) A, B, and C are experimental groups; D is an empty row without adding fluid; E, F, and G are blank control groups; and H is a growth control group;

[0039] (2) Add 100 μL / well of Angelica dahurica extract solution and 100 μL / well of the prepared bacterial suspension to row A. The numbers 1 to 12 correspond to the concentrations of the prepared Angelica dahurica extract solution, ranging from 100 mg / mL to 0.049 mg / mL. Repeat the steps of row A in rows B and C for 3 replicates.

[0040] (3) Add 100 μL / well sterile liquid culture medium and 100 μL / well of the corresponding concentration of Angelica dahurica extract solution to row E; repeat the operation of row E in rows F and G, and repeat three times;

[0041] (4) H row: add 100 μL / well sterile culture medium and 100 μL / well sterile liquid culture medium;

[0042] (5) After the sample is added, place the 96-well plate in a 30°C incubator for 24 hours and observe the results. If the solution in the well plate becomes turbid or white precipitate is generated, it means that bacteria are growing. If the solution in the well is clear without precipitation, it means that this concentration of Angelica dahurica extract can inhibit the growth of aquatic pathogens and can be regarded as the minimum inhibitory concentration. The antibacterial experiments of Aeromonas verumii, Vibrio harveyi and Vibrio parahaemolyticus were performed according to the above steps.

[0043] Figure 1 These are the inoculation and culture results of three aquatic pathogens, Aeromonas veronii, Vibrio harveyi and Vibrio parahaemolyticus. Specifically, Aeromonas veronii has obvious growth characteristics, while Vibrio harveyi and Vibrio parahaemolyticus are in good growth condition. Figure 2 , Figure 3 , Figure 4 The results of the antibacterial activity test of Angelica dahurica extracts at different concentrations (100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL, 1.563 mg / mL, 0.78 mg / mL, 0.39 mg / mL, 0.195 mg / mL, 0.098 mg / mL, 0.049 mg / mL) against Aeromonas vernix, Vibrio harveyi and Vibrio parahaemolyticus, wherein the antibacterial concentration of Angelica dahurica extract against Vibrio harveyi and Vibrio parahaemolyticus is 50 mg / ml, and the antibacterial concentration against Aeromonas vernix is ​​100 mg / ml. This indicates that the Angelica dahurica extract has a significant inhibitory effect on the infection of aquatic pathogens within a certain dosage range.

[0044] Example 2 Qualitative and quantitative analysis of effective active ingredients in Angelica dahurica extract

[0045] 1. Preparation of Angelica dahurica-containing serum

[0046] Take the original medicinal material of Angelica dahurica and grind it finely. Accurately weigh 1000g and pass it through a No. 3 sieve to obtain powder. Add 8L of 95% ethanol and reflux for extraction for 2h. Filter and separate the extract. Repeat the extraction three times and combine the extracts. Concentrate to 250mL by vacuum distillation to obtain a 1:4 concentrated ratio of Angelica dahurica extract (1mL concentrated extract is equivalent to 4.0g of original medicinal material), and store it at -20℃ for use. Six healthy SD male rats aged 2 months were randomly divided into a blank group and an experimental group, with 3 rats in each group. They were gavaged at a dose of 2.0g / kg. The blank group rats were gavaged with an equal volume of 0.5% CMC-Na for one week. After the gavage, blood was collected from the abdominal aorta to prepare Angelica dahurica drug-containing serum, which was stored at -20℃ for use.

[0047] 2. Quantitative analysis of natural active products of Angelica dahurica

[0048] (1) Take out the collected rat drug-containing serum from the -80°C refrigerator, thaw it, and vortex mix it for 10 seconds.

[0049] (2) Pipette 50 μL of the sample into a 2 mL EP tube, add 300 μL of 70% methanol internal standard extract, vortex for 3 min, and centrifuge at 12,000 rpm at 4°C for 10 min.

[0050] (3) After centrifugation, transfer 200 μL of the supernatant to another labeled centrifuge tube and incubate at -20°C for 30 min.

[0051] (4) Centrifuge at 12,000 rpm and 4°C for 3 min, transfer the supernatant to the corresponding liner tube, and perform on-line detection using a high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) platform.

[0052] (5) Obtain metabolite mass spectrometry analysis data of different samples, and perform qualitative and quantitative analysis of substances based on the secondary spectrum matching information of the detection data based on the plant secondary metabolite database.

[0053] Figure 5 The results of the detection of effective active ingredients in Angelica dahurica extract showed that a total of 224 Angelica dahurica small molecule compounds were obtained, which were divided into 7 categories, mainly including alkaloids (27%), phenolic acids (2%), flavonoids (5%), quinones (1%), lignin and coumarins (55%), terpenoids (0.004%) compounds and other components (10%). Figure 6 This is a network diagram of some key compounds in Angelica dahurica extract acting on inflammatory regulatory targets, among which isocortin, alloimperatorin, β-sitosterol and stigmasterol act on inflammatory regulatory targets, exert certain antibacterial activity, and are used to prevent and treat infections caused by aquatic pathogens.

[0054] Table 2. Results of topological analysis of the Angelica dahurica-compound-target network

[0055]

[0056] Example 3 Effect of Angelica dahurica Extract as Feed Additive on Bacterial Diseases of Aquatic Perch

[0057] According to the ratio of adding 1kg of Angelica dahurica extract to 20kg of California bass feed, 10kg of Angelica dahurica extract raw material was weighed and added to 200kg of feed. The California bass was fed for one week to observe the effect of Angelica dahurica extract in treating bacterial diseases of California bass. Angelica dahurica extract and California bass feed were prepared and fed according to the addition ratio of 1:20. The results showed that Angelica dahurica extract had a good therapeutic effect on bacterial infection diseases of California bass, could relieve inflammation of California bass, reduce symptoms such as redness, swelling, and bleeding, and had high safety, excellent cost-effectiveness, and was easy to promote and apply.

[0058] In summary, the Angelica dahurica extract of the present invention has an inhibitory effect on aquatic pathogens and is suitable for preparing anti-aquatic pathogen drugs or feed additives.

[0059] It should be noted that the above-described embodiments are only preferred embodiments of the present invention. For those skilled in the art, without departing from the principles of the present invention, the present invention may be modified, improved and replaced with equivalents, and these modifications, improvements and equivalent replacements are also considered to fall within the scope of protection of the claims of the present invention.

Claims

1. Application of Angelica dahurica extract in the preparation of anti-aquatic pathogen drugs or feed additives.

2. The use of the Angelica dahurica extract according to claim 1 in the preparation of an anti-aquatic pathogen drug or a feed additive, characterized in that: The angelica dahurica extract has antibacterial activity for inhibiting aquatic pathogens.

3. The use of the Angelica dahurica extract according to claim 1 in the preparation of an anti-aquatic pathogen drug or a feed additive, characterized in that: The aquatic pathogens include Aeromonas vermifuge, Vibrio harveyi and Vibrio parahaemolyticus.

4. The use of the Angelica dahurica extract according to claim 1 in the preparation of an anti-aquatic pathogen drug or a feed additive, characterized in that: The active substances in the angelica dahurica extract include isocortin, alloimperatorin, β-sitosterol and stigmasterol.

5. The use of the Angelica dahurica extract according to claim 1 in the preparation of an anti-aquatic pathogen drug or a feed additive, characterized in that: The angelica dahurica extract is used in preventing or treating vibriosis, hemorrhagic septicemia and intestinal inflammation caused by aquatic pathogenic bacteria.

6. Use of the Angelica dahurica extract according to claim 1 in the preparation of an anti-aquatic pathogen drug or a feed additive, characterized in that: The angelica dahurica extract is obtained by extraction and separation from the natural Chinese medicine angelica dahurica.

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