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

Abstract

The application discloses application of Gastrodia elata extract in preparation of a feed additive for resisting Aeromonas hydrophila infection, and belongs to the technical field of water animal disease prevention and treatment. The application first finds that the Gastrodia elata extract has a significant effect of inhibiting Aeromonas hydrophila infection. After the Gastrodia elata extract is added into water animal feed as a feed additive and is fed to the water animal, the Gastrodia elata extract is absorbed into blood circulation system through a digestive tract, up-regulates intestinal immunoglobulin gene expression, improves serum immunoglobulin level, enhances intestinal barrier function, improves animal activity, effectively resists Aeromonas hydrophila infection, reduces disease occurrence, and improves yield and quality of animal breeding. Therefore, the Gastrodia elata extract has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal disease prevention and control technology, specifically relating to the application of Gastrodia elata extract in the preparation of feed additives resistant to Aeromonas hydrophila infection. Background Technology

[0002] Blunthead bream ( Megalobrama amblycephala Blunthead bream is one of my country's important freshwater aquaculture fish species. According to the *China Fisheries Statistical Yearbook*, its total production reached 738.727 tons in 2023, ranking seventh among the 25 freshwater aquaculture fish species listed in the yearbook. In recent years, blunthead bream aquaculture has developed rapidly, bringing significant economic and social benefits. However, high-density intensive farming and environmental degradation have led to frequent outbreaks of various bacterial diseases, causing serious losses to the blunthead bream aquaculture industry. Among these, *Aeromonas hydrophila* (…) is particularly prevalent. Aeromonas hydrophila Bacterial septicemia caused by Aeromonas hydrophila is the most serious disease affecting blunt snout bream, characterized by seasonality, outbreaks, and high mortality. After infection with Aeromonas hydrophila, blunt snout bream exhibit splenomegaly, intestinal and liver congestion, subsequently leading to extraintestinal infection and systemic septicemia.

[0003] Currently, the main treatments for diseases caused by Aeromonas hydrophila are antibiotics commonly used in aquaculture. However, the active use and passive exposure to antibiotics and antimicrobial drugs during prevention and control have led to increasingly prominent multidrug resistance in Aeromonas hydrophila, posing potential risks to both aquaculture and human health. Therefore, there is an urgent need to find green and efficient alternative strategies. Traditional Chinese medicine, due to its advantages of being green, pollution-free, residue-free, and less likely to induce drug resistance, has a long history of application in my country's aquaculture production and is considered the best alternative to antibiotics.

[0004] Gastrodia elata ( Gastrodia elata Gastrodia elata, also known as "Chijian," "Limu," and "Dingfengcao," belongs to the Orchidaceae family, Epidendroidae subfamily, Gastrodieae tribe, and Gastrodinae subtribe. It is a perennial heterotrophic herb. In traditional Chinese medicine, Gastrodia elata is widely used to treat various diseases such as headaches, 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 its traditional anti-epileptic and anticonvulsant effects but also demonstrating multiple health benefits such as neuroprotection, anti-fatigue, anti-inflammation, antioxidant, anti-anxiety and antidepressant effects, regulation of the circulatory system, enhanced memory, and improvement of cardiovascular diseases. Currently, there are no research reports on the inhibition of Aeromonas hydrophila infection by Gastrodia elata. Summary of the Invention

[0005] The purpose of this invention is to provide the application of Gastrodia elata extract in the preparation of feed additives for inhibiting Aeromonas hydrophila infection. This invention is the first to discover that Gastrodia elata extract has a significant inhibitory effect on Aeromonas hydrophila infection. When this Gastrodia elata extract is added to animal feed as a feed additive, it is absorbed through the digestive tract and enters the bloodstream, improving animal vitality, enhancing the intestinal barrier function, effectively resisting Aeromonas hydrophila infection, reducing disease occurrence, and improving the yield and quality of animal husbandry.

[0006] In a first aspect, the present invention provides the use of Gastrodia elata extract in the preparation of feed additives for resistance to Aeromonas hydrophila infection.

[0007] In this invention, the inventors discovered for the first time that Gastrodia elata extract has a significant inhibitory effect on Aeromonas hydrophila infection. Therefore, this Gastrodia elata extract has good application prospects in the preparation of feed additives for preventing Aeromonas hydrophila infection.

[0008] In a second aspect, the present invention provides the use of Gastrodia elata extract in the preparation of medicaments for the prevention and / or treatment of Aeromonas hydrophila infection in aquatic animals.

[0009] In some implementations, the aquatic animals include at least one of blunt snout bream and zebrafish.

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

[0011] It is understood that the Gastrodia elata extract can be extracted using conventional extraction methods in the field, as long as the active components of Gastrodia elata are extracted. Furthermore, the alcohols in the Gastrodia elata ethanol extract can be selected from conventional alcohols in the prior art, preferably anhydrous ethanol, depending on the actual application requirements.

[0012] In some embodiments, the preparation of the aqueous extract of Gastrodia elata includes: crushing dried Gastrodia elata powder into flakes and sieving to obtain fine powder particles; mixing the fine powder particles with water and then extracting; after extraction, centrifuging, then adding an adsorbent to the supernatant and continuing centrifugation, and finally concentrating the supernatant and freeze-drying it under vacuum to obtain the aqueous extract of Gastrodia elata.

[0013] In this invention, the inventors discovered that the aqueous extract of Gastrodia elata has a better inhibitory effect on Aeromonas hydrophila infection.

[0014] In some implementations, the sieve aperture is 90-120 mesh, the mass ratio of Gastrodia elata fine powder particles to water is 1:(20-40), and the extraction specifically includes: extraction for 1-3 hours at a temperature of 50-70℃ and a rotation speed of 140-160 rpm; the adsorbent is diatomaceous earth, and the amount of diatomaceous earth added is 5-15%.

[0015] In some preferred embodiments, the sieve aperture is 100 mesh, the mass ratio of Gastrodia elata fine powder particles to water is 1:30, and the extraction specifically includes: extraction for 2 hours at a temperature of 60°C and a rotation speed of 150 rpm; the adsorbent is diatomaceous earth, and the amount of diatomaceous earth added is 10%.

[0016] In a third aspect, the present invention provides a feed resistant to Aeromonas hydrophila infection, comprising the above-mentioned Gastrodia elata extract.

[0017] In a fourth aspect, the present invention provides a method for preparing feed resistant to Aeromonas hydrophila infection as described above, comprising the following steps: dissolving Gastrodia elata extract in water, mixing and spraying the mixture onto the surface of feed pellets, mixing evenly and allowing it to stand before drying to obtain feed resistant to Aeromonas hydrophila infection.

[0018] In some implementation schemes, the amount of Gastrodia elata extract added to the feed is 0.5-5%.

[0019] In a fifth aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of aquatic animals, comprising the above-mentioned Gastrodia elata extract.

[0020] In some embodiments, the amount of Gastrodia elata extract added to the pharmaceutical composition is 0.5-5%.

[0021] In some embodiments, the pharmaceutical composition may further include a pharmaceutically acceptable carrier.

[0022] In this invention, the term "pharmaceutically acceptable carrier" refers to excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods for dissolving the active ingredient at a desired rate after administration to a subject, or for promoting effective absorption of the active ingredient after administration to a subject. Pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. Pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0023] The pharmaceutical compositions provided by this invention can be prepared using any method known to those skilled in the art, based on the disclosure. Examples include, but are not limited to, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

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

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

[0026] The beneficial effects of this invention are as follows: Unlike the prior art, this invention is the first to discover that Gastrodia elata extract has a significant inhibitory effect on Aeromonas hydrophila infection. When this Gastrodia elata extract is added to the feed of aquatic animals and fed to them, it is absorbed into the blood circulation system through the digestive tract, upregulates the expression of intestinal immunoglobulin genes, increases serum immunoglobulin levels, enhances the intestinal barrier function, improves animal vitality, effectively resists Aeromonas hydrophila infection, reduces the occurrence of diseases, and improves the yield and quality of animal husbandry. Attached Figure Description

[0027] Figure 1 This is a graph showing the survival rate of juvenile blunt snout bream after 96 hours of viral challenge in different treatment groups in Application Test Example 1 of this invention;

[0028] Figure 2 This is a graph showing the disease activity index scores of different treatment groups 24 hours after viral challenge in test example 1 of this invention.

[0029] Figure 3 This is a HE staining result of the intestines of juvenile blunt snout bream after challenge with different treatment groups in Application Test Example 1 of this invention;

[0030] Figure 4The graph shows the expression levels of immunoglobulin genes in the intestinal tissue of zebrafish after 30 days of feeding in different treatment groups in test example 2 of this invention. (A) shows the expression level of IgM gene, (B) shows the expression level of IgZ gene, (C) shows the expression level of IgD gene, and (D) shows the expression level of PIgR gene.

[0031] Figure 5 The figure shows the immune response of zebrafish to Aeromonas hydrophila infection after challenge in different treatment groups in test example 2 of this invention. In this figure, (A) is a schematic diagram of the experimental process, (B) is the survival rate of zebrafish, (C)-(I) are the expression levels of IgM, IgZ, IgD, PIgR, IL-4, IL-10 and TGF-β genes, respectively, and (J) is the serum IgM and IgZ protein levels. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0034] In this invention, the nutritional components of the basic feed supplement are shown in Table 1 below.

[0035] Table 1 Nutritional composition of basic feed

[0036]

[0037] Example 1

[0038] A method for preparing a gastrodia elata aqueous extract includes the following steps:

[0039] 1) Crush the dried Gastrodia elata slices through a 100-mesh sieve to obtain fine powder particles of Gastrodia elata;

[0040] 2) Take 5g of the fine powder particles of Gastrodia elata prepared in step 1) and 150g of water and add them to a 500mL distillation flask. Shake well until a suspension is formed. Place it on a rotary evaporator (water bath temperature 60℃), without vacuuming, and extract for 2h at a speed of 150rpm.

[0041] 3) After extraction, centrifuge at 10,000 rpm for 10 min, then add 10% diatomaceous earth to the obtained supernatant, shake well, and obtain a mixture;

[0042] 4) Centrifuge the mixture obtained in step 3) at 10,000 rpm for 10 min. Finally, concentrate the supernatant using a rotary evaporator and freeze-dry it under vacuum to obtain the water extract of Gastrodia elata.

[0043] A method for preparing feed resistant to Aeromonas hydrophila infection includes the following steps: First, weigh out water extracts of Gastrodia elata at dosages of 0.5%, 1%, 2.5%, and 5%, respectively. Mix with water and spray onto the surface of feed pellets with a particle size of 2 mm. After thorough mixing, allow to stand at room temperature for 2 hours, then dry in an oven at 50°C for 12 hours to obtain feeds with water extract contents of 0.5%, 1%, 2.5%, and 5%, respectively. Store the feeds at -4°C until just before feeding.

[0044] Application Test Case 1

[0045] Three hundred and sixty juvenile crucian carp weighing approximately 5g were randomly divided into four groups: control group (0.5%), medium-dose group (2.5%), and high-dose group (5%), with three replicates per group and 30 fish per replicate. Fish were temporarily housed for one week before the experiment and fed a basal diet. After the experiment began, the low-dose, medium-dose, and high-dose groups were fed diets containing 0.5%, 2.5%, and 5% of the Gastrodia elata aqueous extract prepared in Example 1, respectively. The control group was fed the basal diet. Fish were fed twice daily, at 09:00 and 18:00, at a dose of 3% of their body weight. After one week, the fish were administered a median lethal dose (2 × 10⁻⁶) via intraperitoneal injection. 7 CFU / each Aeromonas hydrophila was used to challenge the bacteria. Survival rates were recorded at 96-hour intervals after challenge, with samples taken 24 hours post-challenge for dissection and evaluation. The disease activity index (DAI) score was calculated, and the scoring criteria are shown in Table 2. Wastewater was discharged daily during the experiment, and the water volume was replenished afterward. Approximately one-third of the water was replaced every 3 days. During the experiment, the water temperature was maintained at 25-30℃, ammonia nitrogen concentration <2 mg / L, nitrite concentration <0.01 mg / L, dissolved oxygen content >5.0 mg / L, and pH at 7.5-8.0.

[0046] Table 2 Disease Activity Index Scoring Criteria

[0047]

[0048] Among them, the 96-hour survival rate results are as follows: Figure 1 As shown.

[0049] from Figure 1 The results show that the survival rate at 96 hours was 45% in the control group, 50% in the low-dose group (0.5%), 60% in the medium-dose group (2.5%), and 80% in the high-dose group (5%), all significantly higher than the control group. The time to death in the control group was significantly earlier than in the high-dose group, occurring 4 hours after infection, while death in the high-dose group occurred after 23 hours. The cumulative mortality rate in the control group peaked at 13 hours, while in the high-dose group it peaked at 23 hours. These results indicate that adding 5% of Gastrodia elata aqueous extract to the feed can significantly improve the survival rate of juvenile blunt snout bream infected with Aeromonas hydrophila.

[0050] The results of the autopsy and scoring of samples taken 24 hours after the challenge are as follows: Figure 2 As shown.

[0051] 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. However, the pathological activity index of the high-dose group (5%) was significantly lower than that of the control group. The results indicate that adding 5% of Gastrodia elata water extract to the feed can significantly reduce the pathological activity index of blunt snout bream infected with Aeromonas hydrophila and improve the vitality of the fish.

[0052] Furthermore, HE staining was used to observe the hindgut in the dissected fish, and the results were as follows: Figure 3 As shown.

[0053] from Figure 3 The results showed that in the control group, infection with Aeromonas hydrophila resulted in shortened intestinal villi, tissue defects, extensive inflammatory cell infiltration in the submucosa, and thinning of the muscle layer. As the concentration of Gastrodia elata aqueous extract increased to 5%, the intestinal villi length gradually increased, the tissue structure remained intact with minimal inflammatory cell infiltration, and the muscle layer thickness returned to normal. These results indicate that adding 5% Gastrodia elata aqueous extract to the feed can enhance the intestinal barrier function and maintain intestinal health.

[0054] Application Test Example 2

[0055] One hundred and eighty two-month-old wild-type zebrafish weighing approximately 1g were randomly divided into two groups: a control group (Control) and a feeding group (GE), with three replicates per group and 30 fish per replicate. The fish were temporarily housed for one week before the experiment and fed a basal diet. After the experiment began, the feeding group was fed a diet containing 1% Gastrodia elata aqueous extract prepared in Example 1, while the control group was fed the basal diet. Feeding was done twice daily, at 09:00 and 18:00, with a feed amount of 3% of the fish's body weight. After 30 days of feeding, intestinal tissue was collected from 10 fish in each group, and the expression levels of immunoglobulin genes were detected by qPCR. The results are as follows: Figure 4As shown. The remaining 20 fish were infected with Aeromonas hydrophila via intraperitoneal injection (3 × 10⁻⁶). 6 CFU / tail), survival rate at 72-hour intervals was calculated 12 hours after challenge. Serum was collected two days post-infection and immunoglobulin expression levels were detected using Western blot. Intestinal tissue was also collected and immunoglobulin gene expression levels were detected using qPCR. Results are as follows: Figure 5 As shown in the figure. During the experiment, wastewater was discharged daily, and the water volume was replenished after discharge. The water was changed every 3 days, with about 1 / 3 of the water being replaced. During the experiment, the water temperature was 25-30℃, the ammonia nitrogen concentration was <2mg / L, the nitrite concentration was <0.01mg / L, the dissolved oxygen content was >5.0mg / L, and the pH was 7.5-8.0.

[0056] from Figure 4 The results show that after feeding zebrafish with a feed containing 1% Gastrodia elata water extract for 30 days, the expression of intestinal immunoglobulin genes (IgM, IgZ, IgD, and PIGR genes) was significantly upregulated. The results indicate that oral administration of Gastrodia elata water extract can improve the expression level of immunoglobulin genes in zebrafish.

[0057] from Figure 5 It can be seen that after infection with Aeromonas hydrophila, the mortality rate at 72 hours was 52% in the control group, while the mortality rate at 72 hours in the fed group was significantly lower than that in the control group, at 20%. Figure 5 B). Intestinal qPCR analysis showed that 48 hours post-infection, immunoglobulins and anti-inflammatory factors were significantly upregulated in the feeding group (B). Figure 5 C-5I), and Western blot analysis showed that the expression levels of IgM and IgZ proteins in the serum of the feeding group were significantly increased. Figure 5 J). The results showed that the aqueous extract of Gastrodia elata could exert a protective effect against Aeromonas hydrophila infection by upregulating immunoglobulin gene expression and increasing immunoglobulin levels.

[0058] In summary, this invention is the first to discover that Gastrodia elata extract has a significant inhibitory effect on Aeromonas hydrophila infection. When this Gastrodia elata extract is added to the feed of aquatic animals, it is absorbed through the digestive tract and enters the blood circulation system, upregulating the expression of intestinal immunoglobulin genes, increasing serum immunoglobulin levels, improving animal vitality, enhancing the intestinal barrier function, effectively resisting Aeromonas hydrophila infection, reducing the occurrence of diseases, and improving the yield and quality of animal husbandry.

[0059] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0060] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of Gastrodia elata extract in the preparation of feed additives for the prevention of aquatic animal diseases, characterized in that, The aquatic animal disease is caused by Aeromonas hydrophila infection; The gastrodia extract is a gastrodia water extract, and the preparation of the gastrodia water extract includes: pulverizing and sieving dried gastrodia slices to obtain gastrodia fine powder particles; and extracting the gastrodia fine powder particles by mixing them with water. The mass ratio of the fine powder of Gastrodia elata to the water is 1:(20-40), and the extraction specifically includes: extracting for 1-3 hours at a temperature of 50-70℃ and a rotation speed of 140-160rpm. The aquatic animals include at least one of blunt snout bream and zebrafish.

2. The application of Gastrodia elata extract in the preparation of drugs for the prevention of aquatic animal diseases, characterized in that, The aquatic animal disease is caused by Aeromonas hydrophila infection; The gastrodia extract is a gastrodia water extract, and the preparation of the gastrodia water extract includes: pulverizing and sieving dried gastrodia slices to obtain gastrodia fine powder particles; and extracting the gastrodia fine powder particles by mixing them with water. The mass ratio of the fine powder of Gastrodia elata to the water is 1:(20-40), and the extraction specifically includes: extracting for 1-3 hours at a temperature of 50-70℃ and a rotation speed of 140-160rpm. The aquatic animals include at least one of blunt snout bream and zebrafish.

3. The application according to claim 1 or 2, characterized in that, The preparation of the gastrodia elata aqueous extract also includes: After extraction, centrifugation was performed, and then an adsorbent was added to the supernatant. Centrifugation was continued, and finally the supernatant was concentrated and dried under vacuum to obtain the gastrodia elata water extract.

4. The application according to claim 3, characterized in that, The pulverizing and sieving process uses a sieve with a mesh size of 90-120, the adsorbent is diatomaceous earth, and the amount of diatomaceous earth added is 5-15%.

5. The application according to claim 1, characterized in that, The feed additive is obtained by dissolving Gastrodia elata extract in water, mixing the mixture, spraying it onto the surface of feed pellets, mixing thoroughly, letting it stand, and then drying it.

6. The application according to claim 2, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

7. The application according to claim 2, characterized in that, The dosage form of the drug includes at least one of solid dosage form, semi-solid dosage form, and liquid dosage form.

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