Echinacea purpurea extract as well as preparation method and application thereof

Abstract

The invention provides an echinacea extract as well as a preparation method and application thereof. The extraction process of the echinacea purpurea extract is optimized through single factor and response surface experiments, the echinacea purpurea extract in which the ratio of the chicoric acid to the mono-caffeoyl tartaric acid can reach 34.1% is prepared, and the ratio of the chicoric acid can reach 20.8%. The prepared echinacea purpurea extract can remarkably improve the weight gain rate and the specific growth rate of largemouth bass, meanwhile, the survival rate and the disease resistance of the largemouth bass against aeromonas veronii infection can be improved, the risk of drug residues is avoided, and the echinacea purpurea extract is suitable for green aquaculture.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to a purple echinacea extract, its preparation method, and its application. Background Technology

[0002] Largemouth bass (Micropterus salmoides), a high-quality freshwater economic fish, saw an annual output of 888,000 tons and a value exceeding 35 billion yuan in my country in 2023. However, its intensive aquaculture faces severe challenges from bacterial diseases. Aeromonas veronii-induced ulceration, septicemia, and enteritis lead to mass mortality, hindering the industry's development. Antibiotics are widely used in the prevention and control of bacterial diseases in aquaculture, playing a crucial role in controlling disease transmission and increasing yields. However, long-term, excessive, and irrational use of antibiotics has led to problems such as drug resistance, drug residues, and ecological pollution, endangering food safety and human health. Therefore, developing safe, effective, and environmentally friendly antibiotic alternatives is urgently needed. Echinacea purpurea contains abundant polyphenols, alkylamides, and polysaccharides, but its application in aquaculture is limited, especially in largemouth bass, where no reports have been found. Research on the growth-promoting and disease-controlling effects of Echinacea purpurea in largemouth bass is crucial for developing green feed additives, ensuring sustainable industrial development, and safeguarding food security. Summary of the Invention

[0003] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide a echinacea extract, its preparation method, and its application. This invention, by optimizing the echinacea extract extraction process, provides a safe and efficient echinacea extract feed additive for largemouth bass, which can significantly improve the growth performance, survival rate, and disease resistance of largemouth bass.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] The first aspect of the present invention provides a method for preparing echinacea extract, characterized in that the preparation method includes the following steps: adding ethanol to echinacea and performing ultrasonic extraction to obtain the echinacea extract; the ultrasonic extraction temperature is 40-60°C; the ultrasonic extraction power is 280-420W; and the ultrasonic extraction time is 30-60 minutes.

[0006] In some embodiments of the present invention, the step further includes purifying the product after ultrasonic extraction.

[0007] In some embodiments of the present invention, the purification step includes filtering the product after ultrasonic extraction at room temperature, centrifuging to obtain the supernatant, concentrating under reduced pressure using a rotary evaporator, and freeze-drying to obtain the echinacea extract.

[0008] In some embodiments of the present invention, the ambient temperature is 20–25°C.

[0009] In some embodiments of the present invention, the ultrasonic extraction is performed 1 to 3 times.

[0010] In some embodiments of the present invention, the power of the ultrasonic extraction is 300-400W.

[0011] In some embodiments of the present invention, the power of the ultrasonic extraction is 340-360W.

[0012] In some embodiments of the present invention, the power of the ultrasonic extraction is 350W.

[0013] In some embodiments of the present invention, the ultrasonic extraction time is 30 to 45 minutes.

[0014] In some embodiments of the present invention, the ultrasonic extraction time is 35 to 40 minutes.

[0015] In some embodiments of the present invention, the ultrasonic extraction time is 37 minutes.

[0016] In some embodiments of the present invention, the temperature of the ultrasonic extraction is 45–55°C.

[0017] In some embodiments of the present invention, the temperature of the ultrasonic extraction is 50-55°C.

[0018] In some embodiments of the present invention, the temperature of the ultrasonic extraction is 50°C.

[0019] In some embodiments of the present invention, the amount of ethanol added to echinacea is 300-500 ml of ethanol per gram of echinacea.

[0020] In some embodiments of the present invention, the amount of ethanol added to echinacea is 350-450 ml of ethanol per gram of echinacea.

[0021] In some embodiments of the present invention, the amount of ethanol added to echinacea is 380-420 ml of ethanol per gram of echinacea.

[0022] In some embodiments of the present invention, the amount of ethanol added to echinacea is 400 ml of ethanol per gram of echinacea.

[0023] In some embodiments of the present invention, the concentration of the ethanol is 30%-50%.

[0024] In some embodiments of the present invention, the concentration of the ethanol is 30%-40%.

[0025] In some embodiments of the present invention, the concentration of the ethanol is 35%-40%.

[0026] In some embodiments of the present invention, the concentration of the ethanol is 36%.

[0027] In some embodiments of the present invention, the number of ultrasound sessions is 1 to 3.

[0028] In some embodiments of the present invention, the number of ultrasound sessions is 1 to 2.

[0029] In some embodiments of the present invention, the number of ultrasound sessions is 1.

[0030] In some embodiments of the present invention, the echinacea is the whole plant of echinacea or a mixture of one or more of the stems, leaves, flowers and roots.

[0031] In some embodiments of the present invention, the echinacea is dried, pulverized, and sieved.

[0032] In some embodiments of the present invention, the sieving is performed through a 50-70 mesh sieve.

[0033] In some embodiments of the present invention, the sieving is performed through a 60-mesh sieve.

[0034] In some embodiments of the present invention, the centrifugation conditions are 8000-12000 rpm for 5-15 minutes.

[0035] In some embodiments of the present invention, the centrifugation conditions are 10,000 rpm for 10 minutes.

[0036] In some embodiments of the present invention, the temperature for vacuum concentration in the rotary evaporator is 55–65°C.

[0037] In some embodiments of the present invention, the temperature for vacuum concentration in the rotary evaporator is 60°C.

[0038] In some embodiments of the present invention, the rotary evaporator is used for vacuum concentration at a speed of 50 to 150 rpm.

[0039] In some embodiments of the present invention, the rotary evaporator is used for vacuum concentration at a speed of 100 rpm.

[0040] In some embodiments of the present invention, the rotary evaporator is used for vacuum concentration for 15 to 45 minutes.

[0041] In some embodiments of the present invention, the rotary evaporator is used for vacuum concentration for 20 to 40 minutes.

[0042] In some embodiments of the present invention, the rotary evaporator is used for vacuum concentration for 30 minutes.

[0043] In a second aspect, the present invention provides an echinacea extract prepared by the above preparation method, wherein the echinacea extract contains ≥30% chicoric acid and monocaffeoyl tartaric acid.

[0044] In some embodiments of the present invention, the content of chicoric acid and monocaffeoyl tartaric acid in the echinacea extract is 30-45%.

[0045] In some embodiments of the present invention, the chicoric acid content in the echinacea extract is ≥20%.

[0046] In some embodiments of the present invention, the chicoric acid content in the echinacea extract is 20-30%.

[0047] In some embodiments of the present invention, the content of monocaffeoyl tartaric acid in the echinacea extract is ≥10%.

[0048] In some embodiments of the present invention, the content of monocaffeoyl tartaric acid in the echinacea extract is ≥13%.

[0049] In some embodiments of the present invention, the content of monocaffeoyl tartaric acid in the echinacea extract is 13-20%.

[0050] In some embodiments of the present invention, the echinacea extract further contains at least one of the following: 1-3% naringenin, 1-3% rutin, 1-3% quercetin, 0.1-1% chlorogenic acid, 0.1-0.5% caffeic acid, and 0.01-0.1% ferulic acid.

[0051] A third aspect of the present invention provides the application of the above-mentioned echinacea extract in improving the growth performance of largemouth bass.

[0052] In some embodiments of the present invention, the improvement in growth performance includes improving weight gain or a specific growth rate.

[0053] A fourth aspect of the present invention provides the use of the above-mentioned echinacea extract in the preparation of products for the prevention and / or treatment of Aeromonas vesiculosus infection in largemouth bass.

[0054] In some embodiments of the present invention, the product includes feed, feed additives, or pharmaceuticals.

[0055] In some embodiments of the present invention, the application includes adding the above-mentioned echinacea extract at a concentration of 450-1500 mg / kg to feed.

[0056] In some embodiments of the present invention, the application includes adding the above-mentioned echinacea extract at a concentration of 750-1050 mg / kg to feed.

[0057] In some embodiments of the invention, the prevention and / or treatment include improving the survival rate of largemouth bass infected with Aeromonas vesiculosus and reducing the Aeromonas vesiculosus load.

[0058] In some embodiments of the present invention, the prevention and / or treatment also include alleviating symptoms such as surface bleeding, abdominal swelling, and ulceration caused by Aeromonas verrucosa infection in fish.

[0059] A fifth aspect of the present invention provides a method for improving the growth performance of largemouth bass, the method comprising feeding largemouth bass with the above-mentioned echinacea extract.

[0060] In some embodiments of the present invention, the improvement in growth performance includes an increase in weight gain or a specific growth rate.

[0061] The beneficial effects of this invention are:

[0062] This invention provides a method for preparing echinacea extract. The extraction process of echinacea extract was optimized through single-factor and response surface methodology experiments. The yield of the prepared echinacea extract can reach 15.2%, and the proportion of chicoric acid and monocaffeoyl tartaric acid in the obtained echinacea extract can reach 34.1%, with chicoric acid accounting for 20.8% and monocaffeoyl tartaric acid accounting for 13.3%. The extraction process of the echinacea extract is simple to operate, low in cost, and easy to promote.

[0063] This invention provides a echinacea extract prepared by an optimized method. Feeding largemouth bass with this echinacea extract significantly improves their weight gain and specific growth rate, with a maximum weight gain of 315.25% and a specific growth rate of 42.20%. It also significantly improves the survival rate of largemouth bass infected with Aeromonas vulgaris, significantly reduces the bacterial load in the liver and intestines, and alleviates symptoms such as external bleeding, abdominal swelling, and ulceration caused by Aeromonas vulgaris infection. The echinacea extract poses no risk of drug residue when fed to largemouth bass, meets green aquaculture standards, and is of great significance for the prevention or treatment of Aeromonas vulgaris infection in largemouth bass. Attached Figure Description

[0064] Figure 1 The figure shows the results of a single-factor optimization experiment on the weight of echinacea powder and the volume of ethanol for optimizing the process of echinacea extract. In the figure, a, b, c, d, and e, as well as the combinations of letters ab, bc, and cd, represent the statistical differences of the corresponding results. If the letters marked on the two groups are the same or the combination of letters in one group contains letters from the other group, it means that the two values ​​are not statistically significant. Otherwise, they are statistically significant.

[0065] Figure 2 Figure 1 shows the results of a single-factor optimization experiment to optimize the ethanol concentration in the process of echinacea extract. In the figure, a, b, c, d, and e, as well as the combination letters ab, represent the statistical differences of the corresponding results. If the letters marked on the two groups are the same or the combination letters of one group contain letters from the other group, it means that the two values ​​are not statistically significant. Otherwise, they are statistically significant.

[0066] Figure 3 The figure shows the results of a single-factor optimization experiment to optimize the extraction temperature of Echinacea extract. In the figure, a, b, c, and d, as well as the combinations of letters ab, bc, and abc, represent the statistical differences of the corresponding results. If the letters marked on the two groups are the same or the combination of letters in one group contains letters from the other group, it means that the two values ​​are not statistically significant. Otherwise, they are statistically significant.

[0067] Figure 4 The figure shows the results of a single-factor optimization experiment to optimize the extraction time of Echinacea extract. In the figure, a, b, and the combination of letters ab represent the statistical differences of the corresponding results. If the letters marked on the two groups are the same or the combination of letters in one group contains letters from the other group, it means that the two values ​​are not statistically significant. Otherwise, they are statistically significant.

[0068] Figure 5 Figure 1 shows the results of a single-factor optimization experiment to optimize the ultrasonic power for the processing of Echinacea extract. In the figure, a, b, c and the combination letters ab and bc represent the statistical differences of the corresponding results. If the letters marked on the two groups are the same or the combination letters of one group contain letters from the other group, it means that the two values ​​are not statistically significant. Otherwise, they are statistically significant.

[0069] Figure 6 Figure 1 shows the results of a single-factor optimization experiment to optimize the number of extractions for Echinacea extract. In the figure, 'a' represents the statistical difference of the corresponding result, and the two sets of letters indicate that the two values ​​are not statistically significant.

[0070] Figure 7 The response surface methodology results for optimizing the process of echinacea extract by adjusting ethanol concentration and extraction time are shown in the figure.

[0071] Figure 8 The results of the response surface methodology for optimizing the process of echinacea extract by adjusting ethanol concentration and extraction temperature are shown in the figure.

[0072] Figure 9 The results of the response surface methodology for optimizing the extraction time and temperature of Echinacea extract are shown in the figure.

[0073] Figure 10 This is a bar chart showing the content ratio of the main components of Echinacea extract.

[0074] Figure 11 This is a schematic diagram illustrating the effect of echinacea extract on the clinical symptoms and pathological features of largemouth bass in an experiment demonstrating bacterial challenge.

[0075] Figure 12 The figure shows the effect of echinacea extract on the mortality rate of largemouth bass in the bacterial challenge experiment of the example.

[0076] Figure 13 The figure shows the effect of echinacea extract on the intestinal bacterial load of largemouth bass in the challenge experiment of the example; in the figure, Aeromonas represents Aeromonas veronii.

[0077] Figure 14 The figure shows the effect of echinacea extract on the bacterial load in the liver of largemouth bass in the challenge experiment of the example; in the figure, Aeromonas represents Aeromonas veronii. Detailed Implementation

[0078] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0079] Example 1: Extraction process of Echinacea extract

[0080] This embodiment provides an extraction process for echinacea extract. The echinacea was obtained from the echinacea germplasm resource nursery of South China Agricultural University (23°9′55″N; 113°21′6″E). The above-ground parts of the annual echinacea, namely the stems, leaves and flowers, were harvested on June 20, 2022. The specific steps are as follows.

[0081] (1) Wash the purple echinacea, air dry for 24 hours, then transfer it to an oven at 60°C to dry to constant weight. Dry the above-ground parts of the purple echinacea, crush them, and pass them through a 60-mesh sieve for later use.

[0082] (2) Take the above-mentioned pulverized and sieved Echinacea powder (g) and ethanol volume (mL) at a ratio of 1:400, add 30% ethanol to Echinacea, and perform ultrasonic extraction for 40 min at a power of 350W and a temperature of 50℃.

[0083] (3) The obtained product was filtered at room temperature and centrifuged at 10,000 rpm for 10 min. The supernatant was then collected.

[0084] (4) The supernatant obtained in step (3) was concentrated under reduced pressure for 30 minutes using a rotary evaporator at 60°C and 100 rpm, and then freeze-dried to obtain echinacea extract, which was stored at -20°C.

[0085] (5) The components of Echinacea extract were detected by HPLC, and the main polyphenol components were quantitatively analyzed.

[0086] Example 2: Optimization of Echinacea Extract Extraction Process

[0087] This embodiment provides an optimization of the extraction process of echinacea extract from Example 1. Single-factor and response surface methodology were used to optimize the process conditions affecting the extraction rate of echinacea polyphenols. Details are as follows.

[0088] A single-factor optimization experiment was conducted on the echinacea powder weight to ethanol volume ratio, ethanol concentration, ultrasonic extraction temperature, ultrasonic extraction time, ultrasonic power, and number of ultrasonic extractions in the echinacea extract extraction process of Example 1. The specific experimental design is as follows:

[0089] (1) Effect of material-liquid ratio (g of echinacea powder / mL of ethanol) on the extraction rate of echinacea polyphenols: The experiment was conducted at material-liquid ratios of 1:40, 1:80, 1:100, 1:200, 1:300, 1:400 and 1:500.

[0090] (2) Effect of ethanol concentration (%) on the extraction rate of Echinacea polyphenols: Experiments were conducted using ethanol solutions of different concentrations (0, 10%, 30%, 50%, 70%, 90%).

[0091] (3) Effect of extraction temperature (°C) on the extraction rate of Echinacea polyphenols: Experiments were conducted under different extraction temperatures (30°C, 40°C, 50°C, 60°C, 70°C, 80°C).

[0092] (4) Effect of extraction time (min) on the extraction rate of Echinacea polyphenols: Experiments were conducted under different extraction times (5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min).

[0093] (5) Effect of ultrasonic power (W) on the extraction rate of polyphenols from Echinacea purpurea: Experiments were conducted under different ultrasonic powers (280W, 350W, 420W, 490W, 560W, 630W).

[0094] (6) Effect of extraction times on the extraction rate of Echinacea polyphenols: Experiments were conducted under different ultrasonic times (1, 2, 3 times).

[0095] The results of the single-factor optimization experiment are as follows Figure 1-6 As shown, Figure 1 The results of the single-factor optimization experiment on the solid-liquid ratio of echinacea powder weight to ethanol volume for the extraction process of echinacea extract showed that the total polyphenol content and the contents of chicoric acid and monocaffeoyl tartaric acid were better in the solid-liquid ratio (echinacea powder g / ethanol volume mL) of 1:400 and 1:500 than in other groups. Figure 2 The results of the single-factor optimization experiment on the extraction process of Echinacea extract with ethanol concentration show that the total polyphenol content and the contents of chicoric acid and monocaffeoyl tartaric acid are better than those of other groups when the ethanol concentration is 30% and 50%. Figure 3 The results of the single-factor optimization experiment on extraction temperature of Echinacea extract showed that the total polyphenol content was better at extraction temperatures of 50℃, 60℃, 70℃ and 80℃ than in other groups. Figure 4 The results of the single-factor optimization experiment on the extraction time of Echinacea extract showed that the total polyphenol content and the contents of chicoric acid and monocaffeoyl tartaric acid were lower than those of other groups when the extraction time was 5 minutes. The total polyphenol content and the contents of chicoric acid and monocaffeoyl tartaric acid were not significantly different when the extraction time was 10, 20, 30, 40, 50 and 60 minutes. Figure 5 The results of the single-factor optimization experiment of ultrasonic power in the extraction process of Echinacea extract show that for an ultrasonic power of 700W, the total polyphenol content, chicoric acid, and monocaffeoyl tartaric acid content are all higher when the ultrasonic power is 350W (50% of the total power). Figure 6 The results of a single-factor optimization experiment on the number of extractions in the extraction process of Echinacea extract show that the number of extractions has little effect on the total polyphenol content, chicoric acid, and monocaffeoyl tartaric acid content.

[0096] Based on the results of the single-factor experiments above, the extraction effects of ethanol concentration, ultrasonic power, material-to-liquid ratio, extraction temperature, extraction time, and number of ultrasonic cycles were analyzed. Finally, ethanol concentration, extraction temperature, and extraction time were selected as the three factors with relatively large influence on polyphenol yield for response surface methodology. Based on the three selected significant factors, an experimental design was designed according to the Box-Behnken design principle, as shown in Table 1 below.

[0097] Table 1 Response Surface Experimental Design

[0098]

[0099] Meanwhile, since the total polyphenol content showed a consistent trend with the content of chicoric acid and monocaffeoyl tartaric acid, the total polyphenol content was subsequently selected as the indicator component.

[0100] Based on the response surface methodology results, the extraction model was established as follows: Y = 18.46 - 1.52A + 0.20B - 0.43C + 0.1017AB - 0.2375AC - 0.1494BC - 0.7332A 2 -0.1149B 2 +0.4077C 2 Where Y is the total polyphenol yield, A is the ethanol concentration, B is the ultrasonic extraction time, and C is the ultrasonic extraction temperature. The model was tested using regression equation variance analysis, and the test results are shown in Table 2 below. The established model is significant overall, the model fits the data well, and it can explain the changes in the response variable.

[0101] Table 2. Analysis of Variance for Regression Equations

[0102]

[0103]

[0104] Response surface methodology results are as follows Figure 7-9 As shown, Figure 7 The results are from a response surface methodology experiment on the relationship between ethanol concentration and extraction time. Figure 8 The results are from a response surface methodology experiment on the relationship between ethanol concentration and extraction temperature. Figure 9 Based on the response surface methodology results of extraction time and temperature, the optimal levels of each influencing factor for the optimized extraction process of echinacea polyphenols were determined: echinacea powder to ethanol volume ratio of 1:400, ultrasonic extraction temperature of 50℃, ultrasonic extraction power of 350W, ultrasonic extraction time of 37min, ethanol concentration of 36%, and ultrasonic extraction once.

[0105] Example 3: Component Analysis of Echinacea Extract

[0106] According to Example 2, the optimized Echinacea was extracted using this process, and the specific steps are as follows.

[0107] (1) Wash the purple echinacea, air dry for 24 hours, then transfer it to an oven at 60°C to dry to constant weight. Dry the above-ground parts of the purple echinacea, crush them, and pass them through a 60-mesh sieve for later use.

[0108] (2) Take the above-mentioned pulverized and sieved Echinacea powder (g) and ethanol volume (mL) at a ratio of 1:400, add 36% ethanol to Echinacea, and perform ultrasonic extraction for 37 minutes at a power of 350W and a temperature of 50℃.

[0109] (3) The obtained product was filtered at room temperature and centrifuged at 10,000 rpm for 10 min. The supernatant was then collected.

[0110] (4) The supernatant obtained in step (3) is concentrated under reduced pressure for 30 minutes using a rotary evaporator at 60°C and 100 rpm, and then freeze-dried to obtain echinacea extract, which is stored at -20°C.

[0111] (5) The components of Echinacea extract were detected by HPLC, and the main polyphenol components were quantitatively analyzed.

[0112] The final yield of Echinacea extract was 15.2%. The results of HPLC quantitative analysis of the extract are as follows: Figure 10 As shown, chicoric acid and monocaffeoyl tartaric acid are the most abundant components of echinacea extract, accounting for 34.1% of the total extract. Specifically, chicoric acid accounts for 20.8% and monocaffeoyl tartaric acid for 13.3%. Other components include naringenin (2.0%), rutin (1.9%), quercetin (1.3%), chlorogenic acid (0.45%), caffeic acid (0.15%), and ferulic acid (0.08%). This indicates that chicoric acid and monocaffeoyl tartaric acid are likely the main active ingredients in echinacea extract.

[0113] Example 4: Effects of different concentrations of Echinacea extract on growth performance and disease resistance of largemouth bass

[0114] This embodiment demonstrates the effects of different concentrations of Echinacea extract on the growth performance and resistance to Aeromonas veronii infection in largemouth bass. The largemouth bass (6-8cm; 5.0±0.5g) used in this embodiment were purchased from Guangzhou Huanan Aquatic Seedling Co., Ltd. The strain used in the challenge experiment was Aeromonas veronii, derived from the Aquatic Disease Research Laboratory of the Animal Health Institute, Guangdong Academy of Agricultural Sciences. All animal husbandry and experimental procedures were approved by the Animal Ethics Center Committee of South China Agricultural University and complied with animal ethics and welfare guidelines. Specific experimental steps are as follows.

[0115] 1. The echinacea extract obtained from the optimized process in Example 3 was dissolved in water and added to the commercial feed of largemouth bass at concentration gradients of 450, 750, 1050, and 1500 mg / kg. The feed was then dried to produce echinacea extract additive feed. The commercial feed of largemouth bass was purchased from Wuxi Tongwei Biotechnology Co., Ltd., Jiangsu Province (Product Standard No.: Q / 320205KAEM01-2021).

[0116] 2. After two weeks of acclimatization rearing, a group experiment was conducted. 420 healthy, disease-free, and uninjured largemouth bass were randomly divided into 6 groups, with 3 replicates per group and 20 fish per replicate. Specific feeding treatments for different groups are as follows:

[0117] (1) Control group (CK): After being fed with basal feed for 8 weeks, 0.2 mL of sterile PBS solution was injected intraperitoneally.

[0118] (2) Challenge group (ACK): After being fed a basal diet for 8 weeks, 0.2 mL of 2.5 × 10⁻⁶ bacteria were injected intraperitoneally. 9 A. veronii bacterial suspension at CFU / mL;

[0119] (3) Echinacea extract challenge group 1 (AEP1): After feeding with a basal diet containing echinacea extract (EP1: 450 mg / kg) for 8 weeks, 0.2 mL of 2.5 × 10⁻⁶ mg / kg of echinacea extract was injected intraperitoneally. 9 A. veronii bacterial suspension at CFU / mL;

[0120] (4) Echinacea extract challenge group 2 (AEP2): After feeding with a basal diet containing echinacea extract (EP2: 750 mg / kg) for 8 weeks, 0.2 mL of 2.5 × 10⁻⁶ mg / kg of echinacea extract was injected intraperitoneally. 9 A. veronii bacterial suspension at CFU / mL;

[0121] (5) Echinacea extract challenge group 3 (AEP3): After feeding with a basal feed containing echinacea extract (EP3: 1050 mg / kg) for 8 weeks, 0.2 mL of 2.5 × 10⁻⁶ mg / kg of echinacea extract was injected intraperitoneally. 9 A. veronii bacterial suspension at CFU / mL;

[0122] (6) Echinacea extract challenge group 4 (AEP4): After feeding with a basal diet containing echinacea extract (EP4: 1500 mg / kg) for 8 weeks, 0.2 mL of 2.5 × 10⁻⁶ mg / kg of echinacea extract was injected intraperitoneally. 9 A. veronii bacterial suspension at CFU / mL.

[0123] (7) Chicoric acid challenge group (ACA): After feeding with chicoric acid (CA: 100 mg / kg) mixed with basal fish feed for 8 weeks, 0.2 mL of chicoric acid (CA: 100 mg / kg) was injected intraperitoneally. 9 A. veronii bacterial suspension at CFU / mL;

[0124] All largemouth bass in the experimental groups were fed twice every 24 hours, at 9:00 and 17:00, with a feed amount of approximately 4% of the fish's body weight. During the rearing period, the water flow rate in each circular tank (diameter: 1.2m; height: 1.0m) was set at 800L / h. The water temperature was maintained at 26.0±1.0℃, pH at 7.4-7.8, and dissolved oxygen at 6.0mg / L to ensure good water quality. After 8 weeks of rearing, four fish from each experimental group were taken for weight and length measurements, and the weight gain rate and specific growth rate were calculated. Forty-eight hours after injection of bacterial solution, the survival rate of each group was calculated. Simultaneously, four fish from each tank were anesthetized with eugenol, and appropriate amounts of liver and intestines were accurately weighed and soaked in 0.9% physiological saline, stored at 4℃. The formulas for weight gain rate, survival rate, and specific growth rate are shown below:

[0125]

[0126] Subsequently, the bacterial load of Aeromonas vera in the liver and intestines of largemouth bass in each experimental group was statistically analyzed. The specific experimental steps are as follows:

[0127] (1) After accurately weighing the liver and intestinal samples, add steel balls and grind them in a tissue grinder (30s, 60Hz, 5 times) until they are uniformly ground.

[0128] (2) After grinding and mixing, the sample was diluted 10, 100, 1000 and 10000 times with sterile 0.9% physiological saline in a sterile laminar flow hood. The samples of each dilution gradient were plated. The liver sample was cultured on MH solid medium and the intestinal sample was cultured on RS agar medium.

[0129] (3) Each plate was incubated at 37°C for 24 hours, and single colonies were counted. There were 12 replicates for each group.

[0130] The formula for calculating bacterial load is as follows:

[0131]

[0132] The results of the growth performance test are shown in Table 3 below. Through the measurement and analysis of the initial weight of largemouth bass, it was found that compared with the CK group, EP2 and EP3 were used as feed additives for largemouth bass for 8 weeks and had the highest final weight, which were 30.52g / fish and 31.96g / fish, respectively. Their weight gain rate (315.25%, 289.49%) and specific growth rate (40.53%, 42.20%) were also the highest.

[0133] Table 3. Effects of Echinacea extract on weight gain and specific growth rates of largemouth bass.

[0134]

[0135]

[0136] Note: a, b, and the combination of letters ab represent statistically significant differences in values. If the letters marked on the two groups are the same or the combination of letters in one group contains letters from the other group, it means that the two values ​​are not statistically significant. If the letters marked on the two values ​​are different or not included, it means that the two values ​​are statistically significant (p<0.05).

[0137] Results of the challenge experiment are as follows Figure 11-14 As shown, Figure 11 The study investigated the effects of echinacea extract on the clinical symptoms and pathological features of largemouth bass during a challenge experiment. The results showed that in the AEP3 group, symptoms such as skin bleeding, abdominal swelling, and ulceration in largemouth bass were alleviated. Figure 12 The effect of echinacea extract on the mortality rate of largemouth bass in the challenge experiment was investigated. The results showed that the survival rate of AEP2 and AEP3 groups was the highest, both at 77%, and the survival rate was 46% higher than that of the control group ACK. Figure 13-14 To investigate the effects of echinacea extract on liver and intestinal bacterial load, experimental results showed that the AEP2 and AEP3 groups significantly reduced intestinal bacterial load. Figure 13 ) and liver ( Figure 14 Aeromonas verrucosa load.

[0138] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

Claims

1. A method for preparing echinacea extract, characterized in that, The preparation method includes the following steps: adding ethanol to echinacea and performing ultrasonic extraction to obtain echinacea extract; the ultrasonic extraction temperature is 40-60℃; the ultrasonic extraction power is 280-420W; the ultrasonic extraction time is 30-60 minutes; preferably, the ultrasonic extraction is performed 1-3 times; preferably, the step further includes purifying the product after ultrasonic extraction.

2. The preparation method according to claim 1, characterized in that, The amount of ethanol added to echinacea is 300-500 ml of ethanol per gram of echinacea; preferably, the concentration of the ethanol is 30%-50%.

3. The preparation method according to claim 1, characterized in that, The echinacea mentioned refers to the whole plant of echinacea or a mixture of one or more of its stems, leaves, flowers, and roots.

4. The echinacea extract prepared by the method according to any one of claims 1 to 3, characterized in that, The echinacea extract contains ≥30% chicoric acid and monocaffeoyl tartaric acid.

5. The echinacea extract according to claim 4, characterized in that, The chicoric acid content in the echinacea extract is ≥20%.

6. The use of the echinacea extract according to any one of claims 4 to 5 in improving the growth performance of largemouth bass.

7. The application according to claim 6, characterized in that, The improvement in growth performance includes increasing weight gain or a specific growth rate.

8. The use of the echinacea extract according to any one of claims 4 to 5 in the preparation of products for the prevention and / or treatment of Aeromonas verrucosa infection in largemouth bass.

9. The application according to claim 8, characterized in that, The products include feed, feed additives, or pharmaceuticals.

10. A method for improving the growth performance of largemouth bass, characterized in that, The method includes feeding largemouth bass with the echinacea extract according to any one of claims 4 to 5.

Images