A traditional Chinese medicine composition for alleviating fatty liver of largemouth bass, preparation method and application thereof

Abstract

The present invention belongs to the technical field of fish medicines and discloses a traditional Chinese medicine composition for alleviating fatty liver disease in largemouth bass. The composition comprises the following components by weight: 20 parts of bupleurum root, 20 parts of kudzu root, 10 parts each of scutellaria baicalensis, white peony root, pinellia tuber, ginger, and immature bitter orange, 6 parts of rhubarb, 20 parts of smilax glabra, 20 parts of dandelion, 10 parts of platycodon grandiflorum, 10 parts of cimicifuga, and jujube; wherein 4 jujubes are added to every 20g of bupleurum root. The drug of the present invention has the advantages of good therapeutic effect, low cost, convenient consumption, and no side effects for treating fatty liver disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of fish medicines, and in particular to a traditional Chinese medicine composition for alleviating fatty liver of largemouth seabass, a preparation method and application thereof. Background Art

[0002] Active ingredients in Chinese herbal medicines can attract aquatic animals, improve their growth performance, enhance their immune function, prevent diseases, reduce fat deposition, and enhance product quality, offering promising applications in aquaculture. The research and development of Chinese herbal additives is still in its infancy, and the following challenges remain: First, the composition of Chinese herbal medicines is complex, with potential for synergistic or antagonistic interactions between different components, and the mechanisms of action remain unclear. Second, processing techniques are not advanced enough, with traditional mechanical crushing methods used for Chinese herbal medicines resulting in inefficient utilization of active ingredients. Third, the metabolic profiles of different Chinese herbal additives vary significantly, leading to a lack of comprehensive and reliable safety assessments. Future efforts require strengthening basic research on Chinese herbal medicines, incorporating modern medical technologies and integrating pharmacological and toxicological research methods to conduct in-depth analysis of their active ingredients and uncover the mechanisms of interaction between them. Advanced production processes and equipment should be introduced to facilitate refined production and develop diverse and specific Chinese herbal additives. Furthermore, safety assessments should be strengthened, with the development of scientifically standardized formulations for Chinese herbal grass and feed additives. A database for basic Chinese herbal medicine data query and typical formulation design should be established to advance research on the application of Chinese herbal additives in aquaculture.

[0003] The largemouth bass (Micropterus salmoides) is a renowned and highly prized aquaculture species in my country. It belongs to the order Perciformes, suborder Perciformes, family Heliopsidae, and genus Micropterus. It is a eurythermal carnivorous fish native to the freshwater waters of North America, commonly known in the Chinese market as the California bass. It is even considered the fifth largest fish in my country. However, due to insufficient anti-lipid factors in their livers and low sugar tolerance, largemouth bass are congenitally diabetic, resulting in extremely low starch utilization. Long-term consumption of high-sugar, high-fat diets can lead to liver dysfunction, slowed growth, and even death. Furthermore, due to the rising price of fish meal, the use of plant protein as a partial replacement for fish meal in fish feeds has become commonplace. Plant protein ingredients contain a high sugar content, resulting in high sugar content in feeds. To reduce costs, fish farmers often use feeds high in plant protein. This can lead to liver dysfunction, poor stress tolerance, and significant production reductions in largemouth bass, resulting in economic losses and hindering the development of the largemouth bass aquaculture industry. Therefore, in the breeding process of largemouth black bass, the maintenance of liver and gallbladder is of great significance to the increase of largemouth black bass production. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a traditional Chinese medicine composition for alleviating fatty liver of largemouth seabass, a preparation method and application.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] A traditional Chinese medicine composition for alleviating fatty liver in largemouth seabass, comprising the following components in parts by weight:

[0007] 20 parts of Bupleurum, 20 parts of Puerariae, 10 parts each of Scutellaria, White Peony, Pinellia, Ginger, and Citrus aurantium, 6 parts of Rhubarb, 20 parts of Smilax, 20 parts of Taraxacum, 10 parts of Platycodon, 10 parts of Cimicifuga, and jujube;

[0008] Among them, add 4 dates to every 20g of Bupleurum.

[0009] The preparation method of the above-mentioned Chinese medicine composition comprises the following steps:

[0010] After the various components are evenly mixed, they are crushed, passed through a 100-mesh sieve, and evenly mixed to obtain powder, which is the traditional Chinese medicine composition for alleviating fatty liver of largemouth seabass.

[0011] Use of the above-mentioned traditional Chinese medicine composition in the preparation of medicines for treating fatty liver and / or hyperlipidemia.

[0012] Furthermore, the medicine is a lotion, ointment or powder.

[0013] The advantages and positive effects achieved by the present invention are:

[0014] 1. The medicine of the present invention has the advantages of good therapeutic effect, low cost, convenient consumption and no side effects when used to treat fatty liver.

[0015] 2. The Chinese herbal medicine additive of the present invention not only contains rich nutrients, but also some of its active ingredients can improve the palatability of feed, increase the food intake of fish and shrimp, and improve feed utilization rate. The application of composite Chinese herbal medicine as a feed additive to improve the disease resistance and anti-stress response of largemouth seabass has become a development trend. So far, there are not many studies on the use of Chinese herbal medicine to prevent largemouth seabass diseases and anti-stress. There is no Chinese herbal medicine feed additive for promoting growth, preventing diseases and resisting stress for largemouth seabass. Therefore, the application technology of Chinese herbal medicine additives for largemouth seabass feed is improved, and a green, environmentally friendly and efficient largemouth seabass growth-promoting and disease-preventing Chinese herbal medicine composite prescription is developed to meet the needs of white shrimp farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Graph showing the effects of different fat levels on the morphology of liver cells in largemouth bass (H&E staining); a: control group; b: L16 group; c: L22 group. DETAILED DESCRIPTION

[0017] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0018] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0019] A traditional Chinese medicine composition for alleviating fatty liver in largemouth seabass, comprising the following components in parts by weight:

[0020] 20 parts of Bupleurum, 20 parts of Puerariae, 10 parts each of Scutellaria, White Peony, Pinellia, Ginger, and Citrus aurantium, 6 parts of Rhubarb, 20 parts of Smilax, 20 parts of Taraxacum, 10 parts of Platycodon, 10 parts of Cimicifuga, and jujube;

[0021] Among them, add 4 dates to every 20g of Bupleurum.

[0022] The preparation method of the above-mentioned Chinese medicine composition comprises the following steps:

[0023] After the various components are evenly mixed, they are crushed, passed through a 100-mesh sieve, and evenly mixed to obtain powder, which is the traditional Chinese medicine composition for alleviating fatty liver of largemouth seabass.

[0024] Use of the above-mentioned traditional Chinese medicine composition in the preparation of medicines for treating fatty liver and / or hyperlipidemia.

[0025] Preferably, the drug is in the form of lotion, ointment or powder.

[0026] Specifically, the relevant preparation and detection are as follows:

[0027] 1 Materials and Methods

[0028] 1.1 Preparation of compound Chinese herbal medicine (Example 1)

[0029] Weigh 20g of bupleurum, 20g of kudzu root, 10g each of scutellaria, white peony root, pinellia, ginger, and immature bitter orange, 6g of rhubarb, 20g of smilax, 20g of dandelion, 10g of platycodon, 10g of cimicifuga, and 4 jujubes. Crush them, pass through a 100-mesh sieve, and mix them evenly to obtain a powder. This is a traditional Chinese medicine composition for alleviating fatty liver disease in largemouth seabass.

[0030] Comparative Example 1

[0031] The preparation method is exactly the same as that of Example 1, except that no Bupleurum is added.

[0032] Comparative Example 2

[0033] The preparation method is exactly the same as that of Example 1, except that: Scutellaria baicalensis is not added.

[0034] 1.2 Methods

[0035] This study used juvenile largemouth bass as the research subjects. In the first phase, 360 robust and uniformly sized juvenile largemouth bass were randomly divided into three groups: a normal control group, model group 1, and model group 2. Each group consisted of three replicates, with 40 fish per group. The normal control group was fed a basal diet with a 10% fat content, while the high-fat group 1 was fed a high-fat diet with a 16% fat content, and the high-fat group 2 was fed a high-fat diet with a 22% fat content. All groups of largemouth bass were maintained normally.

[0036] After the modeling was successful, 120 largemouth bass were modeled and randomly divided into two groups of 120 similar-sized model largemouth bass, namely the model group and the Chinese medicine group, with 3 repetitions in each group. In the Chinese medicine group, 120 g / kg of the powder of Example 1 was added to a high-fat diet. The experiment was divided into two phases, each phase was 30 days, and the experimental feed was fed intermittently, that is, the experimental feed was fed for 7 days before the first phase, and the high-fat diet was fed for 23 days after the second phase. The experimental feed was fed for 15 days before the second phase, and the high-fat diet was fed for 15 days after the second phase. The daily feeding amount was 2% of the largemouth bass body weight, and the fish body weight, body length and other indices were measured on the 7th, 30th, 45th and 60th days of the experiment. Before sampling, the fish were fasted for 24 hours. Twelve largemouth seabass were randomly collected, and their body weight and length were measured. After blood was collected from the tail vein of the largemouth seabass, the largemouth seabass were placed on ice packs for dissection, and their viscera were removed and washed with 4°C pre-cooled physiological saline. The water on the viscera was absorbed with filter paper, and the weight of the viscera was accurately weighed. Then, the hepatopancreas was separated and the weight of the hepatopancreas was accurately weighed. The liver and intestine of a random fish were placed in fixative for later sectioning. Finally, all other samples were stored in a -80°C refrigerator for testing.

[0037] 2 Results Analysis

[0038] 2.1 Establishment of fatty liver model

[0039] 2.1.1 Effects of feeding different fat content diets on the growth performance of juvenile largemouth bass

[0040] Table 1 shows the effects of diets with varying fat content on the growth performance of largemouth bass. Results showed that, compared with the control group, both the L16 and L22 diets increased final body weight after 60 days of feeding, with the L22 group experiencing the greatest weight gain. While the L16 group showed no significant difference in final body length compared to the control group, the L22 group showed significant differences in final body length compared to both the control group and the L16 group. In terms of weight gain rate and specific growth rate, the L22 group showed significant differences compared to both the control group and the L16 group, significantly exceeding both groups. During the experimental feeding period, no largemouth bass died in any of the groups, indicating no significant differences in survival rates.

[0041] Table 1 Effects of different fat levels on the growth of largemouth bass

[0042]

[0043] 2.1.2 Effects of feeding different fat content diets on the body size of juvenile largemouth bass

[0044] The effects of diets with varying fat contents on the growth performance of largemouth bass are shown in Table 2. The results showed that, compared with the control group, feeding the L22 diet for 60 days significantly improved the visceral and hepatosomatic indices of largemouth bass. Significant differences were observed between the L16 and L22 groups and the control group in terms of empty shell rate and hepatosomatic index. During the experimental feeding period, no largemouth bass died in any of the groups, indicating no significant differences in survival rates.

[0045] Table 2 Effects of different fat levels on body size of largemouth bass

[0046]

[0047] 2.1.3 Effects of different fat levels on liver cell morphology in largemouth bass

[0048] After feeding the largemouth bass for 60 days, the liver of the largemouth bass was taken and fixed and sectioned and stained with H&E. The results are shown in Figure 1 . It was found that the liver cell structure of the control group was relatively complete, the intercellular space was small, the nucleus did not show obvious rupture but nuclear pyknosis had occurred, and the liver cells had obvious granular changes. Compared with the control group, the liver of the largemouth bass in the 16% fat water group showed obvious vacuoles, relatively obvious cell edges, intact cell structure, fatty degeneration of the liver cells, focal necrosis, and focal necrosis of the liver cell nuclei with nuclear pyknosis, nuclear rupture and nuclear dissolution. The liver of the largemouth bass in the 22% fat level group showed severe congestion of the hepatic sinusoids, a reticular structure of the liver, and nearly round vacuoles of varying sizes in the cytoplasm. The hepatocyte nuclei had been squeezed under the cell membrane. The number of lipid droplets and vacuoles was large, almost covering the entire section, and the cell structure was unclear.

[0049] 2.1.4 Discussion

[0050] 2.1.4.1 Effects of different fat levels on growth performance and fatty liver in largemouth bass

[0051] The present invention uses a fat level of 9% as the fat level of the control group feed. At the same time, fat levels of 16% and 22% are set. After feeding the largemouth sea bass for 60 days, its growth and body shape indicators are measured to establish a fatty liver model that is easier to obtain. In this experiment, it was found that the L22 group grew faster, had the lowest empty shell rate, and had the highest liver and visceral body indexes after 60 days of high-fat feeding. The autopsy results showed that after 60 days of simultaneous feeding, both the L16 and L22 groups had severe fatty degeneration, of which the L22 group was the most serious. A large number of studies have shown that fatty degeneration of the liver is positively correlated with the fat content in the feed. HE staining showed that on the 60th day, a large number of vacuoles appeared in the liver of the largemouth sea bass in the control group, while the liver cells in the L22 group were arranged in a grid, accompanied by an increase in the degree of fibrosis. The L22 group meets the conditions for fatty liver.

[0052] 2.1.4.2 Effects of different fat levels on antioxidant and lipid metabolism in largemouth bass

[0053] After 60 days of continuous feeding, the antioxidant and oxidative indices of largemouth bass increased, and the L22 group was the largest, indicating that after long-term feeding of high-fat feed, the liver of largemouth bass was damaged to a certain extent and nutritional fatty liver was produced.

[0054] 2.1.4.3 Summary

[0055] After 60 days of continuous feeding of a high-fat diet containing 22% lipids, largemouth bass livers underwent a color change from bright red to whitish and from normal to enlarged. Hepatic steatosis gradually worsened with increasing fat content, ultimately developing into fatty liver disease. This study met the criteria for severe fatty liver disease in largemouth bass liver color change, hepatocyte morphology, and hepatocyte vacuolation. Therefore, continuous feeding of a high-fat diet containing 22% lipids successfully established a fatty liver model in largemouth bass.

[0056] 2.2 Effects of feeding compound Chinese herbal medicine on largemouth bass with fatty liver disease

[0057] 2.2.1 Effects of compound Chinese herbal medicine on the growth of largemouth bass

[0058] As can be seen from Table 3, on the 30th day of the experiment, the Example 1 group promoted the growth of the body weight of largemouth seabass, but there was no significance compared with the control group. In terms of weight gain rate and specific growth rate, the Example 1 group was significant compared with the control group, and there was a significant improvement. In terms of fatness, it was found that the fatness of the largemouth seabass fed with Chinese herbal medicine was less than that of the control group, but there was no significance. On the 60th day of the experiment, the body weight of the Example 1 group was higher than that of the control group, but there was no significance. In terms of weight gain rate and specific growth rate, the Example 1 group was significant compared with the control group, and there was a significant improvement. In terms of fatness, it was found that the fatness of the largemouth seabass fed with the Example 1 group was less than that of the control group, but there was no significance. Comparative Examples 1 and 2 were lower than the Example 1 group in terms of weight gain rate, growth rate, fatness, etc.

[0059] It can also be seen that the Bupleurum and Scutellaria in the Chinese herbal medicine of the present invention have a synergistic effect, which can synergistically improve the relevant properties of the prepared Chinese herbal medicine, such as increasing the weight gain rate and specific growth rate of largemouth sea bass.

[0060] Table 3 Effects of Chinese herbal medicine formula on the growth of largemouth bass fed high-fat diet

[0061]

[0062]

[0063] 2.2.2 Effects of compound Chinese herbal medicine on antioxidant enzymes in largemouth bass

[0064] As shown in Table 4, the MDA content in the control group decreased over time, while SOD activity showed a trend of first increasing and then decreasing over time, with a turning point on the 30th day. CAT activity showed a trend of first increasing and then decreasing over time, with a turning point on the 45th day. GSH-Px activity remained stable, with no significant change. The MDA content in Group 1 showed a downward trend after 7 days of herbal feeding, but showed an upward trend after 15 days. SOD activity continued to increase. CAT activity continued to increase until it showed a downward trend on the 45th day. GSH-Px showed a significant upward trend after both 7 and 15 days of herbal feeding. The MDA content in Groups 1 and 2 of Comparative Examples showed a downward trend after 7 days of feeding, but the change was not as significant as in Group 1 of Example 1. After 15 days of feeding, the MDA content in Group 1 of Comparative Example 1 decreased, while that in Group 2 increased slightly. The SOD content in Groups 1 and 2 of Comparative Examples showed a continuous upward trend, with a smaller change than that in Group 1 of Example 1. The CAT activity value showed a significant change on the 45th day, showing a rapid upward trend. The GSH-Px activity values ​​of the control group 1 and the control group 2 increased on the 7th and 15th days, but the magnitude of the change was not as large as that of the example 1 group.

[0065] It can also be seen that the Bupleurum and Scutellaria in the Chinese herbal medicine of the present invention have a synergistic effect, which can synergistically improve the relevant properties of the prepared Chinese herbal medicine.

[0066] Table 4 Effects of Chinese herbal medicine formula on antioxidant enzymes in the liver of largemouth bass

[0067]

[0068]

[0069] 2.2.3 Discussion

[0070] 2.2.3.1 Effects of compound Chinese herbal medicine on the growth of largemouth bass

[0071] Chinese herbal medicines contain abundant organic components such as proteins, sugars, and fats, as well as numerous minerals. These ingredients play a significant role in balancing nutrition, improving growth performance, and regulating physiological functions. They are also a factor in promoting fish growth. By rationally combining Chinese herbal medicines, they can be formulated into herbal supplements that promote fish feeding and enhance growth. For example, in a growth experiment with pearl grouper, a water extract of Scutellaria baicalensis was added to their feed. Over a two-month period, the grouper treated with the supplemental extract showed a 5-10% increase in length and weight compared to the untreated grouper. The main reasons for the growth-boosting effects of Chinese herbal supplements on fish are likely related to two factors. The first is that Chinese herbal medicines act as attractants, increasing fish's appetite, boosting their feeding rate, and boosting their appetite. The feeding process of fish consists of three stages: the first stage is awareness of food; the second stage is seeking and swimming toward the food; and the third stage is consuming the food and determining whether it is edible. During this process, aquatic animals are not only stimulated by the physical stimuli of the food (such as size, luster, color, and hardness), but also by chemical stimuli from the soluble components of the feed, which are commonly known as attractants. Fish have an extremely sensitive sense of smell, and compared to visual stimuli, taste stimulation is a more effective food attractant in aquaculture. Studies have shown that different fish species have different odor preferences. Carnivorous fish prefer animal baits with strong fishy smells, while omnivorous fish such as grass carp and crucian carp prefer plant baits with aromatic odors. Secondly, Chinese herbal medicines can affect fish feed utilization and place a strain on other fish functions, such as the liver. For example, adding a compound containing red dates, astragalus, and Chinese yam to rainbow trout feed increases the spleen index of rainbow trout, affecting the liver. This study found that Chinese herbal medicine treatments tended to improve the growth performance of juvenile largemouth sea bass.

[0072] 2.2.3.2 Effects of compound Chinese herbal medicine on antioxidant enzymes in largemouth bass

[0073] Under normal conditions, the oxidation and antioxidant systems in animals are in a dynamic equilibrium state. However, if affected by internal and external factors, this balance will be broken, and the body will produce excessive reactive oxygen free radicals, thereby causing an increase in lipid peroxides. The antioxidant system of fish is mainly composed of two parts. One is the non-enzymatic system, including vitamin C, etc. The other is the enzyme system mainly including SOD, CAT, GSH-Px, etc. SOD has the function of scavenging superoxide free radicals in the body, that is, the stronger the SOD activity in the tissue, the lower the expression of free radicals, and the lower the degree of tissue damage [9]. CAT is an important component of the body that scavenges oxygen free radicals. Oxygen free radicals are an important substance that is generated during the body's metabolism and stress resistance. Under the oxidative stress conditions of pollutants, organisms can enhance their ability to scaveng reactive oxygen species by regulating the expression of antioxidant enzymes, thereby reducing damage to the body. Their activity changes due to oxidative stress. GSH-Px can scaveng hydrogen peroxide in the body. Their activity can reflect the degree to which the body scavenges reactive oxygen free radicals. At the same time, GSH-Px is related to liver damage. MDA is the primary degradation product of polyunsaturated fatty acid peroxidation. It directly reflects the level of oxidative stress in the body, and its content indirectly reflects the level of reactive oxygen free radicals and the level or rate of lipid peroxidation in tissue cells. In this experiment, the SOD activity of the control group initially increased and then decreased, while the MDA content decreased, indicating that the largemouth bass liver was already damaged and the body's antioxidant system was disrupted. When the Example 1 group was fed, the MDA content did not change significantly after 7 days of feeding, but it increased significantly after 15 days of feeding. This indicates that Chinese herbal medicine for largemouth bass promotes oxidative stress in largemouth bass, but long-term feeding is necessary. SOD, CAT, and GSH-Px activity values ​​were significantly increased when the Example 1 group was fed compared to the control group, indicating that the Example 1 group improved the antioxidant capacity of largemouth bass. Furthermore, after 7 days of feeding, the activity of the Example 1 group increased slightly, but not significantly. After 15 days of feeding, the activity value increased significantly, significantly enhancing the antioxidant capacity of largemouth bass, indicating that Example 1 is suitable for a feeding frequency of 15 days per month. When feeding the control groups 1 and 2, the MDA content did not change significantly after 7 days of feeding. Although it increased after 15 days, the difference was not significant compared with the control group. The SOD, CAT, and GSH-Px activity values ​​increased in the control groups 1 and 2, but the changes were not significant, indicating that both Bupleurum and Scutellaria are indispensable in the Example 1 group. It can be seen that the Bupleurum and Scutellaria in the Chinese herbal medicine of the present invention have a synergistic effect, which can synergistically improve the relevant properties of the prepared Chinese herbal medicine.

[0074] 2.2.3.4 Summary

[0075] Under the staged administration mode, the Chinese herbal medicines in Example 1 promoted the growth of largemouth bass and improved the antioxidant capacity and immune capacity of largemouth bass. The effect was better when the feeding frequency was 15 days / month than 7 days / month.

[0076] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A Chinese medicine composition for alleviating fatty liver in largemouth seabass, characterized by: Made from the following components in parts by weight: 20 parts of Bupleurum, 20 parts of Puerariae, 10 parts each of Scutellaria, White Peony, Pinellia, Ginger, and Citrus aurantium, 6 parts of Rhubarb, 20 parts of Smilax, 20 parts of Taraxacum, 10 parts of Platycodon, 10 parts of Cimicifuga, and jujube; Among them, add 4 dates to every 20g of Bupleurum.

2. The method for preparing the Chinese medicine composition according to claim 1, wherein: Here are the steps: After the various components are evenly mixed, they are crushed, passed through a 100-mesh sieve, and evenly mixed to obtain powder, which is the traditional Chinese medicine composition for alleviating fatty liver of largemouth seabass.

3. Use of the Chinese medicine composition according to claim 1 in preparing a feed additive for alleviating fatty liver of largemouth seabass.

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