Gleditsia sinensis seed ethanol extract as well as preparation method and application thereof in aquatic animal immunization

Abstract

The invention relates to a Gleditsia sinensis seed ethanol extract and a preparation method and application thereof in aquatic animal immunization, the preparation method of the Gleditsia sinensis seed ethanol extract comprises the following steps: crushing Gleditsia sinensis seeds, adding 75-80% ethanol solution according to a solid-to-liquid ratio of 1: 8-1: 10, and extracting; during extraction, heating is performed to 80-85 DEG C, reflux extraction is performed 2-3 times, and each time lasts for 1.5-2 hours; combining all extracting solutions, and filtering under reduced pressure to obtain clear filtrate; and concentrating the filtrate to 1 / 5 of the original volume under reduced pressure by using a rotary evaporator, and freeze-drying the concentrated solution to obtain faint yellow powder. The gleditsia sinensis seed ethanol extract is applied to improvement of the immune function of aquatic animals. The invention provides a novel immunopotentiator, namely the gleditsia sinensis seed extract, for aquaculture. The immunopotentiator is low in cost, simple in preparation process, remarkable in immunopotentiation effect, safe, environment-friendly and suitable for being mixed with feed for feeding aquatic animals.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal immunization, specifically relating to an ethanol extract of Gleditsia sinensis seeds, its preparation method, and its application in aquatic animal immunization. Background Technology

[0002] In intensive aquaculture, disease outbreaks are one of the main factors restricting the healthy development of the industry. For a long time, the overuse of antibiotics and chemical drugs has led to problems such as drug residues, increased drug resistance in pathogens, and environmental pollution. Vaccines and immune enhancers are effective means of disease prevention. Compared with vaccines, immune enhancers have advantages such as broad-spectrum activity.

[0003] Currently, there are many types of immune enhancers for fish, but most suffer from high costs or complex preparation processes. Therefore, it is of great significance to develop an immune enhancer for fish that is widely available in raw materials, easy to prepare, highly effective, and safe and environmentally friendly.

[0004] Gleditsia japonica is a plant belonging to the genus Gleditsia in the legume family. It is also known as mountain soapberry, soapberry tree, or soap pod tree, and is widely distributed in my country. Currently, Gleditsia japonica is mainly used as a cosmetic additive and in industrial cleaning agents. There are no reports on the systematic development of Gleditsia japonica extract as an immune enhancer for fisheries, nor on elucidating its application methods.

[0005] This invention develops the ethanol extract of Gleditsia sinensis into an immune enhancer, providing a new, low-cost, simple-to-prepare, highly effective, safe, and environmentally friendly immune enhancer suitable for mixing into feed for aquatic animals. Summary of the Invention

[0006] The purpose of this invention is to provide an ethanol extract of Gleditsia sinensis seeds, its preparation method, and its application in aquatic animal immunity. Feeding aquatic animals with feed containing the ethanol extract of Gleditsia sinensis seeds can enhance their immunity and is suitable for preparing immune enhancers for aquatic animals.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides a method for preparing an ethanol extract of Gleditsia sinensis seeds, which includes the following steps: Wash the seeds of *Gleditsia sinensis* clean, dry them, crush them, and pass them through a 20-40 mesh sieve to obtain a uniform powder. Put the powder into an extraction tank and add 75%-80% ethanol solution at a solid-liquid ratio of 1:8-1:10. Then, heat to 80-85℃ and reflux for extraction 2-3 times, each time for 1.5-2 hours. Combine all the extracts, filter under reduced pressure to obtain a clear filtrate. Concentrate the filtrate under reduced pressure to 1 / 5 of its original volume using a rotary evaporator (55-60℃). Freeze-dry the concentrate to obtain a pale yellow powder.

[0008] The present invention also provides an ethanol extract of Gleditsia sinensis seeds prepared by the preparation method described above.

[0009] The application of the ethanol extract of Gleditsia sinensis seeds in improving the immune function of aquatic animals.

[0010] The application described above, specifically the method for enhancing the immune function of aquatic animals using the ethanol extract of *Gleditsia sinensis* seeds, is as follows: The ethanol extract of *Gleditsia sinensis* seeds is mixed with a corresponding basic feed for aquatic animals to obtain an aquatic animal feed containing the ethanol extract of *Gleditsia sinensis* seeds. This aquatic animal feed is then used to feed the corresponding aquatic animals. If the basic feed is a sea bass basic feed, the resulting aquatic animal feed can be fed to sea bass. If the basic feed is a shrimp basic feed, the resulting aquatic animal feed can be fed to shrimp.

[0011] When mixing the ethanol extract of Gleditsia sinensis seeds with the basic feed, add 0.5-1.5g of Gleditsia sinensis extract per kilogram of basic feed. When feeding aquatic animals with the obtained aquatic animal feed, the amount of feed should be 2-4% of the animal's body weight; the feeding period should be 10-15 days.

[0012] The present invention also provides a method for preparing aquatic animal feed, which includes the following steps: dissolving the ethanol extract of Gleditsia sinensis seeds in purified water, diluting it to an appropriate volume, spraying it evenly onto the surface of the basic feed corresponding to the aquatic animals, stirring evenly, and air-drying at room temperature or drying at a temperature range of 50-60℃; wherein, 0.5-1.5g of Gleditsia sinensis seed ethanol extract is added per kilogram of basic feed.

[0013] The present invention also provides an aquatic animal feed prepared by the preparation method described above.

[0014] The application of the ethanol extract of Gleditsia sinensis seeds in the preparation of immune enhancers for aquatic animals.

[0015] Compared to existing technologies, the advantages of this invention are: Currently, there is no known application of *Gleditsia sinensis* seed extract in fish immune enhancers. This invention provides a novel immune enhancer for aquaculture—*Gleditsia sinensis* seed extract. This immune enhancer is low-cost, simple to prepare, has significant immune-enhancing effects, is safe and environmentally friendly, and is suitable for mixing with feed for aquatic animals. Experimental results show that feeding an appropriate amount of *Gleditsia sinensis* seed ethanol extract can significantly improve the immune function of sea bass and shrimp, and enhance their resistance to pathogen infection. Attached Figure Description

[0016] Figure 1 This is the cumulative mortality rate of spotted bass infected with Vibrio alginolyticus after being fed different doses of the extract of this invention; Figure 2 Expression levels of immune-related genes in the spleen tissue of spotted perch in the high-dose group; Figure 3 The levels of serum biochemical indicators in the high-dose group of spotted bass; Figure 4 The mortality rate of shrimp infected with Vibrio parahaemolyticus; Caption: The difference is statistically significant (P < 0.05). Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings and embodiments: Example 1: Selection of extraction solvent for extracting seeds of Gleditsia sinensis Preparation of aqueous and ethanolic extracts of Gleditsia sinensis seeds and their comparative immunomodulatory effects Water and ethanol are both commonly used extraction solvents. To select a suitable extraction solvent, we prepared extracts of *Gleditsia sinensis* seeds using water and 80% ethanol, respectively, and evaluated the effects of the two extracts on the resistance of fish to pathogen infection. The experimental methods are as follows: 1.1 Preparation of Aqueous Extract Wash 500g of *Gleditsia sinensis* seeds quickly with water, sun-dry or dry in a 60℃ oven, pulverize, and pass through a 20-40 mesh sieve to obtain a uniform powder. Add the powder to an extraction tank, add 4L of purified water, heat to 80-85℃, and reflux twice, 2 hours each time. Combine the extracts, filter under reduced pressure to obtain a clear filtrate. Concentrate the filtrate to 1L under reduced pressure using a rotary evaporator.

[0018] 1.2 Preparation of Ethanol Extract Wash 500g of *Gleditsia sinensis* seeds quickly with water, sun-dry or dry in a 60℃ oven, pulverize, and pass through a 20-40 mesh sieve to obtain a uniform powder. Place the powder into an extraction vessel, add 4L of 80% ethanol solution, heat to 80-85℃, and reflux twice, 2 hours each time. Combine the extracts, filter under reduced pressure to obtain a clear filtrate. Concentrate the filtrate to 1L under reduced pressure using a rotary evaporator.

[0019] 1.3 Evaluation of the efficacy of the two extracts against the pathogen infection of spotted sea bass In the experiment evaluating the disease resistance effects of aqueous and alcoholic extracts of *Gleditsia sinensis* seeds on fish, oral administration was used to precisely control the dosage of extract ingested by each experimental fish. The specific experimental method was as follows: Sixty experimental perch (approximately 50g in weight) were divided into three groups (control group, aqueous extract group, and alcoholic extract group), with 20 fish in each group. Each fish in the control group was administered 80µl of purified water, each fish in the aqueous extract group was administered 80µl of the aqueous extract, and each fish in the alcoholic extract group was administered 80µl of the alcoholic extract, for a total of 7 days. One week after the end of the administration, each fish was intraperitoneally injected with *Vibrio alginolyticus* (2×10⁻⁶).5 The mortality rate of experimental fish was observed and recorded for each group (cfu / tail), and the relative immune protection rate (RPS) was calculated. The calculation formula is: RPS (%) = 100 × (1 - mortality rate of experimental group / mortality rate of control group).

[0020] The experimental results showed that the cumulative mortality rate was 65.0% in the control group, 30.0% in the ethanol extract group, and 55.0% in the water extract group. The relative immune protection rate of the ethanol extract group was 53.85%, showing a significant effect. In contrast, the relative immune protection rate of the water extract group was only 15.38%, indicating no significant effect. Therefore, ethanol solution was chosen as the extraction solvent.

[0021] Example 2: Preparation of ethanol extract from Gleditsia sinensis seeds Wash the seeds of *Gleditsia sinensis* clean, dry them, crush them, and pass them through a 20-40 mesh sieve to obtain a uniform powder. Put the powder into an extraction tank, add a 75%-80% ethanol solution (solid-liquid ratio 1:8-1:10), heat to 80-85℃, and reflux for extraction 2-3 times, each time for 1.5-2 hours. Combine all extracts, filter under reduced pressure to obtain a clear filtrate. Concentrate the filtrate under reduced pressure to 1 / 5 of its original volume using a rotary evaporator (55-60℃), and freeze-dry the concentrate to obtain a pale yellow powder. Specifically: Wash 1 kg of *Gleditsia sinensis* seeds quickly with water, sun-dry or dry in a 60℃ oven, pulverize, and pass through a 20-40 mesh sieve to obtain a uniform powder. Place the powder into an extraction tank, add 8 L of a 75%-80% ethanol solution (solid-liquid ratio 1:8-1:10), heat to 80-85℃, and reflux twice, each time for 2-3 hours. Combine the extracts, filter under reduced pressure to obtain a clear filtrate. Concentrate the filtrate under reduced pressure to 2-3 L using a rotary evaporator (55-60℃), and freeze-dry the concentrate to obtain 52.31 g of pale yellow powder.

[0022] Example 3: Preparation of aquatic animal feed containing ethanol extract of Gleditsia sinensis seeds Dissolve the ethanol extract of *Gleditsia sinensis* seeds in purified water, dilute to an appropriate volume, and spray evenly onto the surface of the corresponding basic feed for aquatic animals. Stir well and air dry at room temperature or dry at a low temperature (below 60℃). Add 0.5-1.5g of *Gleditsia sinensis* seed ethanol extract per kilogram of basic feed. Specifically: Preparation of 5 kg of sea bass feed containing ethanol extract of Gleditsia sinensis seeds: Weigh 4.0 g of ethanol extract of Gleditsia sinensis seeds, dissolve in 200 ml of purified water, then dilute to 900-1000 ml, shake well, spray evenly onto the surface of 5 kg of sea bass pellet feed, stir evenly, and air dry at room temperature or dry at low temperature (below 60℃). The base feed mentioned here is sea bass formulated feed (Fujian Tianma Technology Group Co., Ltd.).

[0023] Preparation of 5 kg of shrimp feed containing ethanol extract of Gleditsia sinensis seeds: Dissolve 5 g of Gleditsia sinensis seed ethanol extract in 500 ml of purified water, then dilute to 900-1000 ml, shake well, and spray evenly onto the surface of 5 kg of shrimp pellet feed. Mix well and air dry at room temperature or dry at low temperature (below 60℃). The base feed mentioned here is compound feed for Litopenaeus vannamei (Fujian Tianma Technology Group Co., Ltd.).

[0024] Example 4: Feeding of Aquatic Animals Feed the aquatic animals with a feed containing ethanol extract of Gleditsia sinensis seeds. The amount of feed should be 2-4% of the animal's body weight, depending on the individual animal's feeding habits. Feeding should be done for 10-15 days. Specifically: 4.1 Feeding spotted bass with the prepared spotted bass feed: Approximately 1000 spotted bass (weighing about 10-15g) are raised in one net cage. Based on the daily feed amount of 2-3% of the fish's body weight, 0.3-0.45kg of feed is given to one net cage per day for 5 consecutive days, followed by a 2-day break, and then another 5 consecutive days of feeding.

[0025] 4.2 Feeding shrimp with the prepared shrimp feed: Each pond can hold approximately 1000 shrimp (weighing about 15-20g). Based on the daily feed amount of 3-4% of the shrimp's body weight, each net cage should be fed 0.5-0.8kg of feed per day for 14 consecutive days.

[0026] Example 5: Application Effect Evaluation Experiment Experiment 1: Evaluation of the effects of different doses of ethanol extract from Gleditsia sinensis seeds on the immune function and disease resistance of perch. Experimental Methods: 360 spotted bass (approximately 20-25g in weight) were divided into four groups (control group, low-dose group, medium-dose group, and high-dose group), with three replicates per group and 30 fish per replicate. The three experimental groups were fed a basic spotted bass diet containing low, medium, and high doses of the extract (i.e., ethanol extract of *Gleditsia sinensis* seeds), respectively, at amounts of 0.25g, 0.50g, and 1.0g of the extract per kilogram of the basic diet. The control group was fed a basic spotted bass diet without any additives. Each fish was fed 25g of the corresponding diet daily for 5 consecutive days, followed by a 2-day pause, and then another 5 consecutive days of feeding. One week after the feeding period ended, two fish from each fish were collected for blood and spleen tissue. Simultaneously, 20 fish from each fish were injected intraperitoneally with *Vibrio alginolyticus* (1×10⁻⁶). 5The mortality rate was observed and recorded for each group (cfu / tail), and the relative immune protection rate (RPS) was calculated using the formula: RPS (%) = 100 × (1 - mortality rate of experimental group / mortality rate of control group). The basal feed for the spotted bass was formulated feed for bass (Fujian Tianma Technology Group Co., Ltd.). In addition, based on the results of the infection experiment, the expression levels of immune-related genes in the spleen tissue and serum biochemical indicators were measured in the experimental group with the highest protection rate. Immune-related genes included interleukin-2 (IL2), interleukin-8 (IL8), histocompatibility complex I (MHC I), histocompatibility complex II (MHC II), Toll-like receptor 1 (TLR1), and Toll-like receptor 3 (TLR3). Serum biochemical indicators included superoxide dismutase (SOD), lysozyme (LZM), and complement C3.

[0027] Experimental results: (1) After infection with Vibrio alginolyticus, the average mortality rate of the control group was 67.22%, while the average mortality rates of the low-dose, medium-dose, and high-dose groups were 65.24%, 54.13%, and 31.13%, respectively. The mortality rates of the medium-dose and high-dose groups were significantly lower than those of the control group (P<0.05), with relative immune protection rates of 19.47% and 53.69%, respectively. (See attached results) Figure 1 (2) The expression levels of immune-related genes IL2, IL8, MHCⅠ, MHCⅡ, TLR1, and TLR3 in spleen tissue of the high-dose group were significantly higher than those in the control group (P<0.05). See the results below. Figure 2 (3) The serum SOD, LZM, and C3 levels in the high-dose group were significantly higher than those in the control group (P<0.05). The experimental results showed that feeding an appropriate amount of ethanol extract from *Gleditsia sinensis* seeds could significantly improve the immune function of perch and enhance its resistance to pathogen infection. See the results below. Figure 3 .

[0028] Experiment 2: Evaluation of the effect of ethanol extract of Gleditsia sinensis seeds on the disease resistance of Litopenaeus vannamei. Experimental Methods: Two hundred shrimp (approximately 20-25g in weight) were dispersed into six plastic tanks, each containing 0.5 tons of water and 50 shrimp per tank. Three tanks served as the experimental group, fed a basic shrimp diet supplemented with ethanol extract of *Gleditsia sinensis* seeds. The other three tanks served as the control group, fed a basic shrimp diet without any additives. Each tank was fed 25g of the corresponding diet daily for 14 consecutive days. After feeding, each shrimp was injected with *Vibrio parahaemolyticus* (1×10⁻⁶). 5 (CFU / tail), observe and record the mortality of shrimp in each group, and calculate the relative immune protection rate. The basic shrimp feed was a compound feed for Litopenaeus vannamei (Fujian Tianma Technology Group Co., Ltd.).

[0029] Experimental Results: After injection of Vibrio parahaemolyticus, the average mortality rate of shrimp in the control group was 51.66%, while that in the experimental group was 32.28%, significantly lower than that in the control group (P<0.05), with a relative immune protection rate of 37.51%. These results indicate that appropriate feeding with ethanol extract of Gleditsia sinensis seeds can significantly improve the shrimp's resistance to pathogen infection; see results below. Figure 4 .

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an ethanol extract of *Gleditsia sinensis* seeds, characterized in that: It includes the following steps: Wash the seeds of *Gleditsia sinensis* clean, dry them, crush them, and pass them through a 20-40 mesh sieve to obtain a uniform powder. Put the powder into an extraction tank and add 75%-80% ethanol solution at a solid-liquid ratio of 1:8-1:

10. Then, heat to 80-85℃ and reflux for extraction 2-3 times, each time for 1.5-2 hours. Combine all the extracts, filter under reduced pressure to obtain a clear filtrate. Concentrate the filtrate under reduced pressure to 1 / 5 of its original volume using a rotary evaporator. Freeze-dry the concentrate to obtain a pale yellow powder.

2. An ethanol extract of Gleditsia sinensis seeds prepared by the method described in claim 1.

3. The application of the ethanol extract of Gleditsia sinensis seeds as described in claim 2 in improving the immune function of aquatic animals.

4. The application according to claim 3, characterized in that: The method for improving the immune function of aquatic animals using the ethanol extract of *Gleditsia sinensis* seeds is as follows: The ethanol extract of the seeds of Gleditsia sinensis is mixed with the corresponding basic feed for aquatic animals to obtain aquatic animal feed containing the ethanol extract of the seeds of Gleditsia sinensis, and then the aquatic animals are fed with the aquatic animal feed.

5. The application according to claim 4, characterized in that: When mixing the ethanol extract of Gleditsia sinensis seeds with the basic feed, add 0.5-1.5g of Gleditsia sinensis extract per kilogram of basic feed.

6. The application according to claim 4, characterized in that: When feeding aquatic animals with the obtained aquatic animal feed, the amount of feed should be 2-4% of the animal's body weight; the feeding period should be 10-15 days.

7. A method for preparing aquatic animal feed, characterized in that: It includes the following steps: Dissolve the ethanol extract of Gleditsia sinensis seeds as described in claim 2 in purified water, dilute to an appropriate volume, spray evenly onto the surface of the basic feed corresponding to the aquatic animals, stir evenly, and air dry at room temperature or dry at a temperature range of 50-60℃; wherein, 0.5-1.5g of ethanol extract of Gleditsia sinensis seeds is added per kilogram of basic feed.

8. An aquatic animal feed prepared by the method described in claim 7.

9. The use of the ethanol extract of Gleditsia sinensis seeds as described in claim 2 in the preparation of an immune enhancer for aquatic animals.

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