Application of gastrodia elata extract in preparation of feed additive for resisting aeromonas hydrophila infection

Abstract

The invention discloses an application of a gastrodia elata extract in preparation of a feed additive for resisting aeromonas hydrophila infection, and belongs to the technical field of prevention and treatment of diseases of aquatic animals. The gastrodia elata extract is found to have a remarkable effect of inhibiting aeromonas hydrophila infection for the first time, and after the gastrodia elata extract serving as a feed additive is added into aquatic animal feed for feeding, the gastrodia elata extract enters a blood circulation system through absorption of digestive tracts, gene expression of immune globulin in intestinal tracts is up-regulated, the level of immune globulin in serum is increased, and the effect of inhibiting aeromonas hydrophila infection is achieved. The feed additive has the advantages of enhancing the intestinal barrier effect, improving the animal activity, effectively resisting the infection of aeromonas hydrophila, reducing the occurrence of diseases, and improving the yield and quality of animal breeding. Therefore, the gastrodia elata extract has a relatively good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of prevention and control of aquatic animal diseases, and particularly relates to the application of gastrodia extract in the preparation of a feed additive for resisting Aeromonas hydrophila infection. Background Art

[0002] Megalobrama amblycephala ( Megalobrama amblycephala ) is one of the important freshwater cultured fish species in China. According to the "China Fisheries Statistical Yearbook", its total output reached 738.727 tons in 2023, ranking seventh among the 25 freshwater cultured fish species statistically recorded in this yearbook. In recent years, the aquaculture industry of Megalobrama amblycephala has developed rapidly, bringing significant economic and social benefits. However, high-density intensive farming and environmental deterioration have led to frequent occurrence of various bacterial diseases, causing serious losses to the aquaculture industry of Megalobrama amblycephala. Among them, the bacterial septicemia caused by Aeromonas hydrophila ( Aeromonas hydrophila ) is the most serious disease that harms Megalobrama amblycephala, which has the characteristics of seasonality, explosiveness, and high mortality. After Megalobrama amblycephala is infected with Aeromonas hydrophila, it shows splenomegaly, congestion of the intestine and liver, and then induces extraintestinal infection and systemic septicemia of fish.

[0003] At present, the main treatments for diseases caused by Aeromonas hydrophila are some commonly used antibiotics in aquaculture. However, in the process of prevention and control, the active use and passive exposure of antibiotics and antibacterial drugs have made the problem of multiple drug resistance of Aeromonas hydrophila increasingly prominent, bringing potential risks to both aquaculture and human health. Therefore, there is an urgent need to seek green and efficient alternative strategies. Chinese herbal medicines have a long history of application in China's aquaculture production due to their advantages of being green, pollution-free, residue-free, and not easily generating drug resistance, and are considered the best substitutes for antibiotics.

[0004] Gastrodia elata ( Gastrodia elata ) is also known as "Red Arrow", "Separated Mother", "Wind-Stabilizing Grass", etc. It belongs to the Orchidaceae family, Epidendroidae subfamily, Gastrodieae tribe, and Gastrodinae subtribe, and is a perennial heterotrophic herb. In the field of traditional Chinese medicine, Gastrodia elata has been widely used to treat various diseases such as headache, dizziness, and epilepsy. Modern pharmacology has further revealed the multi-target and multi-pathway pharmacological mechanisms of Gastrodia elata and its active ingredients, not only consolidating the traditional effects of anti-epilepsy and anti-convulsion, but also showing multiple health benefits such as neuroprotection, anti-fatigue, anti-inflammatory, antioxidant, anti-anxiety and anti-depression, regulating the circulatory system, enhancing memory, and improving cardiovascular diseases. At present, there is no research report on the inhibition of Aeromonas hydrophila infection by Gastrodia elata. Summary of the Invention

[0005] The present invention aims to provide the application of gastrodia extract in the preparation of a feed additive for resisting Aeromonas hydrophila infection. In the present invention, it is first discovered that the gastrodia extract has a significant effect of inhibiting Aeromonas hydrophila infection. After adding the gastrodia extract as a feed additive to animal feed and feeding it, it is absorbed into the blood circulation system through the digestive tract, improving animal vitality, enhancing the intestinal barrier function, effectively resisting the invasion of Aeromonas hydrophila, reducing the occurrence of diseases, and improving the yield and quality of animal breeding.

[0006] In the first aspect, the present invention provides the application of gastrodia extract in the preparation of a feed additive for resisting Aeromonas hydrophila infection.

[0007] In the present invention, the inventor first studies and discovers that the gastrodia extract has a significant effect of inhibiting Aeromonas hydrophila infection. Therefore, the gastrodia extract has good application prospects in the preparation of a feed additive for resisting Aeromonas hydrophila infection.

[0008] In the second aspect, the present invention provides the application of gastrodia extract in the preparation of a drug for preventing and / or treating Aeromonas hydrophila infection in aquatic animals.

[0009] In some embodiments, the aquatic animals include at least one of Megalobrama amblycephala and zebrafish.

[0010] In some embodiments, the gastrodia extract includes at least one of a water extract of gastrodia and an alcohol extract of gastrodia.

[0011] It can be understood that the gastrodia extract can be extracted by conventional extraction methods in the art as long as it can ensure the extraction of the active components in gastrodia. And the alcohol substances in the alcohol extract of gastrodia can be selected from conventional alcohol substances in the prior art according to actual usage needs, preferably anhydrous ethanol.

[0012] In some embodiments, the preparation of the water extract of gastrodia includes: crushing and sieving dry gastrodia powder tablets to obtain fine gastrodia powder particles; mixing the fine gastrodia powder particles with water and then extracting; after the extraction is completed, centrifuging, then adding an adsorbent to the obtained supernatant, continuing to centrifuge, and finally concentrating the obtained supernatant, and obtaining the water extract of gastrodia after vacuum freeze-drying.

[0013] In the present invention, the inventor studies and discovers that the water extract of gastrodia has a better inhibitory effect on Aeromonas hydrophila infection.

[0014] In some embodiments, the aperture of the crushing and sieving is 90-120 mesh, the mass ratio of the fine gastrodia powder particles to water is 1:(20-40), and the extraction specifically includes: extracting for 1-3 h under the conditions of a temperature of 50-70 °C and a rotation speed of 140-160 rpm; the adsorbent is diatomaceous earth, and the addition amount of diatomaceous earth is 5-15%.

[0015] In some preferred embodiments, the aperture of the sieving for pulverization is 100 mesh, and the mass ratio of the fine gastrodia powder particles to water is 1:30. The extraction specifically includes: under the conditions of a temperature of 60°C and a rotation speed of 150 rpm, extracting for 2 h; the adsorbent is diatomaceous earth, and the addition amount of the diatomaceous earth is 10%.

[0016] In a third aspect, the present invention provides a feed for preventing Aeromonas hydrophila infection, comprising the above-mentioned gastrodia extract.

[0017] In a fourth aspect, the present invention provides a preparation method of a feed for preventing Aeromonas hydrophila infection as described above, comprising the following steps: dissolving the gastrodia extract in water, spraying the mixture onto the surface of feed particles after mixing, drying after mixing and standing still to obtain the feed for preventing Aeromonas hydrophila infection.

[0018] In some embodiments, the addition amount of the gastrodia extract in the feed is 0.5 - 5%.

[0019] In a fifth aspect, the present invention provides a pharmaceutical composition for preventing and / or treating aquatic animals, comprising the above-mentioned gastrodia extract.

[0020] In some embodiments, the addition amount of the gastrodia extract in the pharmaceutical composition is 0.5 - 5%.

[0021] In some embodiments, the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0022] In the present invention, the term "pharmaceutically acceptable carrier" refers to excipients widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide methods to enable the active ingredient to dissolve at a desired rate after the subject receives the administration, or to promote the effective absorption of the active ingredient after the subject receives the administration of the composition. Pharmaceutical excipients can be inert fillers or provide certain functions, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. Pharmaceutical excipients can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, glidants, wetting agents, gelling agents, absorption retardants, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0023] The pharmaceutical composition provided by the present invention can be prepared by any method known to those skilled in the art according to the disclosed content. For example, including but not limited to conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or freeze-drying processes.

[0024] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of solid preparations, semi-solid preparations, and liquid preparations.

[0025] The pharmaceutical composition provided by the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be in a controlled-release or sustained-release dosage form (such as liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, cachets, soft gelatin capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, injection solutions, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, injection suspensions, and injection emulsions. Examples of other suitable preparations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories, and lozenges.

[0026] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention for the first time discovers that gastrodia extract has a significant effect of inhibiting Aeromonas hydrophila infection. After adding the gastrodia extract as a feed additive to the feed of aquatic animals and feeding it, it is absorbed into the blood circulation system through the digestive tract, up-regulates the expression of intestinal immunoglobulin genes, increases the serum immunoglobulin level, enhances the intestinal barrier function, improves the animal vitality, effectively resists the infection of Aeromonas hydrophila, reduces the occurrence of diseases, and improves the yield and quality of animal breeding. Description of the Drawings

[0027] Figure 1 It is a result graph of the survival rate of juvenile blunt snout bream after 96 h of challenge in different treatment groups in Application Test Example 1 of the present invention; Figure 2 It is a result graph of the disease activity index score after 24 h of challenge in different treatment groups in Application Test Example 1 of the present invention; Figure 3 It is a result graph of the intestinal HE staining of juvenile blunt snout bream after challenge in different treatment groups in Application Test Example 1 of the present invention; Figure 4 It is a result graph of the expression levels of immunoglobulin genes in the intestinal tissues of zebrafish after 30 days of feeding in different treatment groups in Application Test Example 2 of the present invention. Among them, (A) is the result graph of the expression level of IgM gene, (B) is the result graph of the expression level of IgZ gene, (C) is the result graph of the expression level of IgD gene, and (D) is the result graph of the expression level of PIgR gene; Figure 5This is the immune response result graph of zebrafish against Aeromonas hydrophila infection after challenge in different treatment groups in Application Test Example 2 of the present invention. Among them, (A) is a schematic diagram of the experimental process, (B) is the zebrafish survival rate result, and (C)-(I) are the expression quantity results of IgM, IgZ, IgD, PIgR, IL-4, IL-10, and TGF-β genes respectively, and (J) is the protein levels of IgM and IgZ in the serum. Detailed implementation mode

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0029] For the experimental methods without specific conditions noted in the embodiments, they are usually carried out according to the conventional conditions and the conditions described in the manual, or according to the conditions recommended by the manufacturer. For the general equipment, materials, reagents, etc. used, if there is no special description, they can all be obtained from commercial channels.

[0030] In the present invention, the nutritional components of the basic feed additive are shown in Table 1 below.

[0031] Table 1 Nutritional component table of the basic feed

[0032] Example 1 A preparation method of a water extract of Gastrodia elata, comprising the following steps: 1) Crush the dried Gastrodia elata slices through a 100-mesh sieve to obtain Gastrodia elata fine powder particles; 2) Take 5 g of the Gastrodia elata fine powder particles prepared in step 1) and 150 g of water and add them to a 500 mL distillation flask, shake well until it becomes a suspension, place it on a rotary evaporator (water bath temperature 60 °C), without vacuum pumping, and extract for 2 h under the condition of a rotation speed of 150 rpm; 3) After the extraction is completed, centrifuge at 10000 rpm for 10 min, then add 10% diatomaceous earth to the obtained supernatant, shake well to obtain a mixed solution; 4) Centrifuge the mixed solution prepared in step 3) at 10000 rpm for 10 min again, and finally concentrate the obtained supernatant using a rotary evaporator and obtain the water extract of Gastrodia elata after vacuum freeze-drying.

[0033] A preparation method of a feed for resisting Aeromonas hydrophila infection, comprising the following steps: First, according to the addition amounts of 0.5%, 1%, 2.5%, and 5%, respectively weigh the water extract of Gastrodia elata, add water and mix, then spray them onto the surfaces of feed pellets with a particle size of 2 mm respectively. After fully mixing, let it stand at room temperature for 2 h, and then dry it in an oven at 50 °C for 12 h to obtain feeds with the water extract of Gastrodia elata contents of 0.5%, 1%, 2.5%, and 5% respectively. Place the above feeds in a refrigerator at -4 °C for storage until taken out before feeding.

[0034] Application Test Example 1 Select 360 juvenile blunt snout breams with a body weight of about 5 g, and randomly divide them into 4 groups, namely: control group (Control), low-dose group (0.5%), medium-dose group (2.5%), and high-dose group (5%). Each group has 3 replicates, and each replicate has 30 fish. Before the experiment starts, acclimate them for 1 week and feed them with the basal diet. After the experiment starts, the low-dose group, medium-dose group, and high-dose group are respectively fed with the feeds prepared in Example 1 with the water extract of Gastrodia elata contents of 0.5%, 2.5%, and 5%, and the control group is fed with the basal diet. Feed once at 09:00 and 18:00 every day, and the feeding amount is 3% of the body weight. After 1 week, challenge them by intraperitoneal injection with a half-lethal dose (2×10 7 CFU / each fish) of Aeromonas hydrophila. After the challenge, count the 96 h survival rate at an interval of 12 h, and take samples for dissection and observation scoring 24 h after the challenge to calculate the disease activity index score (DAI). The scoring criteria are shown in Table 2. During the experiment, drain the sewage every day, make up the water volume after draining the sewage, and change the water once every 3 d, and the water change amount is about 1 / 3. During the test period, the water temperature is 25 - 30 °C, the ammonia nitrogen concentration < 2 mg / L, the nitrite concentration < 0.01 mg / L, the dissolved oxygen content > 5.0 mg / L, and the pH is 7.5 - 8.0.

[0035] Table 2 Disease Activity Index Scoring Criteria

[0036] Among them, the results of the 96 h survival rate are as Figure 1 shown.

[0037] From Figure 1It can be seen that the survival rate of the control group at 96 h was 45%, the survival rate of the low-dose group (0.5%) was 50%, the survival rate of the medium-dose group (2.5%) was 60%, and the survival rate of the high-dose group (5%) was 80%, which was significantly higher than that of the control group. The death time of the control group was significantly earlier than that of the high-dose group. Deaths occurred at 4 h after infection in the control group, while deaths in the high-dose group occurred after 23 h. The cumulative mortality rate of the control group reached its peak at 13 h, while that of the high-dose group reached its peak at 23 h after infection. The results showed that adding 5% of the water extract of Gastrodia elata in the feed could significantly improve the survival rate of juvenile blunt snout bream after infection with Aeromonas hydrophila.

[0038] The results of sampling, dissecting and observing the scores 24 h after virus challenge are as Figure 2 shown.

[0039] From Figure 2 it can be seen that there was no significant difference between the low-dose group (0.5%) and the medium-dose group (2.5%) and the control group, but the pathological activity index of the high-dose group (5%) was significantly lower than that of the control group. The results showed that adding 5% of the water extract of Gastrodia elata in the feed could significantly reduce the pathological activity index of blunt snout bream after infection with Aeromonas hydrophila and improve the vitality of fish.

[0040] Furthermore, HE staining was used to observe the posterior intestine of the dissected fish, and the results are as Figure 3 shown.

[0041] From Figure 3 it can be seen that after the control group was infected with Aeromonas hydrophila, the length of the intestinal villi became shorter, the tissue was defective, a large number of inflammatory cells infiltrated the submucosa layer, and the muscle layer became thinner. As the addition amount of the water extract of Gastrodia elata increased to 5%, the length of the intestinal villi gradually increased, the tissue structure was complete, there was basically no infiltration of inflammatory cells, and the thickness of the muscle layer returned to normal. The results showed that adding 5% of the water extract of Gastrodia elata in the feed could enhance the intestinal barrier function and maintain the intestinal health status.

[0042] Application Test Example 2 180 two-month-old wild-type zebrafish with a body weight of about 1 g were selected and randomly divided into 2 groups, namely: the control group (Control) and the feeding group (GE), with 3 replicates in each group and 30 fish in each replicate. One week before the experiment, they were temporarily raised and fed a basal diet. After the experiment started, the feeding group was fed with the feed prepared in Example 1 with a content of 1% of the water extract of Gastrodia elata, and the control group was fed with the basal diet. They were fed once at 09:00 and 18:00 every day, and the feeding amount was 3% of the body weight. After 30 days of feeding, 10 fish intestinal tissues were taken from the feeding group and the control group, and the expression levels of immunoglobulin genes were detected by qPCR. The results are as Figure 4 shown. The remaining 20 fish were infected by intraperitoneal injection of Aeromonas hydrophila (3×10 6CFU / fish), and the survival rate was counted at 72 h after challenge with a 12-h interval. Serum was collected two days after infection, and the expression level of immunoglobulin was detected by western blot. Meanwhile, intestinal tissues were collected and the expression levels of immunoglobulin genes were detected by qPCR. The results are as follows Figure 5 shown. During the experiment, sewage was discharged every day, and the water volume was replenished after sewage discharge. The water was changed once every 3 days, and the water change volume was about 1 / 3. During the experiment, the water temperature was 25-30 °C, the ammonia nitrogen concentration was <2 mg / L, the nitrite concentration was <0.01 mg / L, the dissolved oxygen content was >5.0 mg / L, and the pH was 7.5-8.0.

[0043] It can be seen from Figure 4 that after feeding zebrafish with feed containing 1% of the water extract of Gastrodia elata for 30 days, the expression of intestinal immunoglobulin genes (IgM gene, IgZ gene, IgD gene, PIgR gene) was significantly up-regulated. The results showed that oral administration of the water extract of Gastrodia elata could increase the expression level of immunoglobulin genes in zebrafish.

[0044] It can be seen from Figure 5 that after infection with Aeromonas hydrophila, the 72-h mortality rate of the control group was 52%, and the 72-h mortality rate of the feeding group was significantly lower than that of the control group, which was 20% ( Figure 5 B). Intestinal qPCR analysis showed that 48 h after infection, the immunoglobulins and anti-inflammatory factors in the feeding group were significantly up-regulated ( Figure 5 C-5I), and at the same time, the expression levels of IgM and IgZ proteins in the serum of the feeding group were significantly increased by western blot detection ( Figure 5 J). The results showed that the water extract of Gastrodia elata could play a protective role against Aeromonas hydrophila infection by up-regulating the expression of immunoglobulin genes and increasing the immunoglobulin level.

[0045] In summary, the present invention first discovers that the extract of Gastrodia elata has a significant effect of inhibiting Aeromonas hydrophila infection. After adding the extract of Gastrodia elata as a feed additive to the feed of aquatic animals and feeding it, it is absorbed into the blood circulation system through the digestive tract, up-regulates the expression of intestinal immunoglobulin genes, increases the serum immunoglobulin level, improves the animal vitality, enhances the intestinal barrier function, effectively resists the invasion of Aeromonas hydrophila, reduces the occurrence of diseases, and improves the yield and quality of animal breeding.

[0046] It should be noted that the above embodiments all belong to the same inventive concept. The descriptions of the embodiments have their own focuses. For the parts not described in detail in individual embodiments, reference can be made to the descriptions in other embodiments.

[0047] The embodiments described above only represent the implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Application of Gastrodia elata extract in the preparation of feed additives for resisting Aeromonas hydrophila infection.

2. Use of Gastrodia elata extract in the preparation of drugs for preventing and / or treating Aeromonas hydrophila infection in aquatic animals.

3. The use according to claim 1 or 2, characterized in that: The Gastrodia elata extract comprises at least one of a Gastrodia elata water extract and a Gastrodia elata alcohol extract.

4. The use according to claim 3, characterized in that: The preparation of the Gastrodia elata water extract comprises: crushing and sieving dry Gastrodia elata slices to obtain Gastrodia elata fine powder particles; mixing the Gastrodia elata fine powder particles with water and then extracting; after the extraction is completed, centrifuging, then adding an adsorbent to the obtained supernatant, continuing to centrifuge, and finally concentrating the obtained supernatant, and after vacuum drying, obtaining the Gastrodia elata water extract.

5. The use according to claim 4, characterized in that: The aperture of the crushed and sieved particles is 90-120 meshes, the mass ratio of the Gastrodia elata fine powder particles to the water is 1:(20-40), and the extraction specifically comprises: extracting for 1-3 hours at a temperature of 50-70°C and a rotation speed of 140-160rpm; The adsorbent is diatomaceous earth, and the added amount of the diatomaceous earth is 5-15%.

6. A feed resistant to Aeromonas hydrophila infection, characterized in that: The invention comprises the Gastrodia elata extract as claimed in claim 1.

7. A method for preparing feed resistant to Aeromonas hydrophila infection as claimed in claim 6, characterized in that: The method comprises the following steps: dissolving the gastrodia elata extract in water, spraying the mixture onto the surface of feed particles, mixing the mixture evenly, standing the mixture still and drying the mixture to obtain the feed resistant to Aeromonas hydrophila infection.

8. A pharmaceutical composition for preventing and / or treating Aeromonas hydrophila infection in aquatic animals, characterized in that: The invention comprises the Gastrodia elata extract as claimed in claim 2.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 8, characterized in that The dosage form of the pharmaceutical composition includes at least one of a solid preparation, a semisolid preparation, and a liquid preparation.

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