Compound immunopotentiator as well as preparation method and application thereof

Abstract

The invention discloses a compound immunopotentiator as well as a preparation method and application thereof, and belongs to the technical field of aquaculture. The compound immunopotentiator contains a gallnut water extract, a gallnut alcohol extract, an astragalus membranaceus water extract, an astragalus membranaceus alcohol extract, a rhodiola rosea water extract, a rhodiola rosea alcohol extract and chlorogenic acid. The compound immunopotentiator provided by the invention can significantly improve the resistance of aquaculture animals to pathogen infection, is free of environmental pollution and drug resistance risks, and has good safety and effectiveness. Experiments show that for aquatic products infected with nervous necrosis viruses, the positive rate of the nervous necrosis viruses can be obviously reduced by adding the compound immunopotentiator into feed of the aquatic products; for aquatic products treated by mixed infection of nervous necrosis virus and vibrio harveyi, the death rate of the aquatic products can be obviously reduced by adding the compound immunopotentiator into feed of the aquatic products, so that the compound immunopotentiator disclosed by the invention has a good immunoprotection effect when coping with the mixed infection of virus and bacteria.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to a compound immune enhancer, its preparation method, and its application. Background Technology

[0002] With the intensive and large-scale development of aquaculture, the complexity and instability of the aquaculture environment have intensified. The risk of the proliferation and spread of pathogenic microorganisms such as bacteria, viruses, and parasites in the water has increased sharply, leading to frequent outbreaks of aquatic animal diseases. This has become a bottleneck restricting the development of the industry and causing the economic losses of farmers to increase year by year.

[0003] Currently, aquatic disease control still relies primarily on chemical agents. While antibiotics and pesticides can suppress pathogens in the short term, long-term use leads to increased drug resistance in pathogens, decreased treatment effectiveness, and escalating drug concentrations. More seriously, chemical residues threaten the safety of aquatic products, disrupt aquatic microbial communities, and cause eutrophication and ecological imbalance. Traditional physical detoxification methods, such as ultraviolet light and ozone, are difficult to promote on a large scale due to their limited range of action and the potential to damage aquatic animals.

[0004] While vaccination is a key aspect of green disease control, it faces technical barriers in practice. Aquatic animal pathogens mutate rapidly and are diverse, making vaccine development time-consuming and labor-intensive, and difficult to respond to sudden outbreaks. Furthermore, different farmed species exhibit significant differences in immune responses, and existing immunization methods such as injection and immersion are insufficient in terms of efficiency and protection duration, failing to meet the needs for rapid and long-term disease control. Summary of the Invention

[0005] Given the significant shortcomings of both chemical drug control and vaccine immunization, the development of safe and highly effective immune enhancers, focusing on improving the body's immune function to achieve a synergistic effect of active defense and pathogen clearance, has become a research focus in aquatic disease control. Immune enhancers can regulate and strengthen the body's immune function, improving resistance and clearance of pathogens by activating non-specific immunity or inducing specific immune responses. Compared to chemical drugs, they are characterized by milder effects, no drug resistance, and environmental friendliness. They can stimulate the aquatic animals' own defense system, enabling a shift from "passive treatment" to "active prevention," aligning with the concept of green and healthy aquaculture and providing support for sustainable aquaculture.

[0006] The purpose of this invention is to provide a compound immune enhancer, its preparation method, and its application. By combining gallnut, astragalus, and rhodiola, and using their extracts as a compound immune enhancer, the resistance of farmed aquatic animals to pathogen infection can be significantly improved, without environmental pollution or drug resistance risks, demonstrating good safety and efficacy.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] One of the technical solutions of the present invention is to provide a compound immune enhancer, wherein the compound immune enhancer contains gallnut water extract, gallnut alcohol extract, astragalus water extract, astragalus alcohol extract, rhodiola water extract, rhodiola alcohol extract and chlorogenic acid.

[0009] Preferably, each mL of the compound immune enhancer contains 0.2g of water extract of Galla chinensis, 0.2g of alcohol extract of Galla chinensis, 0.2g of water extract of Astragalus membranaceus, 0.2g of alcohol extract of Astragalus membranaceus, 0.05g of water extract of Rhodiola rosea, 0.05g of alcohol extract of Rhodiola rosea and 0.01g of chlorogenic acid.

[0010] Preferably, the extract of the gallnut alcohol extract is an 80% (v / v) ethanol solution; the extract of the astragalus alcohol extract is an 80% (v / v) ethanol solution; and the extract of the rhodiola rosea alcohol extract is an 80% (v / v) ethanol solution.

[0011] The second technical solution of the present invention provides a method for preparing the above-mentioned compound immune enhancer, comprising the following steps:

[0012] Gallnut, Astragalus, and Rhodiola rosea were extracted separately with water to obtain water extracts of Gallnut, Astragalus, and Rhodiola rosea. Gallnut, Astragalus, and Rhodiola rosea were also extracted separately with alcohol solutions to obtain alcohol extracts of Gallnut, Astragalus, and Rhodiola rosea. The combined alcohol extracts of Gallnut, Astragalus, and Rhodiola rosea, along with chlorogenic acid, were then mixed to obtain the compound immune enhancer.

[0013] Preferably, the gallnut, astragalus, and rhodiola are all 40-mesh powder.

[0014] Preferably, the water extraction temperature is 100°C; the alcohol extraction temperature is 80°C.

[0015] More preferably, the water extraction is performed by combining the extracts after three extractions, with the amount of extract added in each extraction being 10 times the amount of the medicinal material, and the extraction time being 1 hour; the alcohol extraction is performed by combining the extracts after three extractions, with the amount of extract added in each extraction being 10 times the amount of the medicinal material, and the extraction time being 1 hour.

[0016] The third technical solution of the present invention provides an application of the above-mentioned compound immune enhancer in aquaculture.

[0017] Preferably, the compound immune enhancer is used as a feed additive.

[0018] The beneficial technical effects of the present invention are as follows:

[0019] The compound immune enhancer provided by this invention can significantly improve the resistance of aquatic animals to pathogen infection, and has no environmental pollution or drug resistance risks, exhibiting good safety and efficacy. Experiments show that adding this compound immune enhancer to the feed of aquatic animals infected with neuronecrosis virus can significantly reduce the positive rate of neuronecrosis virus; for aquatic animals treated with mixed infection of neuronecrosis virus and Vibrio harveyi, adding this compound immune enhancer to their feed can significantly reduce the mortality rate, indicating that the compound immune enhancer of this invention has a good immune protective effect against mixed viral and bacterial infections.

[0020] The compound immune enhancer provided by this invention can be used as a feed additive in the pond culture of California bass, crucian carp and whiteleg shrimp. It can effectively improve their resistance to pathogens such as Edwardsiella pisciformis, Aeromonas hydrophila, and Vibrio parahaemolyticus, reduce the mortality rate after infection and onset, and at the same time have a positive effect on promoting the growth of aquatic animals and improving their growth performance. It shows good protective effect in pond culture. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The expression levels of C3 and C4 in the spleen tissue of grouper fry by different extracts in Example 1.

[0023] Figure 2 The graph shows the relationship between the number of feeding days and the RGNNV positivity rate of grouper in the experimental and control groups in the experiment evaluating the therapeutic effect of CIM compound on grouper infected with nerve necrosis virus.

[0024] Figure 3 The graph shows the relationship between the number of days of infection and the mortality rate of grouper in the experimental and control groups in an experiment on the immunoprotective effect of CIM compound against mixed infection with neuronecrosis virus and Vibrio harveyi in grouper. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0026] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0027] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] The Chinese medicinal materials used in this invention, namely gallnut, astragalus, and rhodiola, all meet the relevant quality standards of the Pharmacopoeia of the People's Republic of China.

[0031] Before extraction, gallnut, astragalus and rhodiola rosea are all pretreated. The specific steps of the pretreatment are as follows: first, they are baked at 50℃ for 16~24h to make each Chinese medicinal material fully dry. Then, they are crushed using a Chinese herbal medicine pulverizer and passed through a 40-mesh sieve. They are then stored in a dry environment at room temperature (20±10℃) for later use.

[0032] Example 1

[0033] Preparation and immunomodulatory activity analysis of extracts from Gallnut, Astragalus and Rhodiola:

[0034] (1) Preparation of Gallnut Water Extract: Accurately weigh 50g of pretreated gallnut powder, add 10 times the amount of deionized water, and heat in an electric heating mantle for decoction. The initial decoction temperature is set at 100℃, and after boiling, maintain a gentle boil for 1 hour; filter to separate the residue and extract, add 10 times the amount of deionized water to the residue again, and repeat the above decoction process twice. Combine the three extracts, concentrate using a rotary evaporator at 60℃ under reduced pressure, and finally adjust the volume to 1g crude drug / mL, dispense and store at 4℃ for later use.

[0035] (2) Preparation of Gallnut Alcohol Extract: Weigh 50g of pretreated gallnut powder, add 10 times the volume of 80 vol.% ethanol solution, place in a round-bottom flask, connect a reflux condenser, and extract by reflux at 80℃ in a water bath. Each extraction time is 1h, and extraction is performed 3 times. Combine the extracts. Use a rotary evaporator to remove ethanol at 45℃ under reduced pressure. The concentrated extract is then diluted with deionized water to a volume of 1g crude drug / mL, dispensed, and stored in the dark and refrigerated.

[0036] (3) Preparation of Astragalus membranaceus water extract: Accurately weigh 50g of pretreated Astragalus membranaceus powder, add 10 times the amount of deionized water, and heat in an electric heating mantle for decoction. The initial decoction temperature is set at 100℃, and after boiling, maintain a gentle boil for 1 hour; filter to separate the residue and extract, add 10 times the amount of deionized water to the residue again, and repeat the above decoction process twice. Combine the three extracts, concentrate them using a rotary evaporator at 60℃ under reduced pressure, and finally adjust the volume to 1g crude drug / mL, dispense and store at 4℃ for later use.

[0037] (4) Preparation of Astragalus ethanol extract: Weigh 50g of pretreated Astragalus powder, add 10 times the volume of 80 vol.% ethanol solution, place in a round-bottom flask, connect a reflux condenser, and extract by reflux at 80℃ in a water bath. Each extraction time is 1h, and extraction is performed 3 times. Combine the extracts. Use a rotary evaporator to remove ethanol at 45℃ under reduced pressure. The concentrated extract is then diluted with deionized water to a volume of 1g crude drug / mL, dispensed, and stored in the dark and refrigerated.

[0038] (5) Preparation of Rhodiola rosea water extract: Accurately weigh 50g of pretreated Rhodiola rosea powder, add 10 times the amount of deionized water, and heat in an electric heating mantle for decoction. The initial decoction temperature is set at 100℃, and after boiling, maintain a gentle boil for 1 hour; filter to separate the residue and extract, add 10 times the amount of deionized water to the residue again, and repeat the above decoction process twice. Combine the three extracts, concentrate using a rotary evaporator at 60℃ under reduced pressure, and finally adjust the volume to 1g crude drug / mL, dispense and store at 4℃ for later use.

[0039] (6) Preparation of Rhodiola rosea ethanol extract: Weigh 50g of pretreated Rhodiola rosea powder, add 10 times the volume of 80 vol.% ethanol solution, place in a round-bottom flask, connect a reflux condenser, and extract by reflux at 80℃ in a water bath. Each extraction time is 1h, and extraction is performed 3 times. Combine the extracts. Use a rotary evaporator to remove ethanol at 45℃ under reduced pressure. The concentrated extract is then diluted with deionized water to a volume of 1g crude drug / mL, dispensed, and stored in the dark and refrigerated.

[0040] Testing the effects of different extracts of Galla chinensis, Astragalus membranaceus, and Rhodiola rosea on the immune activity of grouper fry.

[0041] The above-mentioned herbal extracts were fed to grouper fry in the form of feed. The activities of alkaline phosphatase (AKP), acid phosphatase (ACP), and superoxide dismutase (SOD) in the liver, as well as the expression levels of immune genes C3 and C4 in the spleen tissue, were measured to clarify the regulatory effects of different extracts on the immune function of grouper fry. The specific experiments are as follows:

[0042] 1. Experimental Materials

[0043] Experimental subjects: Healthy grouper fry of uniform size with an average weight of (20±0.2) g were selected, totaling 630 fish, purchased from the Yangjiang grouper breeding base.

[0044] The extraction methods for the experimental extracts, including Galla chinensis aqueous extract, Galla chinensis alcohol extract, Astragalus membranaceus aqueous extract, Astragalus membranaceus alcohol extract, Rhodiola rosea aqueous extract, and Rhodiola rosea alcohol extract, have been given in Example 1.

[0045] 2. Detection indicators

[0046] The activities of alkaline phosphatase (AKP), acid phosphatase (ACP), and superoxide dismutase (SOD) in the liver of grouper in each group, as well as the expression levels of immune genes C3 and C4 in spleen tissue.

[0047] 3. Experimental methods:

[0048] Experimental grouping and treatment: 630 grouper fry were randomly divided into 7 groups, including 1 control group and 6 experimental groups, with 3 replicates per group and 30 fish per replicate. The control group was fed ordinary commercial feed, while the experimental groups were fed feed mixed with different kinds of Chinese herbal extracts. The types and amounts of feed are shown in Table 1.

[0049] Table 1

[0050]

[0051] 4. Feeding and Management: The experiment was conducted in an indoor recirculating aquaculture system. The water temperature was controlled at (28±1)℃, the salinity at 28~30‰, the dissolved oxygen at ≥6mg / L, and the pH at 7.8~8.2. The fish were fed twice a day (8:00 and 18:00) at a rate of 3%~5% of their body weight. The experimental period was 7 days.

[0052] 5. Sample collection and index determination

[0053] Sample collection: After the experiment, 10 grouper fry were randomly selected from each group, and liver and spleen tissues were taken from each group. The tissues were then flash-frozen in liquid nitrogen and then transferred to a -80℃ freezer for storage.

[0054] Liver enzyme activity assay: Following the instructions of the kit, the activities of AKP, ACP and SOD in the liver were measured using a UV-Vis spectrophotometer.

[0055] Determination of spleen immune gene expression: Total RNA was extracted from spleen tissue using the Trizol method, and cDNA was synthesized by reverse transcription. Using cDNA as a template, the expression levels of immune genes C3 and C4 were determined by real-time quantitative PCR. β-actin was used as an internal reference gene, and the relative expression level was calculated using the 2^(-ΔΔCt) method.

[0056] 6. Experimental Results

[0057] (I) Effects of different extracts on the activities of AKP, ACP, and SOD in the liver of grouper fry

[0058] Table 2 shows the AKP, ACP, and SOD enzyme activities in the liver tissues of different groups of grouper samples:

[0059] Table 2

[0060]

[0061] Table 2 shows that, compared with the control group, the activities of AKP, ACP, and SOD in the liver of grouper fry in each extract-added group were increased to varying degrees, and the differences were statistically significant (P<0.05). Among them, the Astragalus ethanol extract group had the highest liver AKP activity, significantly higher than other extract groups (P<0.05); the Gallnut ethanol extract group had the highest liver ACP activity, significantly different from other groups (P<0.05); and the Rhodiola rosea ethanol extract group had the highest liver SOD activity, significantly higher than other groups (P<0.05). Overall, the water and ethanol extracts of the three traditional Chinese medicines could increase the activities of AKP, ACP, and SOD in the liver of grouper fry.

[0062] (II) Effects of different extracts on the expression levels of C3 and C4 in the spleen tissue of grouper fry

[0063] The expression levels of C3 and C4 in the spleen tissue of grouper fry by different extracts are shown in the figure. Figure 1 .

[0064] Figure 1In the table, control represents the control group, group 1 represents the Galla chinensis water extract group, group 2 represents the Galla chinensis alcohol extract group, group 3 represents the Astragalus membranaceus water extract group, group 4 represents the Astragalus membranaceus alcohol extract group, group 5 represents the Rhodiola rosea water extract group, and group 6 represents the Rhodiola rosea alcohol extract group. Compared with the control group, the expression levels of immune genes C3 and C4 in the spleen of grouper in each extract-added group were increased to varying degrees, and the differences were statistically significant (P<0.05).

[0065] Based on the above experiments, it can be concluded that the water extract of Galla chinensis, the alcohol extract of Galla chinensis, the water extract of Astragalus membranaceus, the alcohol extract of Astragalus membranaceus, the water extract of Rhodiola rosea, and the alcohol extract of Rhodiola rosea can all improve the immune activity of fish and can be applied to the prevention and control of aquatic animal diseases.

[0066] Example 2

[0067] Preparation of immune-enhancing compound and its safety experiment on live grouper.

[0068] The above-mentioned herbal extracts were combined with chlorogenic acid to obtain a compound immune enhancer, which was named "CIM Compound" for ease of description.

[0069] Preparation of chlorogenic acid solution: Take 10g of chlorogenic acid standard (purity ≥98%), dissolve it in 100mL of deionized water to prepare a chlorogenic acid solution of 0.1g / mL, dispense it into containers and store it in the dark and refrigerated.

[0070] All raw materials are compounded according to the proportions in Table 3, and the compounding proportions are as follows:

[0071] Table 3. Combination of Compound Immunostimulants

[0072]

[0073] To investigate the safety of CIM compound and analyze its effect on fish growth performance, this invention conducted tests on grouper. The experimental methods are as follows:

[0074] 1. Experimental materials

[0075] Experimental fish: 240 healthy grouper of similar size (approximately 45±5g in weight) were selected and purchased from a grouper farm in Yangjiang City, Guangdong Province. They were temporarily kept for 7 days before the experiment.

[0076] Experimental diet: The basic diet consisted of commercially available grouper-specific compound feed (Ruby brand produced by Guangdong Yuequn Marine Biotechnology Co., Ltd.). CIM compound was evenly mixed into the basic diet at a ratio of 10g / kg to make the experimental group diet; the control group was fed the basic diet without CIM compound.

[0077] Aquaculture equipment: Prepare several aquaculture tanks (100cm×80cm×80cm in size) and equip them with a circulating water system and aeration devices, etc.

[0078] 2. Feeding experiment of CIM compound

[0079] Two hundred and forty grouper fish were randomly divided into two groups, with three replicates per group and forty fish per replicate. The experimental group was fed a diet supplemented with CIM compound, while the control group was fed a basal diet. The experiment was conducted under the same culture conditions: water temperature maintained at 27±1℃, dissolved oxygen ≥5.5mg / L, and pH between 7.0 and 8.0. Fish were fed twice daily (09:00 and 15:00), with the amount of food given until the fish were about 80% full. Uneaten food was removed promptly, and the water was changed regularly, with one-quarter of the water replaced each time.

[0080] 3. Data Collection and Indicator Measurement

[0081] Health status: Observe the feeding, swimming and body characteristics of the grouper every day, and record any abnormalities and the number of deaths. After the experiment, 12 fish were randomly selected from each group and dissected to observe whether there were any lesions in the internal organs.

[0082] Growth indicators: At the beginning and end of the experiment, the grouper in each replicate group were weighed and the initial weight and final weight were recorded. The weight gain rate was then calculated using the following formula: Weight gain rate (%) = (final weight - initial weight) / initial weight × 100%.

[0083] 4. Experimental Results

[0084] Health status: Throughout the experiment, the survival rate of grouper in both the experimental and control groups was 100%. The grouper in the experimental group showed a strong appetite, good swimming condition, and no abnormalities such as injury or bleeding on their body surface. Dissection of their internal organs revealed no abnormalities in the spleen or kidneys, consistent with the control group. This indicates that feeding grouper with CIM compound at this ratio for 2 weeks did not have any adverse effects on the fish and is highly safe.

[0085] Growth performance: The initial weight of the control group was (44.7±1.1)g, the final weight was (58.2±1.8)g, and the weight gain rate was 30.2%; the initial weight of the experimental group was (45.3±1.2)g, and the final weight was (62.0±1.6)g, with a weight gain rate of 36.8%. It can be seen that feeding grouper with CIM compound at a feed mixing ratio of 10g / kg for 2 weeks can significantly improve the weight gain rate of grouper.

[0086] The efficacy of CIM compound in treating grouper infected with nerve necrosis virus was evaluated using the following experimental methods:

[0087] 1. Preparation of experimental materials

[0088] Grouper fry and grouping: 1000 healthy grouper fry with an average body length of 7cm±1 and an average weight of 15g±5 were selected for the experiment. The fry were purchased from a grouper fry farm in Yangjiang, Guangdong Province, and were temporarily raised for 7 days to acclimatize to the laboratory environment. No deaths or abnormal symptoms were observed during the temporary rearing period. After the temporary rearing period, 800 fry were randomly selected and divided into an experimental group and a control group, with 400 fry in each group, and were raised in the respective aquaculture systems.

[0089] 2. Infection experiment of grouper with nerve necrosis virus (RGNNV)

[0090] Neuronecrosis virus (RGNNV) was isolated from diseased fish tissue in our laboratory. Infection experiments were conducted on control and experimental group samples using intraperitoneal injection. The injection dose of RGNNV was 80 μL, and the viral titer was TCID50: 1 × 10⁻⁶. 5 Twenty-four hours after infection, eight samples from each of the control and experimental groups were randomly selected, and brain tissue was taken for RGNNV positivity testing. The results showed that the positivity rate in both groups was 100%, indicating that all samples were infected with the virus.

[0091] 3. Experimental study on the treatment of infected fish with CIM compound.

[0092] Thirty hours after the infection experiment, the control and experimental groups were fed. The control group was fed regular commercial feed, while the experimental group was fed regular commercial feed mixed with CIM compound at a ratio of 10g:1000g. Feeding was done twice daily at a rate of 1% each time for 14 consecutive days.

[0093] Pathogen positive detection: Starting from the second day of feeding, samples were taken from the control group and the experimental group every day. Ten fish were randomly selected from each group, and brain tissue was taken for RGNNV positive detection. The detection was carried out for 14 consecutive days, and the daily positive rate was counted.

[0094] Positive detection experiments were performed using qRT-PCR. The specific steps were as follows: Total RNA was extracted from tissues using a kit, and the RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions. Using the cDNA as a template, qRT-PCR amplification was performed using specific primers for RGNNV. The reaction volume was 25 μL, including 12.5 μL 2×SYBR Green PCR Master Mix, 0.8 μL upstream primer (10 μM), 0.8 μL downstream primer (10 μM), 1.5 μL cDNA template, and 9.4 μL LNase-Free Water. The reaction conditions were: 95℃ pre-denaturation for 40 s; 95℃ denaturation for 8 s; 58℃ annealing for 35 s, for a total of 38 cycles. A Ct value less than 35 was considered positive, and the pathogen positivity rate was calculated based on the Ct value (number of positive individuals / number of tested individuals × 100%).

[0095] The positive rate statistics during the experiment are shown in Table 4, and the positive rate statistics chart is shown in [the original text]. Figure 2 .

[0096] Table 4

[0097]

[0098] From Table 4 and Figure 2 The results showed that in grouper infected with nerve necrosis virus, after two weeks of treatment with this compound, the virus positivity rate dropped to 20%, which was significantly lower than that of the control group (60%).

[0099] An experiment was conducted to investigate the immunoprotective effect of CIM compound against mixed infection of grouper with neuronecrosis virus and Vibrio harveyi. The experimental method is as follows:

[0100] 1. Preparation of experimental materials

[0101] Grouper and Grouping: 300 healthy groupers with an average body length of 9cm ± 1 and an average weight of 25g ± 5 were purchased from a grouper hatchery in Yangjiang City, Guangdong Province. They were temporarily housed for 10 days before the experiment to acclimatize to the laboratory environment. No deaths or abnormal symptoms were observed during this period. After the temporary housing period, the groupers were randomly divided into two groups of 150 each, and housed in separate rearing systems. One group served as the control group, and the other as the experimental group.

[0102] Experimental feed: The control group feed was conventional commercial feed; the experimental group feed was conventional commercial feed mixed with CIM compound. The mixing ratio of CIM compound was 1g:100g, that is, 1g of CIM compound was mixed into every 100g of conventional commercial feed and used after being thoroughly mixed.

[0103] Pathogen preparation: Neuronecrosis virus (RGNNV) and Vibrio harveyi (VH) were both isolated and purified from diseased grouper tissue in our laboratory. The RGNNV titer was adjusted to TCID50 after culture: 1×10⁻⁶. 6 The concentration of Vibrio harveyi was adjusted to 1×10 after culturing. 7 CFU / ml, store at 4℃ for later use.

[0104] 2. Immunization feeding experiment

[0105] Two groups of grouper were fed: the control group was fed conventional commercial feed, while the experimental group was fed a feed mixed with CIM compound. Feeding was done twice daily (8:30 AM and 4:30 PM), at a rate of 2.5% of the fish's body weight, ensuring sufficient food intake. Feeding continued for two weeks, during which uneaten feed and feces were regularly removed to maintain clean water and consistent environmental conditions: water temperature 28±1℃, dissolved oxygen ≥6mg / L, pH 7.5~8.5, and 1 / 3 of the water was changed daily.

[0106] 3. Mixed infection experiment

[0107] Two weeks after feeding, two groups of grouper were subjected to a mixed infection experiment with neuronecrosis virus (RGNNV) and Vibrio harveyi. Infection was performed via intraperitoneal injection, with each grouper receiving 100 μL of RGNNV and 100 μL of Vibrio harveyi (VH). After infection, both groups of grouper were placed back into their original culture system, maintaining the same environmental conditions.

[0108] 4. Data recording and observation

[0109] After infection, the activity, feeding, and mortality of the two groups of grouper were observed daily at regular intervals, and the number of deaths in each group was recorded in detail. If any dead individuals were found, they were promptly removed and the time of death was recorded. The observation continued for 14 days.

[0110] 5. Experimental Results

[0111] The observation results during the 14-day observation period are shown in Table 5, and the cumulative mortality rate over time is shown in Table 6. Figure 3 .

[0112] Table 5

[0113]

[0114] As shown in Table 5, there was a significant difference in mortality between the control group and the experimental group. In the control group, grouper began to die on the second day after infection, and the number of deaths gradually increased, reaching a total of 53 deaths by the 14th day, with a cumulative mortality rate of 35.33%. In the experimental group, a small number of grouper died on the fourth day after infection, and the number of deaths increased more slowly, reaching a total of 19 deaths by the 14th day, with a cumulative mortality rate of 12.67%.

[0115] From Table 5 and Figure 3 The results showed that after feeding healthy grouper with the compound feed for two weeks, the mortality rate was significantly lower than that of the control group (35.33% in the control group and 12.67% in the compound group). This indicates that the CIM compound has a good immunoprotective effect in dealing with mixed viral and bacterial infections.

[0116] The application experiment of CIM compound in pond aquaculture was conducted using the following methods:

[0117] 1. Materials and Methods

[0118] Application trials of CIM compound were conducted in earthen ponds for California bass farming in Foshan, crucian carp farming in Nansha, and Litopenaeus vannamei farming in Jiangmen. The CIM compound was mixed thoroughly with the feed at a dosage of 1 wt.% and fed twice daily at 8:30 AM and 3:30 PM for 45 consecutive days. During this period, the disease incidence, mortality, and growth of aquatic animals in each experimental pond were recorded in detail.

[0119] 2. Results

[0120] During the experiment, fish-killing Edwardsiella pneumoniae infection occurred in the soil ponds used for California bass farming in Foshan. The mortality rate in the ponds without CIM compound treatment reached 18%, while the mortality rate in the ponds using CIM compound treatment was only 3%. Aeromonas hydrophila infection occurred in the soil ponds used for crucian carp farming in Nansha. The mortality rate in the ponds without CIM compound treatment was 15%, while the mortality rate in the ponds using CIM compound treatment was controlled below 4%. Vibrio parahaemolyticus was detected in the soil ponds used for Litopenaeus vannamei farming in Jiangmen. The mortality rate in the ponds without CIM compound treatment was 20%, while the mortality rate in the ponds using CIM compound treatment was 5%. Meanwhile, in the ponds using CIM compound treatment, California bass, crucian carp, and Litopenaeus vannamei all showed good overall vitality and vigorous feeding. Among them, California bass and crucian carp showed significant weight gain, and Litopenaeus vannamei showed higher uniformity in size and growth rate than the untreated group.

[0121] 3. Conclusion

[0122] When CIM compound is added to pond culture of California bass, crucian carp and whiteleg shrimp, it can effectively improve their resistance to pathogens such as Edwardsiella piscicola, Aeromonas hydrophila, and Vibrio parahaemolyticus, reduce the mortality rate after infection and onset, and also play a positive role in promoting the growth and improving the growth performance of aquatic animals, showing good protective effects in pond culture.

[0123] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A complex immune enhancer, characterized in that, The compound immune enhancer contains water extract of gallnut, alcohol extract of gallnut, water extract of Astragalus membranaceus, alcohol extract of Astragalus membranaceus, water extract of Rhodiola, alcohol extract of Rhodiola and chlorogenic acid.

2. The compound immunopotentiator according to claim 1, characterized in that, The compound immune enhancer contains 0.2 g of water extract of gallnut, 0.2 g of alcohol extract of gallnut, 0.2 g of water extract of Astragalus membranaceus, 0.2 g of alcohol extract of Astragalus membranaceus, 0.05 g of water extract of Rhodiola, 0.05 g of alcohol extract of Rhodiola and 0.01 g of chlorogenic acid per mL of the compound immune enhancer.

3. The compound immunopotentiator according to claim 1, characterized in that, The extract solution of the alcohol extract of gallnut is 80% ethanol solution by volume fraction; the extract solution of the alcohol extract of Astragalus membranaceus is 80% ethanol solution by volume fraction; and the extract solution of the alcohol extract of Rhodiola is 80% ethanol solution by volume fraction.

4. The method for preparing the compound immunopotentiator according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: The water extract of gallnut, the water extract of Astragalus membranaceus and the water extract of Rhodiola are obtained by water extraction of gallnut, Astragalus membranaceus and Rhodiola respectively; the alcohol extract of gallnut, the alcohol extract of Astragalus membranaceus and the alcohol extract of Rhodiola are obtained by alcohol solution extraction of gallnut, Astragalus membranaceus and Rhodiola respectively; and the water extract of gallnut, the water extract of Astragalus membranaceus, the water extract of Rhodiola, the alcohol extract of gallnut, the alcohol extract of Astragalus membranaceus, the alcohol extract of Rhodiola and the chlorogenic acid are mixed to obtain the compound immune enhancer.

5. The production method according to claim 4, characterized by, The gallnut, Astragalus membranaceus and Rhodiola are all 40-mesh powder.

6. The preparation method according to claim 4, characterized in that, The temperature of water extraction is 100 ℃; and the temperature of alcohol extraction is 80 ℃.

7. The production method according to claim 6, wherein The water extraction is three times of extraction, and the extraction liquid is combined after extraction; the addition amount of the extraction liquid is 10 times of the mass of the traditional Chinese medicinal material each time, and the extraction time is 1 h; the alcohol extraction is three times of extraction, and the extraction liquid is combined after extraction; the addition amount of the extraction liquid is 10 times of the mass of the traditional Chinese medicinal material each time, and the extraction time is 1 h.

8. Application of the compound immune enhancer of any one of claims 1-3 in aquaculture.

9. Use according to claim 8, characterized in that, The compound immune enhancer is used as a feed additive.

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