Application of ovateleaf holly bark and rhizoma cyperi compound composition in preparation of medicine for preventing and controlling liver injury of livestock and poultry

Abstract

The invention discloses an application of an ovateleaf holly bark and rhizoma cyperi compound composition in preparation of a medicine for preventing and controlling liver injury of livestock and poultry, and the liver injury refers to mouse acute liver injury induced by lipopolysaccharide (LPS) / D-galactosamine (D-GalN) and compound chick liver injury induced by enrofloxacin (ENR) / LPS. According to the pharmaceutical composition, the levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) in serum can be remarkably reduced through preventive administration or therapeutic administration, liver tissue damage is avoided, the integrity of the liver tissue is maintained, the feed conversion ratio of chicks can be reduced, and a new thought is provided for prevention and treatment of liver injury of livestock and poultry.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a compound of Jiubiying and Xiangfu in the preparation of drugs for preventing and treating liver injury in livestock and poultry. Technical Background

[0002] The liver is the most important metabolic and digestive organ in animals, playing crucial roles in bile secretion, detoxification, immune defense, and blood volume regulation. In animal husbandry, the main causes of liver damage in livestock and poultry fall into four categories: pathogenic damage, drug-induced liver injury, nutritional liver injury, and environmental damage. When the liver is damaged, hepatocytes degenerate, the ability to produce bile weakens or is lost, protein metabolism is disrupted, and detoxification function declines. Mild cases present with slowed growth, anemia, decreased antibody levels, emaciation, gastroenteritis, jaundice, and other symptoms of liver disease. Severe cases can lead to edema, ascites, severe hemolysis and uremia, and even death. The health and growth rate of livestock and poultry are closely related to liver function; liver damage causes significant economic losses to the livestock industry. Therefore, strengthening prevention and control measures to prevent liver damage in livestock and poultry is crucial for the long-term development of the industry. In recent years, with the continuous implementation of the "antibiotic reduction and replacement" policy, finding new natural drugs for preventing and controlling liver damage is particularly important for protecting animal health and improving the quality of livestock and poultry products. Summary of the Invention

[0003] This invention provides the application of a compound composition of Jiubiying and Xiangfu in the preparation of drugs for preventing and treating liver injury in livestock and poultry, so as to protect animal health and improve livestock and poultry products.

[0004] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0005] Application of the combination of Jiubiying and Xiangfu in the preparation of drugs for the prevention and treatment of liver injury in livestock and poultry.

[0006] Furthermore, the Cyperus rotundus mentioned is vinegar-processed Cyperus rotundus.

[0007] Furthermore, the mass ratio of the described "Jiu Bi Ying" to "Zu Xiang Fu" is 1:1.

[0008] Furthermore, the liver injury is LPS / D-GalN-induced acute liver injury in mice and ENR / LPS-induced combined liver injury in chicks.

[0009] Furthermore, the application is manifested in the reduction of serum ALT levels caused by liver injury in livestock and poultry by the combination of Jiubiying and vinegar-processed Cyperus rotundus.

[0010] Furthermore, the application is demonstrated by the combination of Jiubiying and vinegar-processed Cyperus rotundus complex in reducing the increase in serum AST levels caused by liver injury in livestock and poultry.

[0011] Furthermore, the application demonstrated that the combination of Jiubiying and vinegar-processed Cyperus rotundus improved LPS / D-GalN-induced pathological damage to mouse liver tissue.

[0012] Furthermore, the application is manifested in the reduction of feed conversion ratio in chicks by the combination of Jiubiying and vinegar-processed Cyperus rotundus compound.

[0013] Furthermore, the drug comprises an aqueous extract of the compound composition or a lyophilized powder of the aqueous extract thereof, and other pharmaceutically acceptable excipients.

[0014] Further, the method for preparing the aqueous extract of the compound composition is as follows: *Jiu Bi Ying* and *Zui Xiang Fu* are mixed in a 1:1 mass ratio and soaked overnight in 10 times their mass volume of distilled water; the next day, the mixture is brought to a boil over high heat and then simmered over low heat for 60 minutes, and the filtrate is filtered; distilled water is added successively at a ratio of 1:8 and 1:6, and the above operation is repeated. The filtrates are combined, filtered under reduced pressure, and then concentrated to 500 mg / mL using a rotary evaporator. The concentrated solution is dispensed and stored at -20 °C for later use.

[0015] The beneficial effects of this invention are as follows:

[0016] 1. Significantly effective in preventing and controlling liver injury, with both preventative and therapeutic value: The compound composition of this invention (Jiubiying: vinegar-processed Cyperus rotundus = 1:1) can effectively reduce the elevation of serum ALT and AST levels caused by liver injury in livestock and poultry, and precisely improve the state of hepatocyte damage, whether administered preventively or therapeutically. In particular, the preventive administration can also significantly reduce LPS / D-GalN-induced pathological damage to mouse liver tissue, and has a targeted preventive and control effect on LPS / D-GalN-induced acute liver injury in mice and enrofloxacin / LPS-induced complex liver injury in chicks, providing an effective means for the whole-cycle prevention and control of liver injury in livestock and poultry.

[0017] 2. Improve the economic benefits of breeding and optimize the growth performance of livestock and poultry: The compound composition of the present invention can significantly reduce the feed conversion ratio of chicks, which means that feed consumption can be reduced and feed utilization can be improved during the breeding process, helping livestock and poultry to achieve weight gain more efficiently, reducing breeding costs while improving slaughter efficiency, effectively improving the problem of slow growth of livestock and poultry caused by liver damage, and helping farmers improve the overall benefits of large-scale breeding.

[0018] 3. Aligning with the trend of green farming and providing a new natural prevention and control solution: This invention uses a combination of natural Chinese medicinal herbs such as *Cyperus rotundus* and *Cyperus rotundus*, prepared through a traditional water extraction process (a mature and easily industrialized process). The drug components are natural and safe, avoiding the residue and drug resistance problems that may be caused by chemical drugs, and precisely aligning with the current policy direction of "reducing and replacing antibiotics". It not only provides a new natural drug approach for the prevention and control of liver injury in livestock and poultry, but also helps to improve the quality of livestock and poultry products, ensure the safety of animal-derived food, and promote the green and sustainable development of animal husbandry. Attached Figure Description

[0019] Figure 1 The graph shows the preventive reduction of ALT and AST levels in the serum of mice with liver injury by the aqueous extract of the compound composition.

[0020] Figure 2 Figure (200×) showing the preventive effect of the aqueous extract of the compound composition on LPS / D-GalN-injured mice for HE staining evaluation.

[0021] Figure 2 Note: A, blank control group; B, model group; C, positive drug control group; D, low-dose group; E, high-dose group; F, decoction control group.

[0022] Figure 3 The graph shows the therapeutic effect of the aqueous extract of the compound on reducing serum ALT and AST levels in mice with liver injury.

[0023] Figure 4 Figure (200×) showing the therapeutic effect of the aqueous extract of the compound composition on LPS / D-GalN-injured mice for HE staining evaluation.

[0024] Figure 4 Note: A, blank control group; B, model group; C, treatment group.

[0025] Figure 5 A graph showing how the lyophilized powder of the aqueous extract of the compound reduces ALT and AST levels in the serum of chicks with liver damage.

[0026] Figure 6 Figure (400×) showing the effect of HE staining on the effect of the lyophilized powder of the compound composition on chicks with liver injury.

[0027] Figure 6 Note: A, blank control group; B, model group; C, positive drug control group; D, low-dose group; E, high-dose group. Detailed Implementation Plan

[0028] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can more fully understand the technical solution, but the present invention is not limited to the scope of the described embodiments.

[0029] Example 1

[0030] Materials and reagents: Jiubiying (a traditional Chinese medicine), vinegar-processed Cyperus rotundus, LPS, D-GalN, liver and gallbladder granules, and ALT and AST detection kits were purchased from Nanjing Jiancheng.

[0031] Preparation of aqueous extract of the compound composition: *Jiu Bi Ying* and *Zui Xiang Fu* were mixed in a 1:1 mass ratio and soaked overnight in 10 times their mass volume of distilled water. The next day, the mixture was brought to a boil over high heat, then simmered for 60 minutes. The filtrate was filtered. Distilled water was added successively at a ratio of 1:8 and 1:6, and the above operation was repeated. The filtrates were combined, filtered under reduced pressure, and concentrated to 500 mg / mL using a rotary evaporator. The concentrate was dispensed and stored at -20 °C for later use.

[0032] Experimental animals: 36 Kunming mice weighing 16-18 g were purchased from Changsha Tianqin Biotechnology Co., Ltd. They were kept in a 12-hour day-night cycle, with the room temperature maintained at (25±2)℃, clean and sufficient drinking water, and 4 g of fresh mouse food per mouse per day. After one week of acclimatization, they were divided into groups for the experiment.

[0033] Grouping and treatment: 36 mice (n=6 per group) were randomly divided into 6 groups: blank control group, model group, positive drug control group, low-dose group, high-dose group, and treatment group. Specific administration and treatment methods are as follows:

[0034] (1) Blank control group: Pure water was continuously administered by gavage for 7 days, once a day. On the 7th day, 2 hours after gavage, an equal volume of physiological saline was injected into the peritoneum.

[0035] (2) Model group: Pure water was continuously administered by gavage for 7 days, once a day. On the 7th day, LPS / D-GalN (10 μg / kg + 700 mg / kg) was injected intraperitoneally 2 hours after gavage.

[0036] (3) Positive drug control group: The dosage of liver and gallbladder granules in mice was converted according to the instructions for use. The mice were administered the granules by gavage for 7 consecutive days, once a day. On the 7th day, 2 hours after gavage, LPS / D-GalN (10 μg / kg + 700 mg / kg) was injected intraperitoneally.

[0037] (4) Low-dose group: Low-dose decoction was continuously administered by gavage for 7 days, once a day. On the 7th day, 2 hours after gavage, LPS / D-GalN (10 μg / kg + 700 mg / kg) was injected intraperitoneally.

[0038] (5) High-dose group: High-dose decoction was continuously administered by gavage for 7 days, once a day. On the 7th day, 2 hours after gavage, LPS / D-GalN (10 μg / kg + 700 mg / kg) was injected intraperitoneally.

[0039] (6) Control group of decoction: High-dose decoction was administered by gavage for 7 days, once a day. On the 7th day, 2 hours after gavage, an equal volume of physiological saline was injected into the peritoneum.

[0040] Six hours after intraperitoneal injection of LPS / D-GalN or saline, mice were weighed, blood was collected from the ocular vein, whole blood was allowed to stand at room temperature for 2 hours, and the supernatant serum was separated by centrifugation at 3500 rpm for 15 minutes. ALT and AST levels in the serum were measured. The left lobe of the liver was taken, fixed in 4% formaldehyde, and then stained with hematoxylin and eosin (HE) for analysis.

[0041] result:

[0042] Pretreatment with the compound composition significantly reduced the elevated ALT and AST levels in mouse serum due to LPS / D-GalN stimulation, and this effect was dose-dependent. Figure 1 The high-dose group showed better hepatoprotective effects than the positive control drug, hepatobiliary granules, while its ALT and AST levels were not significantly different from those of the blank group. Figure 1 ). Analysis of HE staining results ( Figure 2 As can be seen, the liver tissue in group M completely lost its original tissue structure, filled with erythrocyte infiltration, vacuoles of varying sizes and ballooning degeneration, and hepatocyte nuclei showed pyknosis and necrosis. The high-dose compound composition prevented liver hemorrhage caused by LPS / D-GalN, reduced vacuoles and ballooning degeneration, and maintained the integrity of hepatocyte cords to a certain extent; its preventive effect was far superior to that of the low-dose complex and hepatobiliary granules. The high-dose compound composition alone did not affect the tissue integrity and function of the liver.

[0043] Example 2

[0044] Materials and reagents: Jiubiying (a traditional Chinese medicine), vinegar-processed Cyperus rotundus, LPS, D-GalN, liver and gallbladder granules, and ALT and AST detection kits were purchased from Nanjing Jiancheng.

[0045] Preparation of aqueous extract of the compound composition: *Jiu Bi Ying* and *Zui Xiang Fu* were mixed in a 1:1 mass ratio and soaked overnight in 10 times their mass volume of distilled water. The next day, the mixture was brought to a boil over high heat, then simmered for 60 minutes. The filtrate was filtered. Distilled water was added successively at a ratio of 1:8 and 1:6, and the above operation was repeated. The filtrates were combined, filtered under reduced pressure, and concentrated to 500 mg / mL using a rotary evaporator. The concentrate was dispensed and stored at -20 °C for later use.

[0046] Experimental animals: 18 Kunming mice weighing 16-18 g were purchased from Changsha Tianqin Biotechnology Co., Ltd. They were kept in a 12-hour day-night cycle, with the room temperature maintained at (25±2)℃, clean and sufficient drinking water, and 4 g of fresh mouse food per mouse per day. After one week of acclimatization, they were divided into groups for the experiment.

[0047] Grouping and treatment: Six to eighteen mice were randomly divided into three groups: a blank control group, a model group, and a treatment group. Specific drug administration and treatment methods are as follows:

[0048] (1) Blank control group: an equal volume of normal saline was injected into the peritoneum, and an equal volume of pure water was administered by gavage 1 h later;

[0049] (2) Model group: LPS / D-GalN (10 μg / kg + 700 mg / kg) was injected intraperitoneally, followed by gavage with an equal volume of pure water 1 h later;

[0050] (3) Treatment group: LPS / D-GalN (10 μg / kg + 700 mg / kg) was injected intraperitoneally, followed by oral administration of the aqueous extract 1 h later;

[0051] Six hours after intraperitoneal injection of LPS / D-GalN or saline, mice were weighed, blood was collected from the ocular vein, whole blood was allowed to stand at room temperature for 2 hours, and the supernatant serum was separated by centrifugation at 3500 rpm for 15 minutes. ALT and AST levels in the serum were measured. The left lobe of the liver was taken, fixed in 4% formaldehyde, and then stained with hematoxylin and eosin (HE) for analysis.

[0052] result:

[0053] Therapeutic administration of the compound also significantly reduced the elevated ALT and AST levels in mouse serum due to LPS / D-GalN stimulation. Figure 3 ).Depend on Figure 4 It can be seen that the compound composition completely inhibited liver bleeding, reduced vacuolation and ballooning degeneration, and maintained the integrity of hepatocyte cords to a certain extent.

[0054] Example 3

[0055] Materials and reagents: Jiubiying (a traditional Chinese medicine), vinegar-processed Cyperus rotundus, LPS, enrofloxacin hydrochloride (ENR), liver and gallbladder granules, and ALT and AST detection kits were purchased from Nanjing Jiancheng.

[0056] Preparation of aqueous extract of the compound composition: *Jiu Bi Ying* and *Cyperus rotundus* were mixed in a 1:1 mass ratio and soaked overnight in 10 times their mass volume of distilled water. The next day, the mixture was brought to a boil over high heat, then simmered for 60 minutes. The filtrate was filtered. Distilled water was added sequentially at a ratio of 1:8 and 1:6, and the above operation was repeated. The filtrates were combined, filtered under reduced pressure, and concentrated to 1 g / mL using a rotary evaporator. The filtrate was then pre-cooled overnight at -80℃ and frozen in a vacuum freeze dryer to obtain lyophilized powder, which was stored at -20℃ for later use.

[0057] Experimental animals: 150 one-day-old Guangxi Sanhuang chickens were purchased from Guangxi Fufeng Agricultural and Animal Husbandry Group. They were normally fed, allowed free access to clean food and water. On day 14, 30 chickens were randomly divided into 5 groups: blank control group, model group, low-dose group, high-dose group, and positive control group. Specific drug administration and treatment methods are as follows:

[0058] (1) Blank control group: From day 15, the animals were fed feed without any drugs and had normal food and water; on day 22, they were injected with an equal amount of physiological saline.

[0059] (2) Model group: From day 15, feed without any drugs was given and normal drinking water was provided. On day 20, ENR was mixed into the drinking water (100 mg / Kg) for 3 consecutive days. On day 22, after mixing into the drinking water, LPS was injected intraperitoneally at a dose of 200 μg / Kg.

[0060] (3) Positive drug control group: From day 15, feed without any drugs was given for 7 consecutive days. Liver and gallbladder granules 1g / L were mixed in drinking water. On day 20, ENR (100 mg / Kg) was mixed in drinking water. This treatment was continued for 3 days. On day 22, after mixing in drinking water, LPS was injected intraperitoneally at 200 μg / Kg.

[0061] (4) Low-dose group: From day 15, feed was prepared with a mixture of low-dose compound composition and normal drinking water. On day 20, ENR (100 mg / Kg) was mixed into the drinking water and treated for 3 consecutive days. On day 22, after mixing into the drinking water, LPS was injected intraperitoneally at a dose of 200 μg / Kg.

[0062] (5) High-dose group: From day 10, feed was mixed with high-dose compound composition and normal drinking water was provided. On day 20, ENR (100 mg / Kg) was mixed into the drinking water and treated for 3 consecutive days. On day 22, after mixing into the drinking water, LPS was injected intraperitoneally at a dose of 200 μg / Kg.

[0063] From day 15 to 22, chicks were weighed daily, and feed intake for each group was recorded to calculate the feed conversion ratio. On day 22, chicks were weighed 6 hours after intraperitoneal injection of LPS or saline, euthanized, and blood was collected. Whole blood was allowed to stand at room temperature for 2 hours, then centrifuged at 3500 rpm for 15 minutes to separate the supernatant serum, and ALT and AST levels were measured in the serum. A portion of liver tissue was collected, fixed in 4% saline solution, and stained with hematoxylin and eosin (HE) for analysis.

[0064] result:

[0065]

[0066] Note: All data in the table are expressed as averages; different letters in the upper right corner indicate significant differences between groups, i.e., P < 0.05; the same letter in the upper right corner indicates no difference between groups, i.e., P > 0.05.

[0067] Depend on Figure 5 and Figure 6It is known that adding the compound composition to the diet can prevent liver damage in chicks, and this effect is dose-dependent. High doses of the compound composition significantly reduce the high levels of ALT and AST caused by ENR / LPS stimulation; significantly improve liver tissue damage, prevent vacuolation in hepatocyte cytoplasm, improve the dilation of hepatic sinusoids, and maintain the integrity of hepatocyte cords; adding the compound composition alone to the feed does not affect the integrity and function of liver tissue.

Claims

1. Application of the combination of Jiubiying and Xiangfu in the preparation of drugs for the prevention and treatment of liver injury in livestock and poultry.

2. The application of the compound composition of Jiubiying and Xiangfu as described in claim 1 in the preparation of drugs for preventing and treating liver injury in livestock and poultry, characterized in that, The Cyperus rotundus mentioned is vinegar-processed Cyperus rotundus.

3. The application of the compound composition of Jiubiying and Xiangfu as described in claim 2 in the preparation of drugs for preventing and treating liver injury in livestock and poultry, characterized in that, The mass ratio of the aforementioned Jiubiying to vinegar-processed Cyperus rotundus is 1:

1.

4. The use of the compound composition of Jiubiying and Xiangfu as described in any one of claims 1-3 in the preparation of drugs for preventing and treating liver injury in livestock and poultry, characterized in that, The liver injury referred to was LPS / D-GalN-induced acute liver injury in mice and ENR / LPS-induced combined liver injury in chicks.

5. The application of the compound composition of Jiubiying and Xiangfu as described in claim 2 or 3 in the preparation of drugs for preventing and treating liver injury in livestock and poultry, characterized in that, The application is characterized by the reduction of serum ALT levels caused by liver injury in livestock and poultry by the combination of Jiubiying and vinegar-processed Cyperus rotundus.

6. The application of the compound composition of Jiubiying and Xiangfu as described in claim 2 or 3 in the preparation of drugs for preventing and treating liver injury in livestock and poultry, characterized in that, The application is shown to reduce the increase in serum AST levels caused by liver injury in livestock and poultry by the combination of Jiubiying and vinegar-processed Cyperus rotundus.

7. The application of the compound composition of Jiubiying and Xiangfu as described in claim 2 or 3 in the preparation of drugs for preventing and treating liver injury in livestock and poultry, characterized in that, The application demonstrated that the combination of Jiubiying and vinegar-processed Cyperus rotundus improved LPS / D-GalN-induced pathological damage to mouse liver tissue.

8. The application of the compound composition of Jiubiying and Xiangfu as described in claim 2 or 3 in the preparation of drugs for preventing and treating liver injury in livestock and poultry, characterized in that, The application is manifested in the reduction of feed conversion ratio in chicks by the combination of Jiubiying and vinegar-processed Cyperus rotundus.

9. The use of the compound composition of Jiubiying and Xiangfu as described in any one of claims 1-3 in the preparation of a drug for preventing and treating liver injury in livestock and poultry, characterized in that, The drug comprises an aqueous extract of the compound composition or a lyophilized powder of the aqueous extract thereof, and other pharmaceutically acceptable excipients.

10. The application of the compound composition of Jiubiying and Xiangfu as described in claim 9 in the preparation of drugs for preventing and treating liver injury in livestock and poultry, characterized in that, The method for preparing the aqueous extract of the compound composition is as follows: after mixing Jiubiying and vinegar-processed Xiangfu in a 1:1 mass ratio, soak them overnight in 10 times their mass volume of distilled water. The next day, bring to a boil over high heat, then simmer over low heat for 60 minutes. Filter the filtrate. Add distilled water in a 1:8 and 1:6 ratio, repeating the above steps. Combine the filtrates, filter under reduced pressure, and concentrate the filtrate to 500 mg / mL using a rotary evaporator. Dispense the concentrate and store at -20 °C for later use.

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