Application of kaempferol in preparing products for improving spawning function damage in fish

Abstract

The present invention discloses the application of kaempferol in the preparation of products for improving the spawning function damage of fish. The research of the present invention shows that by feeding Artemia incubated with kaempferol to fish whose spawning function is damaged due to endocrine disruptors, the spawning function damage of fish caused by endocrine disruptors can be effectively restored, ensuring the stable spawning of fish. Therefore, kaempferol can be used to improve the problems of reduced spawning amount of fish and decreased proportion of mature oocytes in ovaries caused by endocrine disruptors, ensuring that the proportion of mature oocytes in ovaries and spawning amount reach the stable state before damage. The present invention has far-reaching significance for maintaining the sustainable utilization of aquatic resources, improving reproductive efficiency, reducing the negative impact of endocrine disruptors on the industry, and promoting the development of healthy breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and more specifically, relates to the application of kaempferol in preparing a product for improving the spawning function damage of fish. Background Art

[0002] As an important source of protein for humans, fish occupies a pivotal economic position in the aquaculture industry. The spawning process is the lifeblood of the aquaculture industry. It is not only directly related to the efficiency and economic benefits of aquaculture production, but also a key factor affecting the sustainable development and ecological balance of the aquaculture industry.

[0003] Endocrine disrupting chemicals (EDCs), such as bisphenols, dioxins, perchlorates, per- and polyfluoroalkyl substances, phthalates and plant hormones, have become new pollutants of global concern. These substances have the characteristics of bioaccumulation, biomagnification and persistence, causing serious damage to the endocrine system. The endocrine system plays a vital role in the energy metabolism, growth and development, reproduction, homeostasis maintenance and osmotic regulation of fish. The spawning process of fish is closely related to the endocrine system, which regulates the development of reproductive organs and the production and maturation of germ cells through the secretion of hormones. At present, endocrine disruptors have entered the environment through various pathways, including wastewater treatment plants, land runoff (derived from industrial activities, pharmaceuticals and household waste) and precipitation. EDCs are widely present in aquatic ecosystems, posing a serious threat to the reproductive health of aquatic organisms such as fish, thereby affecting the development of aquaculture.

[0004] At present, there are relatively limited effective methods to restore the spawning function of fish. The application and research of Chinese herbal medicine ingredients in aquaculture are receiving increasing attention. Many Chinese herbal medicine ingredients, such as polysaccharides, saponins, and flavonoids, show the potential to enhance immunity. Some specific Chinese herbal medicine ingredients, such as Epimedium and Morinda officinalis, are believed to improve the reproductive performance of fish and increase the spawning rate and hatching rate. However, its safety, raw material cost and standardization issues have also restricted its application and development to a certain extent. Among them, kaempferol (Kae) is a significant natural Chinese herbal medicine ingredient and one of the common dietary flavonoids. It is widely present in a variety of plant foods, such as tea, broccoli, delphinium, witch hazel, grapefruit, Brussels sprouts and kaempferol. Its raw material sources are wide, the extraction technology is mature, and no obvious toxic side effects have been found so far, showing its advantages in aquaculture applications. Studies have shown that kaempferol has anti-inflammatory and anti-oxidative stress effects in the treatment of neurological diseases, can reduce the activation of microglia, and play a neuroprotective effect. It can also remove excess free radicals, prevent DNA and cell oxidative damage, and has multiple pharmacological effects such as anti-tumor, anti-inflammatory, and anti-aging. Patent CN110623963A discloses a pharmaceutical composition for treating ovarian cancer and its application, which relates to a pharmaceutical composition containing kaempferol and tripterygium wilfordii as active ingredients, which has a synergistic inhibitory effect in treating human ovarian cancer cells. Patent CN116327789A discloses the application of raspberry extract in the preparation of ovarian function protection products, involving kaempferol-3-O-rutinoside, kaempferol-3-O-sophoroside and other substances that significantly improve the vitality of human ovarian granulosa cells, or significantly improve the level of oxidative stress, or significantly regulate the level of mitochondrial membrane potential, or effectively maintain the level of estrogen in the microenvironment. However, there is no report on the use of kaempferol for the functional recovery of fish ovarian damage. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide an application of kaempferol in the preparation of a product for improving the spawning function damage of fish.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The present invention's research shows that endocrine disruptors can significantly reduce the amount of fish eggs laid, and the proportion of mature oocytes in the ovaries is also significantly reduced. The present invention found that adding a certain concentration of kaempferol to artificial seawater to hatch Artemia, obtaining Artemia containing kaempferol, using the Artemia hatched with kaempferol as feed, feeding it to the sea medaka whose egg-laying function was damaged by endocrine disruptors twice a day, after two weeks of feeding, the egg-laying function of the sea medaka was restored.

[0008] Therefore, the present invention provides the use of kaempferol in preparing a product for improving the spawning function damage of fish.

[0009] Furthermore, the damage to the spawning function of fish is a decrease in the amount of fish eggs laid and / or a decrease in the proportion of mature oocytes in the ovaries.

[0010] Furthermore, the improvement is to restore the spawning ability of fish ovaries and increase the proportion of mature oocytes in fish ovaries.

[0011] There are many reasons for the damage of fish spawning function, mainly environmental pollutants, viral infection, etc. The kaempferol of the present invention has an improving effect on the damage of fish spawning function. The cause of the damage of fish spawning function is not limited. As long as the fish spawning function is abnormal, it is within the scope of the present invention.

[0012] Furthermore, the fish spawning function damage is fish spawning function damage caused by endocrine disruptors.

[0013] Furthermore, the endocrine disruptors are selected from one or more of bisphenols, dioxins, perchlorates, perfluoroalkyl and polyfluoroalkyl substances, phthalates, and plant hormones.

[0014] Preferably, the endocrine disruptor is tetramethyl bisphenol A (TMBPA). The tetramethyl bisphenol A (TMBPA) is an emerging bisphenol endocrine disruptor that can simultaneously bind to human estrogen receptor α (ERα) as an agonist and to human androgen receptor (AR) as an antagonist, thereby posing a potential threat to the reproductive system.

[0015] Furthermore, the product is a medicine or a feed.

[0016] Preferably, the feed is Artemia salina fed with kaempferol. The Artemia salina, also known as brine shrimp, is rich in protein, amino acids and fat and is an indispensable and important bait organism in aquaculture.

[0017] Preferably, the concentration of kaempferol in the feeding is 25-35 mg / L. Artemia feed containing kaempferol is obtained by incubating Artemia with 25-35 mg / L kaempferol for 24 hours. By feeding Artemia incubated with kaempferol to the marine medaka, the spawning function damage caused by endocrine disruptors can be effectively restored, ensuring stable spawning of fish.

[0018] Preferably, the fish is a marine medaka. Oryzias melastigma ) is a model fish with a wide range of salinity adaptability, a short sexual maturity period, stable reproductive ability and excellent traits, and is often used in scientific research.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides the use of kaempferol in the preparation of products for improving the spawning function damage of fish. The present invention shows that by feeding the fish with spawning function damage caused by endocrine disruptors with Artemia incubated with kaempferol, the spawning function damage of fish caused by endocrine disruptors can be effectively restored, ensuring the stable spawning of fish. Therefore, kaempferol can be used to improve the problems of reduced spawning of fish and decreased proportion of mature oocytes in the ovaries caused by endocrine disruptors, ensuring that the proportion of mature oocytes in the ovaries and the spawning amount reach the stable state before damage. The present invention has far-reaching significance for maintaining the sustainable utilization of aquatic resources, improving reproductive efficiency, reducing the negative impact of endocrine disruptors on the industry, and promoting the development of healthy breeding. At the same time, the main source of kaempferol is kaempferol (commonly known as sand ginger), which has a wide range of raw materials, mature extraction technology, and no obvious toxic and side effects have been found so far, showing its advantages in aquaculture applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of ovarian tissue sections of marine medaka in the tetramethyl bisphenol A exposure group and the control group. Figure 1 A in the figure is the ovarian tissue section of the marine medaka in the control group; B is the ovarian tissue section of the marine medaka in the tetramethyl bisphenol A exposure group. Note: PO-primary oocyte; CAO-cortical alveolar oocyte; EVO-early yolk oocyte; LVO / MO-late yolk oocyte / mature oocyte.

[0022] Figure 2 The results of ovarian tissue sections of the marine medaka fed with Artemia hatched with kaempferol and the control group. Figure 2 A in the figure is the ovarian tissue section of the marine medaka in the control group; B is the ovarian tissue section of the marine medaka fed with Artemia hatched by kaempferol. Note: PO-primary oocyte; CAO-cortical alveolar oocyte; EVO-early yolk oocyte; LVO / MO-late yolk oocyte / mature oocyte. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0024] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0025] The marine medaka (Oryzias melastigma) is a model fish with a wide range of salinity adaptability, a short sexual maturity period, stable reproductive capacity and excellent traits, and is often used in scientific research.

[0026] Artemia salina, also known as brine shrimp, is rich in protein, amino acids and fat, and is an indispensable and important bait organism in aquaculture.

[0027] Tetramethyl bisphenol A (TMBPA) is an emerging bisphenol endocrine disruptor, mainly used in the manufacture of flame retardants and tetramethyl polycarbonate plastics. In vitro experiments have shown that TMBPA can bind to human estrogen receptor α (ERα) as an agonist and to human androgen receptor (AR) as an antagonist, posing a potential threat to the reproductive system.

[0028] Kaempferol, CAS No.: 520-18-3, the structure is shown below, purchased from MACKLIN, batch number: C15941008, item number K812226-5g.

[0029]

[0030] Example 1 Exposure experiment of medaka

[0031] 1. Methods

[0032] In order to obtain marine medaka with impaired spawning function, four-month-old marine medaka with normal morphology and uniform body size were selected for exposure experiment. Two groups were set up in the experiment, one was the control group (TMBPA mass concentration was 0) and the other was the exposure group (TMBPA mass concentration was 50μg / L). The cosolvent of TMBPA was dimethyl sulfoxide (DMSO), and the volume fraction of DMSO in each group was 0.01%. Multiple parallel experiments were set up in each group, and 20 fish (half male and half female) were placed in each parallel experiment and cultured in a 6L glass tank. The culture conditions were maintained at 15‰ salinity and (28±1)℃. The exposure time was 7 days, the exposure solution was replaced 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. After the exposure experiment, embryos were collected and the number of eggs laid was counted. At the same time, four female fish were randomly selected for dissection, and the ovarian tissue was removed to make HE sections to analyze the proportion of mature oocytes, thereby evaluating the damage to the spawning function.

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

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

[0035] (2) Dehydration and wax dipping: Place the dehydration box into the dehydrator and dehydrate with graded alcohols in sequence, use xylene for transparency, and dip into paraffin wax.

[0036] (3) Embedding: Embed the wax-soaked tissue in an embedding machine.

[0037] (4) Sectioning: Use a paraffin slicer to slice the embedded wax block into slices with a thickness of 4-6 μm.

[0038] (5) Dewaxing the slices: Place the slices 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% alcohol for 5 min, then wash with tap water.

[0039] (6) Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

[0040] (7) Eosin staining: Dehydrate the sections in 85% and 95% graded alcohol for 5 min each, then stain them in eosin solution for 5 min.

[0041] (8) Dehydration and sealing: Place the slices 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 seal the slices with neutral gum.

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

[0043] 2. Results

[0044] The results of the exposure experiment are as follows Figure 1 As shown, the TMBPA-exposed group ( Figure 1 B) Ovarian tissue sections of medaka compared to the control group ( Figure 1 A), the proportion of late yolk oocytes or mature oocytes is low.

[0045] The results of the number of eggs laid by the marine medaka after 7 days of TMBPA exposure are shown in Table 1. TMBPA can significantly reduce the number of eggs laid by the marine medaka. The results of the percentage of mature oocytes in the ovaries of the marine medaka after 7 days of TMBPA exposure are shown in Table 2. The proportion of mature oocytes in the ovaries also decreased significantly. The proportion of mature oocytes to a certain extent indicates its spawning ability in the next period of time. In summary, TMBPA has damaged the spawning function of the marine medaka.

[0046] Table 1 Egg production / grain after 7-day TMBPA exposure

[0047]

[0048] Table 2 The percentage of mature oocytes in the ovaries after 7 days of TMBPA exposure

[0049]

[0050] **Indicates T-test compared with the control group P <0.01; *** indicates comparison with the control group by T-test P <0.001.

[0051] Example 2 Hatching Artemia containing kaempferol

[0052] Two incubation buckets were prepared for hatching Artemia, one of which used conventional artificial seawater (salinity 30‰) for standard hatching, while the other used artificial seawater (salinity 30‰) containing 30 mg / L kaempferol for hatching. The incubation process was set to 24 hours and the temperature was maintained at 26°C. After the Artemia hatched, they were rinsed with running water to remove the kaempferol residue on their surface.

[0053] The observation results showed that there was no difference in vitality and state between the Artemia hatched using kaempferol and those hatched under normal conditions, indicating that kaempferol had no adverse effects on the growth of Artemia.

[0054] Example 3: Ovarian injury recovery experiment in medaka

[0055] 1. Methods

[0056] The marine medaka with impaired spawning function due to TMBPA exposure in Example 1 were gathered and randomly divided into two groups for subsequent experiments. One group of medaka was fed with normally hatched Artemia as the control group, while the other group was fed with Artemia treated with kaempferol. Four parallel experiments were set up for each group, and 20 fish (half male and half female) were placed in each parallel experiment and cultured in a 6L glass tank. The culture environment no longer contained TMBPA, the salinity was maintained at 15‰, the temperature was (28±1)℃, the light intensity was 1745 Lx, the light-dark cycle was set to 14 hours of light / 10 hours of darkness, and fresh water was replaced every 48 hours, and the daily spawning amount was counted during this period. After a 2-week feeding period (feeding twice a day), 4 female fish were randomly selected from each group for dissection, and the ovarian tissue was removed to make HE sections to analyze the proportion of mature oocytes in the ovaries.

[0057] 2. Results

[0058] The recovery test results are as follows Figure 2As shown, the ovarian tissue sections of the marine medaka fed with Artemia incubated with kaempferol ( Figure 2 B) compared with the control group ( Figure 2 A), the proportion of late yolk oocytes or mature oocytes is high.

[0059] The average egg production of the marine medaka after 14 days of recovery is shown in Table 3. Feeding normally hatched Artemia cannot effectively restore the damaged egg-laying function, and its egg production is still low. However, the egg production of medaka fed with Artemia treated with kaempferol increased significantly, which is very close to the level of the exposed experimental control group. In addition, the percentage of mature oocytes in the ovaries of the marine medaka after 14 days of recovery is shown in Table 4, which is also consistent with the improvement trend of egg production. Based on the above experimental results, the use of Artemia treated with kaempferol as feed to the marine medaka with impaired egg-laying function twice a day can effectively restore the egg-laying function of the marine medaka damaged by endocrine disruptors after two weeks of feeding.

[0060] Table 3 Average egg production / egg after 14 days of recovery

[0061]

[0062] Table 4 The percentage of mature oocytes in the ovaries after 14 days of recovery

[0063]

[0064] **Indicates T-test compared with the control group P <0.01; *** indicates comparison with the control group by T-test P <0.001.

Claims

1. Use of kaempferol as the sole active ingredient in the preparation of a product for improving the spawning function impairment of fish, characterized in that: The fish spawning function damage is caused by endocrine disruptors; the endocrine disruptor is tetramethyl bisphenol A; and the fish is marine medaka.

2. The application according to claim 1, characterized in that: The damage to the fish's spawning function is a decrease in the amount of fish eggs laid and / or a decrease in the proportion of mature oocytes in the ovaries.

3. The application according to claim 1, characterized in that: The improvement is to restore the spawning ability of fish ovaries and increase the proportion of mature oocytes in fish ovaries.

4. The use according to claim 1, characterized in that: The product is a medicine or a feed.

5. The application according to claim 4, characterized in that: The feed is Artemia fed with kaempferol.

6. The use according to claim 5, characterized in that: The concentration of the kaempferol in the feeding is 25-35 mg / L.

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