Use of kaempferol in the preparation of a product for treating liver damage in fish

Abstract

The application discloses application of kaempferol in preparation of products for treating fish liver injury. Researches of the application show that feeding fish with liver injury caused by endocrine disruptors with brine shrimp hatched by kaempferol can effectively recover the fish liver injury caused by the endocrine disruptors. Therefore, kaempferol can be used to improve the fish liver injury caused by the endocrine disruptors, and ensure that the liver function is recovered to the state before the injury. The application has far-reaching significance for keeping sustainable utilization of aquatic resources, reducing negative influence of the endocrine disruptors on the industry, and promoting development of healthy aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to the application of kaempferol in the preparation of products for treating liver damage in fish. Background Technology

[0002] Endocrine disruptors (EDCs) are a class of chemical substances that can mimic, block, or interfere with the normal function of hormones in the human body. Examples include bisphenols, dioxins, perchlorates, perfluorinated and polyfluoroalkyl substances, phthalates, and plant hormones. They have become emerging pollutants of global concern. They are associated with a range of health problems, such as: (1) EDCs having hormone-like effects, interfering with the endocrine function of organisms and leading to abnormal hormone secretion; (2) having carcinogenic effects, particularly potentially increasing the risk of reproductive system cancers; and (3) interfering with the development and function of the nervous system, causing behavioral, learning, and memory disorders. These chemicals are widely present in daily life, including in cosmetics, food and beverage packaging, toys, carpets, and pesticides. Through human activities such as industrial emissions, agricultural activities, urban sewage discharge, and landfill leakage, endocrine disruptors are released into the aquatic environment, posing a serious threat to the survival and health of aquatic organisms and the sustainable development of aquaculture.

[0003] Fish, as one of the main sources of protein for humans, have extremely high economic value in aquaculture. The liver plays a crucial role in the physiological functions of fish. Any damage to the liver can seriously affect the health and survival of fish, such as (1) fish nutritional metabolism disorder: The liver is an important metabolic organ for fish, participating in the synthesis, storage, and transport of starch, sugars, fats, vitamins, etc. When the liver is damaged, the metabolism of these substances will be affected, leading to poor nutrient absorption and slower growth rate; (2) fish immune system disorder: The liver has immune function, which can engulf, isolate, and eliminate various invading and endogenous microorganisms. Liver damage will weaken the immunity of fish, making them more susceptible to pathogens, thereby causing various diseases; (3) fish growth hindrance: Impaired liver function will affect the growth and development of fish, resulting in slow growth rate and smaller size, affecting aquaculture efficiency; (4) increased risk of fish mortality: When subjected to stress stimuli (such as netting, transportation, sudden changes in water temperature, poor water quality, etc.), fish with liver damage are more likely to develop stress hemorrhage syndrome, and even die in large numbers. The fish liver is a key target of endocrine disruptors (EDCs), which can cause liver damage and consequently affect the growth, reproduction, and overall health of fish. Therefore, liver damage in fish caused by EDCs has become a major challenge hindering the healthy development of the aquaculture industry.

[0004] With the increasing use of antibiotics in treating fish diseases, the problems of antibiotic resistance and further environmental pollution have received growing attention. Therefore, traditional Chinese medicine (TCM) is gaining popularity as an alternative to antibiotics. Many active ingredients in TCM, such as polysaccharides, alkaloids, and flavonoids, possess antibacterial, antiviral, immune-enhancing, and damage-repairing properties, showing great potential and broad prospects in aquaculture. They may provide an effective solution to the problem of liver damage in fish caused by lipopolysaccharides (EDCs). For example, patent CN110585216A discloses the application of lupeol in the preparation of drugs for the prevention or treatment of liver damage, specifically, lupeol can prevent or treat liver damage in zebrafish caused by lipopolysaccharides. Kaempferol, a flavonoid compound, is widely found in nature and is abundant in most plant-based foods, such as tea, broccoli, delphinium, witch hazel, grapefruit, Brussels sprouts, and galangal. Kaempferol possesses various pharmacological effects, primarily including good antibacterial, anti-inflammatory, and anticancer activities. It also exhibits pharmacological effects such as diabetes prevention and treatment, antioxidant activity, anti-aging properties, osteoporosis treatment, and liver and myocardial protection. The paper "Kaempferol Induces Hepatocellular Carcinoma Cell Death via Endoplasmic Reticulum Stress-CHOP-Autophagy Signaling Pathway" discloses that kaempferol can significantly reduce the survival rate of human hepatocellular carcinoma cells HepG2 and Huh7, clarifying its inhibitory effect on the proliferation of human hepatocellular carcinoma cells. The article "The Effect and Mechanism of Kaempferol in Improving Acute Liver Injury Induced by Acetaminophen" mentions that kaempferol can improve acute liver injury induced by acetaminophen in mice. However, there are currently no reports on the use of kaempferol in treating liver damage in fish. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of kaempferol in the preparation of products for treating liver damage in fish.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention demonstrates that endocrine disruptors can cause liver damage in fish. The invention found that adding a certain concentration of kaempferol to artificial seawater to hatch Artemia larvae yields kaempferol-containing Artemia larvae. These kaempferol-treated Artemia larvae were then fed twice daily to marine medaka fish suffering from liver damage due to endocrine disruptors. After four weeks of feeding, the liver function of the marine medaka fish recovered.

[0008] Therefore, the present invention provides the use of kaempferol in the preparation of products for treating liver damage in fish.

[0009] Furthermore, the liver injury includes hepatocellular lesions and / or necrosis.

[0010] Furthermore, the liver damage was caused by endocrine disruptors.

[0011] Furthermore, the endocrine disruptor is selected from one or more of bisphenols, dioxins, perchlorates, perfluorinated and polyfluoroalkyl substances, phthalates, and plant hormones.

[0012] Preferably, the endocrine disruptor is tetramethylbisphenol A (TBA). It can interfere with the endocrine system of fish, affecting the liver's metabolic and detoxification functions through various mechanisms such as oxidative stress and inflammatory responses, leading to liver cell damage and abnormal lipid metabolism. For example, it may induce liver cells to produce excessive reactive oxygen species (ROS), leading to cell membrane lipid peroxidation and DNA damage; it may also interfere with the liver's lipid metabolism pathways, causing abnormal fat accumulation in the liver and forming fatty liver.

[0013] Furthermore, the product is a medicine or feed.

[0014] Furthermore, the feed is brine shrimp fed with kaempferol. Brine shrimp (Artemia salina), also known as brine shrimp, is an indispensable feed organism in aquaculture due to its rich protein, amino acid, and fat content, with protein content reaching up to 60% and fat content around 20%. Secondly, brine shrimp have superior vitality and palatability compared to many other types of feed, making them particularly suitable as initial feed for aquatic products. They help improve the feeding activity and growth rate of fish and shrimp larvae, significantly enhancing their physical condition and immunity. As a natural feed, the use of brine shrimp helps reduce the use of chemical feeds and antibiotics, thereby mitigating pollution to the aquaculture environment and promoting the development of eco-friendly aquaculture.

[0015] Preferably, the concentration of kaempferol fed is 25–35 mg / L.

[0016] Preferably, the feeding time for kaempferol is 1-2 days. Using kaempferol at a concentration of 25-35 mg / L, brine shrimp are hatched for 24 hours to obtain brine shrimp feed containing kaempferol. By feeding marine killifish with brine shrimp treated with kaempferol twice daily for four weeks, liver damage caused by endocrine disruptors can be effectively restored.

[0017] Preferably, the fish is the marine killifish (Oryzias melastigma). The marine killifish (Oryzias melastigma) is widely used as a model fish due to its wide salinity tolerance, short sexual maturity cycle, excellent traits, and small size suitable for laboratory culture.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention provides the application of kaempferol in the preparation of products for treating liver damage in fish. The research of this invention shows that feeding fish with liver damage caused by endocrine disruptors with brine-treated brine can effectively restore liver function caused by endocrine disruptors. Therefore, kaempferol can be used to improve liver damage in fish caused by endocrine disruptors, ensuring that liver function returns to its pre-damage state. This invention has profound significance for maintaining the sustainable use of aquatic resources, mitigating the negative impact of endocrine disruptors on the industry, and promoting the development of healthy aquaculture. Furthermore, the main source of kaempferol is galangal (commonly known as sand ginger), a safe, reliable, and cost-effective traditional Chinese medicine ingredient. Its raw material sources are widely available, extraction technology is mature, and no obvious toxic side effects have been found to date, demonstrating its advantages in aquaculture applications. Attached Figure Description

[0020] Figure 1 The images show liver tissue sections from marine medaka fish in the tetramethylbisphenol A (TBA) exposure group and the control group. Figure 1 In the table, A represents a liver tissue section from the control group of marine medaka; B represents a liver tissue section from the tetramethylbisphenol A-exposed group of marine medaka. Note: Blue arrow: nuclear dissolution; black arrow: pitted necrosis; yellow arrow: nuclear aggregation; green arrow: eosinophilic bodies; green circle: vacuolation.

[0021] Figure 2 The images show liver tissue sections from marine medaka fed with Artemia larvae hatched with kaempferol and from a control group. Figure 2 In the table, A represents a liver tissue section from the normally hatched Artemia-fed group (control group); B represents a liver tissue section from the Artemia-fed group hatched with kaempferol. Note: Blue arrow: nuclear dissolution; yellow arrow: nuclear aggregation; red box: structural lesions. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0023] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0024] The marine medaka (Oryzias melastigma) is a model fish species with wide salinity adaptability, short sexual maturity cycle, stable reproductive capacity, and excellent traits, and is often used in scientific research.

[0025] Artemia salina, also known as brine shrimp, is rich in protein, amino acids, and fat, making it an indispensable food organism in aquaculture.

[0026] Tetramethylbisphenol A (TMBPA) is a novel bisphenol endocrine disruptor primarily used in the manufacture of flame retardants and tetramethyl polycarbonate plastics. In vitro studies have shown that TMBPA can bind simultaneously as an agonist to human estrogen receptor α (ERα) and as an antagonist to human androgen receptor (AR), thus posing a potential threat to the reproductive system.

[0027] Kaempferol, CAS No.:520-18-3, structure shown below, purchased from MACKLIN, lot number: C15941008, catalog number K812226-5g.

[0028]

[0029] Example 1: Exposure experiment of marine medaka

[0030] 1. Method

[0031] Four-month-old marine killifish with normal morphology and uniform body size were selected for an exposure experiment to obtain samples of marine killifish with liver damage. Two concentration groups were set up in the experiment: a control group (TMBPA concentration of 0) and an exposure group (TMBPA concentration of 50 μg / L). Dimethyl sulfoxide (DMSO) was used as the co-solvent for TMBPA, and the DMSO volume fraction was 0.01% in all groups. Several parallel experiments were set up for each concentration group, with 20 fish placed in each parallel experiment and cultured in a 6L glass tank. Culture conditions were maintained at approximately 15‰ salinity, (28±1)℃, and an exposure time of 7 days. The exposure solution was changed every 48 hours, the light intensity was 1745 Lx, and the light-dark cycle was set to 14 hours of light / 10 hours of darkness.

[0032] After the experiment, six fish were randomly selected from each concentration group for dissection, and liver tissue was taken to prepare HE sections for histopathological examination.

[0033] The steps for HE sectioning are as follows:

[0034] (1) Fixation: Fix the ovarian tissue with 4% paraformaldehyde for 18-24 hours, trim the tissue at the target site with a scalpel, and place it in a dehydration box.

[0035] (2) Dehydration and wax impregnation: The dehydration box is placed in the dehydration machine and dehydrated with alcohol in sequence, then cleared with xylene and impregnated with paraffin.

[0036] (3) Embedding: The tissue impregnated with wax is embedded in an embedding machine.

[0037] (4) Sectioning: The embedded wax block is sectioned using a paraffin microtome to a thickness of 4-6 μm.

[0038] (5) Dewaxing the sections to water: Place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min in sequence, and then wash with tap water.

[0039] (6) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue back solution, and rinse with running water.

[0040] (7) Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol for 5 min each, and then stained in eosin staining solution for 5 min.

[0041] (8) Dehydration and mounting: The sections were sequentially placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min until transparent, and then mounted with neutral resin.

[0042] (9) Microscopic examination, image acquisition and analysis.

[0043] 2. Results

[0044] The slice results are as follows Figure 1 The results showed that after 7 days of TMBPA exposure, the control group ( Figure 1 A) The liver morphology was normal, and the hepatocytes were tightly packed and evenly distributed; while the exposed group ( Figure 1 B) Significant liver damage was observed, with hepatocytes arranged in a dispersed and disordered manner, exhibiting pathological changes such as nucleolysis, nuclear aggregation, eosinophilic bodies, vacuolation, and pitting necrosis. These results indicate that exposure to TMBPA caused liver damage in marine killifish.

[0045] Example 2: Hatching Artemia containing Kaempferol

[0046] Two hatching tanks were prepared for hatching Artemia larvae. One tank used standard artificial seawater (salinity 30‰) for standard hatching, while the other tank used artificial seawater (salinity 30‰) containing 30 mg / L kaempferol for hatching. The hatching process was set for 24 hours, maintaining a temperature of 26°C. After hatching, the Artemia larvae were rinsed with running water to remove any kaempferol residue from their surface.

[0047] The observation results showed that there was no difference in vitality and condition between Artemia hatched using kaempferol and Artemia hatched under normal conditions, indicating that kaempferol did not have an adverse effect on the growth of Artemia.

[0048] Example 3: Experiment on Liver Injury Recovery in Marine Medaka

[0049] 1. Method

[0050] Marine killifish previously exposed to 50 μg / L TMBPA were collected and randomly divided into two groups to assess the effects of different diets on liver recovery. One group was fed normally hatched brine shrimp, while the other group was fed brine shrimp treated with kaempferol. Each group had four parallel experiments, with 20 fish per parallel, cultured in 6L glass tanks in a TMBPA-free environment. Culture conditions included a salinity of 15‰, a temperature of (28±1)℃, a light intensity of 1745 Lx, and a light / dark cycle of 14 hours light / 10 hours darkness, with fresh water changed every 48 hours. After four weeks of twice-daily feeding, six fish were randomly selected from each group for dissection, and liver tissue was collected for hematologic analysis (HE) sections and histopathological examination.

[0051] 2. Results

[0052] The results are as follows Figure 2 As shown, the group of medaka fed with normally hatched brine shrimp ( Figure 2 A), whose livers still showed damage, including regional lesions, eosinophilic bodies, nucleolysis, and nuclear aggregation; while the group of medaka fed with brine shrimp hatched from kaempferol ( Figure 2 In case B), the liver generally recovered, with no obvious pathological structures, normal liver morphology, and tightly packed, evenly distributed hepatocytes. Based on these results, this method can effectively restore livers damaged by endocrine disruptors.

Claims

1. The application of kaempferol in the preparation of feed for treating liver damage in fish, characterized in that, The liver injury is liver damage caused by an endocrine disruptor; the endocrine disruptor is tetramethylbisphenol A; the liver injury includes hepatocellular lesions and / or necrosis; the feed is brine shrimp fed with kaempferol; the fish is marine medaka.

2. The application according to claim 1, characterized in that, The concentration of kaempferol fed to the animal was 25–35 mg / L.

3. The application according to claim 1, characterized in that, The feeding time for kaempferol is 1 to 2 days.

Images