Application of quercetin in preparation of medicine for preventing and treating iron overload type liver injury and oxidative stress of snakehead
By adding quercetin to snakehead feed, the problems of liver damage and oxidative stress caused by iron overload were solved, significantly improving the liver function and immune response of snakehead and achieving a protective effect on snakehead.
Patent Information
- Application Number
- CN202511518765.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-23
- Publication Date
- 2025-11-28
AI Technical Summary
In aquaculture, iron overload leads to liver damage and oxidative stress in snakehead fish, and current technologies lack effective prevention and control measures.
Quercetin was used as the active ingredient, with an addition dose of 300 mg/kg, to prepare drugs for preventing and treating iron overload-induced liver injury and oxidative stress in snakehead fish. The drugs were prepared in various dosage forms, including tablets, granules, powders, granules, suspensions, and oral liquids. The therapeutic effects were verified through animal experiments.
Quercetin significantly reduced the levels of ROS, MDA, and 8-OHdG in the liver of snakehead fish, increased the activity of SOD and CAT in the liver, enhanced antioxidant capacity, increased the content of non-specific immune markers LZM, C3, C4, and IgM, alleviated oxidative stress, and enhanced immune response.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of aquaculture, and particularly relates to application of quercetin in preparation of a medicine for preventing and treating iron overload type liver damage and oxidative stress of snakehead. BACKGROUND
[0002] In aquaculture, in order to improve growth performance or prevent anemia, iron agents (such as ferrous sulfate, methionine iron) are often added in excess in feed. If the dosage is not precisely controlled, long-term feeding will lead to iron accumulation in the liver. Iron participates in the formation of Hb, Ferritin and Tf, and is also the active center of many enzyme systems. It is an essential mineral for vertebrates, and the body iron level plays an important role in physiological activities such as cell oxygen supply, nutrient absorption, energy metabolism, genetic material synthesis and immune response. However, when the body iron content exceeds the binding capacity of Tf, free iron ions will deposit in organs such as the liver. The liver is the main site for storing iron and is closely related to iron metabolism, and is one of the organs that are most easily damaged when iron overload occurs. Appropriate iron and the active oxygen (ROS) generated by catalysis are beneficial to cell functions such as mitochondrial respiration, transcription factor activation and signal transduction. However, a large amount of ROS produced through the Fenton reaction when iron is overloaded induces oxidative stress, damages liver cells, destroys immune homeostasis, and leads to growth retardation and increased mortality. Therefore, establishing a targeted prevention and control strategy has dual significance for ensuring the quality and safety of aquatic products and the economic benefits of aquaculture.
[0003] Quercetin is a flavonoid compound, and its research in the field of aquatic animals is less. This substance can be extracted from plants such as onions and hawthorns, and is one of the flavonoids with the highest intake in humans and animals. As a powerful antioxidant, quercetin can effectively chelate transition metal ions and scavenge ROS. It can induce the body to synthesize glutathione (GSH): superoxide dismutase (SOD) converts oxygen into hydrogen peroxide (H2O2), which is then catalyzed to decompose into water molecules, and GSH acts as a hydrogen donor in this process. Glutathione peroxidase (GSH-Px) can specifically catalyze the reaction of GSH and H2O2, thereby reducing the generation of peroxide and maintaining normal body function.
[0004] Channa argus, commonly known as black fish, raw fish or snakehead fish, belongs to Channa and is a kind of freshwater economic fish widely distributed in East Asia. In China, its natural distribution covers the Yangtze River Basin and the area north to Heilongjiang, especially Hubei, Jiangxi, Anhui, Henan and Liaoning provinces. Channa argus has strong adaptability, high tolerance to low oxygen, turbid water and water temperature changes, and can directly breathe air through the gill organ, and can survive for several days after leaving the water. As a carnivorous fish, Channa argus grows rapidly, has high meat content, less muscle spines and tender meat, and has the effects of removing blood stasis, activating blood and nourishing and tonifying. It has become a best-selling high-grade aquatic product in the market, and its breeding mode is flexible, with the advantages of "low investment and fast effect".
[0005] In summary, previous studies have reported that Channa argus has a suitable demand for iron, and whether oxidative stress occurs during the establishment of an iron overload-induced liver injury model in Channa argus and whether quercetin has a protective effect when fish are iron overloaded have not been reported. SUMMARY
[0006] Therefore, the main purpose of the present application is to provide the application of quercetin in the preparation of a drug for preventing and treating iron overload type liver injury and oxidative stress in Channa argus.
[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:
[0008] In a first aspect of the present application, the application of quercetin in the preparation of a drug for preventing and treating iron overload type liver injury and oxidative stress in Channa argus is provided, wherein the structural formula of quercetin is , the molecular formula is C 15 H 10 O7, and the CAS number is 117-39-5.
[0009] As a preferred embodiment, the drug comprises an effective dose of quercetin and takes it as an active ingredient.
[0010] As a preferred embodiment, the drug further comprises a pharmaceutically acceptable carrier or excipient, and the dosage form comprises at least one of tablet, granule, powder, powder, suspension, oral liquid.
[0011] As a preferred embodiment, the addition amount of quercetin is 150-450 mg / kg.
[0012] As a more preferred embodiment, the addition amount of quercetin is 300 mg / kg.
[0013] In a second aspect of the present application, a pharmaceutical composition for preventing and treating iron overload type liver injury and oxidative stress in Channa argus is provided, which comprises an effective dose of quercetin and takes it as an active ingredient, wherein the structural formula of quercetin is , the molecular formula is C15 H 10 O7, CAS No. 117-39-5.
[0014] Compared with the prior art, the present application is verified by animal experiments, different gradient quercetin is added in fish compound feed containing ferrous sulfate heptahydrate, the quercetin addition amount is 150-450 mg / kg, the ferrous sulfate heptahydrate content is 5000 mg / kg, the liver damage index, liver iron content, oxidative stress and immune index are evaluated, and the therapeutic effect of quercetin on liver iron overload snakehead is determined, and the beneficial effect is that:
[0015] (1) The present application is verified by experiment, and 300 mg / kg of quercetin can relieve liver damage of snakehead caused by iron overload.
[0016] (2) The present application is verified by experiment, and 300 mg / kg of quercetin can make the liver ROS, malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) levels of snakehead decrease, the liver SOD, liver catalase (CAT), total antioxidant capacity (T-AOC) activity increase, and the CAT, Cu / ZnSOD, glutathione sulfur transferase (GST), GPx gene expression level up-regulate, relieve oxidative stress and enhance antioxidant capacity.
[0017] (3) The present application is verified by experiment, and 300 mg / kg of quercetin can increase the content of serum (LZM), complement 3 (C3), complement 4 (C4) and immunoglobulin M (IgM) of snakehead, and enhance the non-specific immune capacity. BRIEF DESCRIPTION OF DRAWINGS
[0018] Figure 1 The results of activity detection of liver damage biomarkers glutamic oxalate transaminase (AST) and glutamic pyruvic transaminase (ALT) of snakehead in different groups in the examples.
[0019] Figure 2 The results of content detection of serum LZM, C3, C4 and IgM of snakehead in different groups in the examples.
[0020] Figure 3 The results of liver iron content detection of snakehead in different groups in the examples.
[0021] Figure 4 The results of antioxidant enzyme activity and total antioxidant capacity test of snakehead in different groups in the examples.
[0022] Figure 5 The results of liver non-specific immune index test of snakehead in different groups in the examples.
[0023] Figure 6The results of the liver antioxidant gene expression level test of different groups of snakeheads in the examples. DETAILED DESCRIPTION
[0024] In order to more fully understand the technical solutions, objectives and advantages of the present application, the technical effects produced by the present application will be further described in detail and completely below in combination with the drawings and specific examples. Obviously, the described examples are only some of the examples of the present application, not all. It should be noted that other examples obtained by those of ordinary skill in the art without departing from the concept of the present application all belong to the protection scope of the present application.
[0025] The following examples verify that quercetin is used for preventing and treating iron overload type liver damage and oxidative stress of snakeheads, specifically including the following aspects:
[0026] (1) 900 snakeheads of similar specifications were randomly divided into 5 groups, with 6 replicates in each group.
[0027] (2) The snakeheads were fed with 5000 mg / kg ferrous sulfate heptahydrate and / or 4 kinds of daily rations containing different levels of quercetin for 56 days.
[0028] (3) The serum and liver samples of the snakeheads were subjected to kit determination, iron ion content detection, and liver antioxidant gene expression level detection, specifically including:
[0029] The whole blood was extracted from the tail vein using a 1 mL syringe, centrifuged at 3500 rpm for 15 min at 4 ℃, and the upper serum was aspirated and stored in a -20 ℃ refrigerator. The liver was dissected on ice under sterile conditions and stored in a -80 ℃ refrigerator.
[0030] The activities of liver damage biomarkers AST and ALT were detected by microplate method kit (Nanjing Jiancheng Biological Engineering Institute). The contents of serum LZM, C3, C4 and IgM were detected by ELISA kit (Changchun Jingmei Biological Engineering Co., Ltd.).
[0031] The iron content was detected using a Thermo Fisher Scientific inductively coupled plasma emission spectrometer.
[0032] Total RNA was extracted using a Trizol kit and quantified by spectrophotometry. cDNA was synthesized using a reverse transcriptase cDNA synthesis kit. The reaction program was set as follows: 95 ℃ pre-denaturation for 30 seconds, followed by 40 cycles of 95 ℃ denaturation for 5 seconds, annealing for 30 seconds and 72 ℃ extension for 30 seconds. The relative expression level of the gene was determined by 2 -ΔΔCt method, and the genes including β-actin, CAT, CU / ZnSOD, GST, GPx were detected.
[0033] (4) Combine the results obtained from step (3) to determine whether quercetin can prevent and treat iron overload-induced liver damage and oxidative stress in snakehead fish.
[0034] Example 1
[0035] I. Specific Grouping
[0036] (1) Control group (CON): Snakehead fish were fed a basal diet.
[0037] (2) High-iron group (Fe): Snakehead fish were fed a basic diet containing 5000 mg / kg ferrous sulfate heptahydrate (purity ≥99%, Fe ≥20%, Wuhan Disel Biotechnology Co., Ltd.).
[0038] (3) Treatment group (150+Fe): Snakehead fish were fed a basic diet containing 150mg / kg quercetin (purity ≥98%, Xi'an Changyue Biotechnology Co., Ltd.).
[0039] (4) Treatment group (300+Fe): Snakehead fish were fed a basic diet containing 300mg / kg quercetin (purity ≥98%, Xi'an Changyue Biotechnology Co., Ltd.) + 5000mg / kg ferrous sulfate heptahydrate (purity ≥99%, Fe ≥20%, Wuhan Disel Biotechnology Co., Ltd.).
[0040] (5) Treatment group (450+Fe): Snakehead fish were fed a basic diet containing 450mg / kg quercetin (purity ≥98%, Xi'an Changyue Biotechnology Co., Ltd.) + 5000mg / kg ferrous sulfate heptahydrate (purity ≥99%, Fe ≥20%, Wuhan Disel Biotechnology Co., Ltd.).
[0041] The basic diet formula is shown in Table 1.
[0042] Table 1: Basic Feed Formulation and Nutritional Composition
[0043]
[0044] 1. Vitamin premix (mg / kg) -1 Feed: Vitamin A, 7.2; Vitamin B, 1.5; Vitamin B2, 10; Niacin, 30; Calcium pantothenate, 30; Vitamin B6, 20; Biotin, 10; Inositol, 500; Folic acid, 20; Vitamin B12, 10; Vitamin C, 300; Vitamin D3, 10; Vitamin E, 150; Vitamin K3, 10.
[0045] 2. Mineral premix (mg / kg) -1Feed): MgS04-H20, 4000; CuS04-5H20, 19.2; FeS04-7H20, 750; MnS04-H20, 53.2; ZnS04-H20, 165; CoCl2-6H20, 50; KI, 1.3; Na2Se03, 25.
[0046] II. Breeding test
[0047] The snakehead (China, Jiangsu) had the same size and the body weight was (16.32 ± 0.14) g. Before starting the test, the fish were acclimated to the breeding environment for 2 weeks in the aquaculture system of the Aquaculture Nutrition and Feed Laboratory of Jilin Agricultural University, during which time they were provided with continuously aerated freshwater and commercial feed. The 900 fish were randomly divided into 5 groups, and the snakehead was fed three times a day (7:00, 12:00, 17:00) during the breeding process. The water was changed by two-thirds every two days during the experiment. The water temperature was maintained at (26 ± 1) °C, the pH value was 7.2 ± 0.1, the nitrite content was less than 0.05 mg / L, the ammonia content was less than 0.5 mg / L, and the dissolved oxygen content was higher than 5.0 mg / L.
[0048] III. Sample collection
[0049] After 56 days, fish were randomly selected from each tank and anesthetized with MS-222 (Sigma, USA). Whole blood was drawn from the tail vein using a 1 mL syringe, centrifuged at 3500 rpm for 15 min at 4°C, and the upper serum was aspirated and stored in a -20°C freezer. The liver was dissected under sterile conditions on ice and stored in a -80°C freezer.
[0050] Example 2: Detection of serological indicators
[0051] The activities of liver damage biomarkers AST and ALT were detected using a microplate kit (Nanjing Jiancheng Biological Engineering Institute). The contents of serum LZM, C3, C4, and IgM were detected using an ELISA kit (Changchun Jingmei Biological Engineering Co., Ltd.).
[0052] As shown in Table 2, compared with the control group, the activities of AST and ALT in the Fe group increased significantly (P < 0.05), while the activities of AST and ALT in the Fe+P group decreased significantly (P < 0.05) compared with the Fe group. Figure 1 Addition of 300 and 450 mg / kg quercetin to the serum of iron-overloaded O. falciaus The activities of AST and ALT in the Fe+P group decreased significantly (P < 0.05) compared with the Fe group.
[0053] As shown in Table 2, compared with the control group, the activities of AST and ALT in the Fe group increased significantly (P < 0.05), while the activities of AST and ALT in the Fe+P group decreased significantly (P < 0.05) compared with the Fe group. Figure 2 As shown, compared with the control group, the levels of LZM, IgM, C3, and C4 in the Fe group were significantly decreased (P < 0.05), while 300 mg / kg quercetin significantly increased serum LZM activity in iron-overloaded snakehead. 150, 300, and 450 mg / kg quercetin all significantly increased serum IgM activity, and 300 and 450 mg / kg quercetin significantly increased serum C3 activity (P < 0.05). Furthermore, quercetin also showed some improvement in C4 activity.
[0054] Example 3: Detection of liver iron content
[0055] Iron content in liver was determined using inductively coupled plasma atomic emission spectrometry (ICP-MS, Thermo Fisher Scientific, USA). First, organic matrix interference was eliminated through a combined carbonization-ashing pretreatment, followed by digestion with 10 mL of concentrated hydrochloric acid for 16 h. The mixture was heated on a temperature-controlled hot plate (130 °C) until the solution became clear. After purification through a 0.45 μm microporous membrane, the solution was quantitatively transferred to a standard volumetric flask (100 mL), and homogeneity was ensured using a gradient dilution method. Standard working solutions were prepared simultaneously (using gradient dilution technique), and a high-precision calibration curve (linear correlation coefficient R² ≥ 0.9999) was established. Detection was performed using a Thermo Fisher Scientific inductively coupled plasma atomic emission spectrometer.
[0056] like Figure 3 As shown, compared with the control group, the iron content in the liver of the Fe group was significantly increased (P<0.05), while the iron content in the liver of the iron-overloaded snakehead fish was significantly decreased after quercetin supplementation (P<0.05).
[0057] Example 4: Detection of liver oxidative stress indicators
[0058] Liver ROS were detected using a chemiluminescence assay kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd.), while MDA and 8-OHdG levels were detected using an ELISA kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd.).
[0059] like Figure 4 As shown, compared with the control group, the levels of ROS, MDA and 8-OHdG in the liver of Fe group were significantly increased (P<0.05), while the levels of ROS, MDA and 8-OHdG in the liver of iron-overloaded snakehead were significantly decreased compared with the Fe group at 150, 300 and 450 mg / kg quercetin (P<0.05).
[0060] Example 5: Detection of liver antioxidant capacity
[0061] CAT, SOD, GSH-Px, and T-AOC were analyzed using commercial reagent kits from Nanjing Jiancheng Biological Research Institute according to the instructions.
[0062] The gene expression levels of CAT, Cu-Zn / SOD, GST and GPx in the liver samples were analyzed by qPCR technology. Total RNA was extracted using a Trizol kit (Takara, Dalian, China) and quantified by spectrophotometry. cDNA was synthesized using a reverse transcriptase cDNA synthesis kit (Takara, Dalian, China). The qPCR reaction system was prepared as follows: forward and reverse primers (10 mM, 1 µL), SYBR qPCR Mix (10 µL) (Shandong Sikaijie Biotechnology Co., Ltd.), DEPC-treated water (7 µL), and cDNA template (1 µL). The primer sequences are shown in Table 2. The reaction program was set as follows: 95℃ pre-denaturation for 30 seconds, followed by 40 cycles of 95℃ denaturation for 5 seconds, annealing for 30 seconds, and 72℃ extension for 30 seconds. The relative expression level of the gene was determined using 2 -ΔΔCt method.
[0063] Table 2: Primer sequences
[0064]
[0065] As shown in Figure 5 , compared with the control group, the liver CAT, SOD, GPx and T-AOC activities of the Fe group were significantly reduced (P<0.05), while 300 and 450 mg / kg quercetin significantly increased the liver CAT, SOD, GPx and T-AOC activities of the iron-overloaded O. falcatus compared with the Fe group (P<0.05).
[0066] As shown in Figure 6 , compared with the control group, the liver CAT, Cu-Zn / SOD, GST and GPx gene expression levels of the Fe group were significantly down-regulated (P<0.05), while 300 and 450 mg / kg quercetin significantly up-regulated the liver CAT, Cu-Zn / SOD, GST and GPx gene expression levels of the iron-overloaded O. falcatus compared with the Fe group (P<0.05).
[0067] In summary, the serum AST, ALT, ROS, MDA, 8-OHdG levels and liver iron content of the iron-overloaded O. falcatus supplemented with quercetin were significantly decreased, the non-specific immune indicators LZM, IgM, C3 and C4 activities were significantly increased, the antioxidant capacity indicators CAT, SOD, GPx and T-AOC were significantly improved, and the antioxidant gene CAT, CU-Zn / SOD, GST and GPx expression levels were significantly up-regulated, proving that quercetin can be used to prepare drugs for preventing and treating iron-overloaded liver damage and oxidative stress in O. falcatus, and has important application value.
[0068] The above merely provides the preferred embodiments of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the present application shall fall into the scope of the present application.
Claims
1. The application of quercetin in the preparation of drugs for preventing and treating iron overload-induced liver injury and oxidative stress in snakehead fish, wherein the structural formula of quercetin is: The molecular formula is C 15 H 10 O7, CAS number 117-39-5.
2. The application according to claim 1, characterized in that, The drug comprises an effective dose of quercetin and uses it as the active ingredient.
3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients, and the dosage form includes at least one of the following formulations: tablets, granules, powders, granules, suspensions, and oral liquids.
4. The application according to claim 1, characterized in that, The amount of quercetin added is 150-450 mg / kg.
5. The application according to claim 4, characterized in that, The amount of quercetin added is 300 mg / kg.
6. A pharmaceutical composition for preventing and treating iron overload-induced liver injury and oxidative stress in snakehead fish, comprising an effective dose of quercetin as the active ingredient, wherein the structural formula of quercetin is as follows: The molecular formula is C 15 H 10 O7, CAS number 117-39-5.