An active extract of chrysanthemum stem and leaf for improving heavy metal poisoning in fish and a preparation method thereof

Abstract

The application discloses a chrysanthemum stem and leaf active extract with improved fish heavy metal poisoning and a preparation method thereof. The chrysanthemum stem and leaf active extract is compounded by a chrysanthemum stem and leaf crude polysaccharide part and a chrysanthemum stem and leaf phenolic ketone part. The pharmacodynamic experiment results show that the chrysanthemum stem and leaf active extract has a good improvement effect on fish heavy metal poisoning. The chrysanthemum stem and leaf active extract can not only improve pathological changes such as heart and liver granuloma, kidney tubular cast, intestinal cavity expansion and gill lamella shedding caused by heavy metal poisoning, but also improve metabolic dysfunction such as taurine and hypotaurine metabolic disorder, pyruvic acid metabolic abnormality, tricarboxylic acid cycle disorder, glycolysis and gluconeogenesis disorder caused by heavy metal poisoning. The preparation method has reasonable whole preparation process design, can realize waste utilization, extends a chrysanthemum industry chain, expands a chrysanthemum resource utilization approach, is helpful to quality improvement and efficiency increase and green development of the chrysanthemum industry, and provides support for prevention and treatment of heavy metal poisoning.

Description

Technical Field

[0001] This invention relates to a resource utilization product of non-medicinal parts of natural medicinal and edible Chinese medicine, and particularly to an active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish and its preparation method. Background Technology

[0002] Heavy metals generally refer to those with a density greater than 4.5 g / cm³. 3 Of the 45 metals mentioned, heavy metals in the context of environmental pollution mainly refer to mercury, cadmium, lead, chromium, and metalloids such as arsenic, which are highly toxic to organisms. Lead is a naturally occurring heavy metal in the Earth's crust. Its mining, smelting, manufacturing, and recycling activities, as well as its widespread use in various products, have caused extensive environmental pollution, human exposure, and significant public health problems. Lead can affect various organs in the body, such as the nervous, hematopoietic, endocrine, immune, and skeletal systems, and impair cognitive function, neurobehavioral, and learning and memory functions. Severe poisoning can lead to dementia. Clinically, acute lead poisoning is usually treated with metal chelating agents and hemodialysis to lower blood lead levels, but these methods have low safety and effectiveness, are expensive, lack specificity, and have a tendency to rebound, making them unsuitable for treating lead poisoning in aquatic animals such as fish and shellfish. Therefore, developing practical and economical treatments for lead poisoning not only contributes to food safety but also aligns with national environmental and ecological policies.

[0003] Chrysanthemum, a major Chinese medicinal and edible herb, is rich in active secondary metabolites such as flavonoids, phenolic acids, lactones, and polysaccharides, as well as nutrients such as amino acids, nucleotides, and monosaccharides. Our team's previous research found that the large amount of waste chrysanthemum stems and leaves generated during chrysanthemum production are also rich in various resource-based chemical components, possessing a wide range of biological activities, including preventing and treating eye diseases, fever combined with cough and inflammation, liver damage, and improving intestinal function (CN201910513995 Active extract of chrysanthemum stems and leaves for preventing and treating eye diseases and its preparation method; CN201910597938 Active extract of chrysanthemum leaves for preventing and treating fever combined with cough and inflammation and its preparation method; CN201910563890 Chrysanthemum leaf extract for preventing and treating liver damage and its application; CN201910552867 Multi-component granules of chrysanthemum stems and leaves for improving intestinal function and its preparation method). In addition to the aforementioned bioactivities, the abundant flavonoids and phenolic acids in chrysanthemum stem and leaf extracts can, on the one hand, chelate free lead ions in water, reducing the amount of lead entering the body. On the other hand, the phenolic acids and polysaccharides in chrysanthemum stem and leaf extracts can regulate the body's metabolism and immune function, thereby alleviating the damage caused by heavy metal poisoning. This invention extends the chrysanthemum industrial chain, expands the utilization pathways of chrysanthemum resources, contributes to improving the quality and efficiency of the chrysanthemum industry and promoting green development, and provides support for the prevention and treatment of heavy metal poisoning, thus contributing to the implementation of energy conservation and emission reduction policies. Summary of the Invention

[0004] Objective of the Invention: The objective of this invention is to provide an active extract of chrysanthemum stems and leaves that can improve tissue damage and metabolic dysfunction in fish caused by heavy metal poisoning, including damage to the gills, intestines, and liver. Another objective of this invention is to provide a method for preparing the aforementioned active extract of chrysanthemum stems and leaves.

[0005] Technical solution: In order to achieve the above objectives, the present invention adopts the following technical solution.

[0006] An active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish is obtained by the following preparation method:

[0007] (1) Take the waste chrysanthemum stems and leaves after the medicinal chrysanthemum is harvested, dry them in the sun, cut them into sections and crush them appropriately, add distilled water, extract them 1 to 3 times, filter them, combine the extracts, and concentrate them under reduced pressure to obtain the water extract of chrysanthemum stems and leaves for later use.

[0008] (2) Take the water extract of chrysanthemum stems and leaves from step (1), add anhydrous ethanol to make the alcohol content 50% to 95%, let stand overnight, and centrifuge to obtain the crude polysaccharide part (A) of chrysanthemum stems and leaves for later use;

[0009] (3) Take the remaining chrysanthemum stem and leaf residue after the water extract of chrysanthemum stem and leaf in step (1), add 55% to 95% ethanol, extract 1 to 3 times, filter and combine to obtain chrysanthemum stem and leaf ethanol extract for later use.

[0010] (4) Take the ethanol supernatant remaining from the preparation of crude polysaccharide of chrysanthemum stems and leaves in step (2), combine it with the ethanol extract of chrysanthemum stems and leaves in step (3), and concentrate it under reduced pressure to obtain chrysanthemum stem and leaf phenol ketone extract (B) for later use.

[0011] (5) Take the crude polysaccharide part (A) of chrysanthemum stem and leaf from step (2) and the phenolic ketone part (B) of chrysanthemum stem and leaf from step (4) and combine them to obtain the active extract of chrysanthemum stem and leaf.

[0012] As a preferred embodiment, the active extract of chrysanthemum stems and leaves that improves heavy metal poisoning in fish is extracted under the following conditions in step (1): waste chrysanthemum stems and leaves after harvesting medicinal chrysanthemum are dried, cut into sections and crushed to a suitable degree, and then 20 times, 10 times and 8 times the amount of distilled water are added in sequence. The extraction is carried out 3 times, each time for 0.5 to 2 hours, filtered, and the three extracts are combined.

[0013] As a preferred embodiment, the active extract of chrysanthemum stems and leaves that improves heavy metal poisoning in fish is extracted in step (1) by cold soaking, percolation, reflux extraction, microwave extraction, ultrasonic extraction, or supercritical extraction. Reflux extraction is particularly preferred.

[0014] As a preferred embodiment, the active extract of chrysanthemum stems and leaves that improves heavy metal poisoning in fish has an ethanol content of 50%, 65%, 80%, or 95% in step (2). 80% is particularly preferred.

[0015] As a preferred embodiment, the active extract of chrysanthemum stems and leaves that improves heavy metal poisoning in fish has an ethanol content of 55%, 65%, 75%, 85%, or 95% in step (3). 75% is particularly preferred.

[0016] As a preferred embodiment, the active extract of chrysanthemum stems and leaves that improves heavy metal poisoning in fish is extracted in step (3) by cold soaking, percolation, reflux, microwave extraction, ultrasonic extraction, or supercritical extraction. Percolation is particularly preferred.

[0017] As a preferred embodiment, the active extract of chrysanthemum stems and leaves that improves heavy metal poisoning in fish has a compounding ratio of crude polysaccharide fraction (A) and phenolic ketone fraction (B) of chrysanthemum stems and leaves in step (5) of A:B = 2:1, 1:1, 1:2, or 1:3. A:B = 1:2 is particularly preferred.

[0018] An active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish is obtained by the following preparation method:

[0019] (1) Take the discarded stems and leaves of medicinal chrysanthemum after harvesting, dry them in the sun, cut them into sections and crush them appropriately, and put them into an extraction tank. First, add 20 times the amount of distilled water and soak overnight, then heat and reflux for 2 hours. Take the residue and add 10 times and 8 times the amount of distilled water respectively for the second and third extractions, then heat and reflux for 1 hour and 0.5 hours respectively. Filter, combine the three extracts, and concentrate under reduced pressure to obtain an aqueous extract of chrysanthemum stems and leaves for later use.

[0020] (2) Take the water extract of chrysanthemum stems and leaves obtained in step (1), add anhydrous ethanol to make the alcohol content 80%, stir thoroughly, let stand overnight, and centrifuge to obtain the crude polysaccharide part (A) of chrysanthemum stems and leaves for later use.

[0021] (3) Take the remaining chrysanthemum stem and leaf residue after the extraction of chrysanthemum stem and leaf water extract in step (1), add 10 times the amount of 75% ethanol to soak overnight and percolate under normal pressure; add 8 times and 6 times the amount of 75% ethanol to percolate under normal pressure for the second and third times respectively, combine the percolates to obtain chrysanthemum stem and leaf ethanol extract for later use.

[0022] (4) Take the ethanol supernatant remaining from the preparation of crude polysaccharide of chrysanthemum stems and leaves in step (2), combine it with the ethanol extract of chrysanthemum stems and leaves in step (3), and concentrate it under reduced pressure to obtain chrysanthemum stem and leaf phenol ketone extract (B) for later use.

[0023] (5) Take the crude polysaccharide part (A) of chrysanthemum stem and leaf from step (2) and the phenolic ketone part (B) of chrysanthemum stem and leaf from step (4) and mix them in a ratio of A:B = 1:2 to obtain the active extract of chrysanthemum stem and leaf.

[0024] This invention analyzes the components of each raw material in the active extract of chrysanthemum stems and leaves: using glucose as a reference standard, the absorbance was measured at 490 nm using the phenol-sulfuric acid method and ultraviolet-visible spectrophotometry. This method showed that the content of neutral polysaccharides in the prepared crude polysaccharide fraction of chrysanthemum stems and leaves was 65.19%. Using glucuronic acid as a reference standard, the absorbance was measured at 512 nm using the carbazole-sulfuric acid method and ultraviolet-visible spectrophotometry. This method showed that the content of acidic polysaccharides in the prepared crude polysaccharide fraction of chrysanthemum stems and leaves was 11.94%. The sum of the contents of neutral polysaccharides and total acidic polysaccharides in the prepared crude polysaccharide fraction of chrysanthemum stems and leaves was greater than or equal to 77.13%.

[0025] Using rutin as a reference standard, the absorbance was measured at 510 nm using the aluminum nitrate-sodium nitrite colorimetric method and ultraviolet-visible spectrophotometry. This method revealed that the total flavonoid content in the prepared chrysanthemum stem and leaf active fraction was 25.93%. Using gallic acid as a reference standard, the absorbance was measured at 760 nm using the Folin-phenol method, revealing that the total phenolic acid content in the prepared chrysanthemum stem and leaf active fraction was 47.27%. Therefore, the combined content of total flavonoids and total phenolic acids in the prepared chrysanthemum stem and leaf active fraction is greater than or equal to 73.20%, indicating a high content of effective components.

[0026] The present invention relates to the application of the active extract of chrysanthemum stems and leaves, which has the effect of improving heavy metal poisoning in fish, in the preparation of fish medicines and functional feeds that improve heavy metal poisoning.

[0027] The active extract of chrysanthemum stems and leaves, which has the effect of improving heavy metal poisoning in fish, as described in this invention, is prepared into medicines and feeds using pharmaceutically and food-acceptable carriers.

[0028] A pharmaceutical preparation or functional feed, which is made into granules, tablets, pills, powders, etc., by combining an active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish with a pharmaceutically and food-acceptable carrier.

[0029] Beneficial effects: The active extract of chrysanthemum stems and leaves provided by this invention, which improves heavy metal poisoning in fish, develops and utilizes new non-medicinal parts of chrysanthemum stems and leaves based on the original application of chrysanthemum medicinal resources. Compared with existing technologies, it has the following advantages:

[0030] (1) This invention uses chrysanthemum stems and leaves, traditional waste generated during the production of medicinal chrysanthemum, as raw materials to prepare its active extract for improving heavy metal damage in fish. This realizes the transformation of waste into treasure, extends the chrysanthemum industry chain, expands the utilization of chrysanthemum resources, helps improve the quality and efficiency of the chrysanthemum industry and promotes green development, and provides support for the prevention and treatment of heavy metal poisoning. It also helps to achieve energy conservation and emission reduction policies and has good economic, social and ecological benefits.

[0031] (2) Experimental results show that the active extract of chrysanthemum stem and leaf provided by the present invention can not only improve the pathological changes such as cardiac and hepatic granulomas, renal tubular casts, intestinal dilatation and gill lamellae caused by heavy metal poisoning, but also improve metabolic dysfunction such as taurine and hypoturine metabolism disorders, pyruvate metabolism abnormalities, tricarboxylic acid cycle disorders, glycolysis and gluconeogenesis disorders caused by heavy metal poisoning.

[0032] (3) The present invention selects the best preparation process through comparison and optimization. It is highly operable and automated. The content and transfer rate of the crude polysaccharide and phenol ketone of chrysanthemum stem and leaf obtained by separation are high, and the effective components in the raw materials can be well preserved. The preparation process consumes less energy, has low production cost, and is environmentally friendly. Attached Figure Description

[0033] Figure 1 Effects of active extracts from chrysanthemum stems and leaves on serum alanine aminotransferase (ALT) (A), aspartate aminotransferase (AST) (B), blood urea nitrogen (BUN) (C), and creatinine (CRE) (D) in goldfish poisoned with lead. Note: Compared with the blank control group NC: *p<0.05, **p<0.01, ***p<0.001; Compared with the model group MO: #p<0.05, ##p<0.01, ###p<0.001.

[0034] Figure 2 Effects of active extracts from chrysanthemum stems and leaves on the pathological characteristics of the heart tissue of goldfish poisoned by lead.

[0035] Figure 3 Effects of active extracts from chrysanthemum stems and leaves on the histopathological characteristics of liver tissue in goldfish poisoned by lead.

[0036] Figure 4 Effects of active extracts from chrysanthemum stems and leaves on the histopathological characteristics of the kidney tissue of goldfish poisoned by lead.

[0037] Figure 5 Effects of active extracts from chrysanthemum stems and leaves on the pathological characteristics of brain tissue in goldfish poisoned by lead.

[0038] Figure 6 Effects of active extracts from chrysanthemum stems and leaves on the histopathology of intestinal tissue in goldfish poisoned by lead.

[0039] Figure 7 Effects of active extracts from chrysanthemum stems and leaves on the histopathological characteristics of goldfish gill tissue in lead poisoning.

[0040] Figure 8 Scoring plot (A) and S-Plot (B) of the effects of active extracts from chrysanthemum stems and leaves on liver metabolism in goldfish poisoned by lead.

[0041] Figure 9 Effects of active extracts from chrysanthemum stems and leaves on the liver metabolic pathways of goldfish poisoned by lead. Detailed Implementation

[0042] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0043] The amounts of medicinal materials, distilled water, and ethanol mentioned in this invention refer to weight-to-volume ratios. Ethanol concentration refers to volume concentration.

[0044] Example 1

[0045] A method for preparing an active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish, comprising the following steps:

[0046] (1) Take the discarded stems and leaves of medicinal chrysanthemum after harvesting, dry them in the sun, cut them into sections and crush them appropriately, and put them into an extraction tank. First, add 20 times the amount of distilled water and soak overnight, then heat and reflux for 2 hours. Take the residue and add 10 times and 8 times the amount of distilled water respectively for the second and third extractions, then heat and reflux for 1 hour and 0.5 hours respectively. Filter, combine the three extracts, and concentrate under reduced pressure to obtain an aqueous extract of chrysanthemum stems and leaves for later use.

[0047] (2) Take the water extract of chrysanthemum stems and leaves obtained in step (1), add anhydrous ethanol to make the alcohol content 95%, stir thoroughly, let stand overnight, and centrifuge to obtain the crude polysaccharide part of chrysanthemum stems and leaves (A) for later use.

[0048] (3) Take the chrysanthemum stem and leaf residue remaining after the water extract of chrysanthemum stems and leaves in step (1), add 10 times the amount of 95% ethanol and soak overnight, then heat and reflux for 2 hours; take the residue and add 8 times and 6 times the amount of 95% ethanol respectively for the second and third extractions, then heat and reflux for 1 hour and 0.5 hours respectively. Filter, combine the three extracts, and concentrate under reduced pressure to obtain the water extract of chrysanthemum stems and leaves for later use;

[0049] (4) Take the ethanol supernatant remaining from the preparation of crude polysaccharide of chrysanthemum stems and leaves in step (2), combine it with the ethanol extract of chrysanthemum stems and leaves in step (3), and concentrate it under reduced pressure to obtain chrysanthemum stem and leaf phenol ketone extract (B) for later use.

[0050] Using glucose as a reference standard, the absorbance of the prepared chrysanthemum stem and leaf crude polysaccharide fraction (A) was determined by UV-Vis spectrophotometry at 490 nm using the phenol-sulfuric acid method. The content of neutral polysaccharides was found to be 65.27%. Using glucuronic acid as a reference standard, the absorbance of the prepared chrysanthemum stem and leaf crude polysaccharide fraction (A) was determined by UV-Vis spectrophotometry at 512 nm using the carbazole-sulfuric acid method. The content of acidic polysaccharides was found to be 11.09%. The sum of the contents of neutral polysaccharides and total acidic polysaccharides in the prepared chrysanthemum stem and leaf crude polysaccharide fraction was greater than or equal to 76.36%.

[0051] Using rutin as a reference standard, the absorbance was measured at 510 nm using the aluminum nitrate-sodium nitrite colorimetric method and ultraviolet-visible spectrophotometry. The total flavonoid content in the prepared chrysanthemum stem and leaf phenolic extract (B) was 21.32%. Using gallic acid as a reference standard, the absorbance was measured at 760 nm using the Folin-phenol method. The total phenolic acid content in the prepared chrysanthemum stem and leaf phenolic extract (B) was 43.17%, and the total phenolic ketone content was greater than or equal to 64.49%.

[0052] Example 2

[0053] A method for preparing an active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish, comprising the following steps:

[0054] (1) Take the waste chrysanthemum stems and leaves after the medicinal chrysanthemum is harvested, dry them in the sun, cut them into sections and crush them appropriately. Add 20 times the amount of distilled water to soak overnight and percolate under normal pressure. Add 10 times the amount of distilled water and 8 times the amount of distilled water to percolate under normal pressure for the second and third time respectively. Combine the percolates and concentrate them under reduced pressure to obtain the water extract of chrysanthemum stems and leaves for later use.

[0055] (2) Take the water extract of chrysanthemum stems and leaves from step (1), add anhydrous ethanol to make the alcohol content 95%, let stand overnight, and centrifuge to obtain the crude polysaccharide part (A) of chrysanthemum stems and leaves for later use;

[0056] (3) Take the remaining chrysanthemum stem and leaf residue after the extraction of chrysanthemum stem and leaf water extract in step (1) and soak it overnight with 10 times the amount of 95% ethanol and percolate at normal pressure; add 8 times the amount of 95% ethanol and 6 times the amount of 95% ethanol respectively for the second and third time and percolate at normal pressure. Combine the percolates to obtain chrysanthemum stem and leaf ethanol extract for later use.

[0057] (4) Take the ethanol supernatant remaining from the preparation of crude polysaccharide of chrysanthemum stems and leaves in step (2), combine it with the ethanol extract of chrysanthemum stems and leaves in step (3), and concentrate it under reduced pressure to obtain chrysanthemum stem and leaf phenol ketone extract (B) for later use.

[0058] Using glucose as a reference standard, the absorbance was measured at 490 nm using the phenol-sulfuric acid method and ultraviolet-visible spectrophotometry. This method showed that the content of neutral polysaccharides in the prepared chrysanthemum stem and leaf crude polysaccharide fraction (A) was 58.56%. Using glucuronic acid as a reference standard, the absorbance was measured at 512 nm using the carbazole-sulfuric acid method and ultraviolet-visible spectrophotometry. This method showed that the content of acidic polysaccharides in the prepared chrysanthemum stem and leaf crude polysaccharide fraction (A) was 8.45%. The sum of the contents of neutral polysaccharides and total acidic polysaccharides in the prepared chrysanthemum stem and leaf crude polysaccharide fraction was greater than or equal to 67.01%.

[0059] Using rutin as a reference standard, the absorbance was measured at 510 nm using the aluminum nitrate-sodium nitrite colorimetric method and ultraviolet-visible spectrophotometry. The total flavonoid content in the prepared chrysanthemum stem and leaf phenolic extract (B) was 21.87%. Using gallic acid as a reference standard, the absorbance was measured at 760 nm using the Folin-phenol method. The total phenolic acid content in the prepared chrysanthemum stem and leaf phenolic extract (B) was 46.75%, and the total phenolic ketone content was greater than or equal to 68.62%.

[0060] Example 3

[0061] A method for preparing an active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish, comprising the following steps:

[0062] (1) Take the discarded stems and leaves of medicinal chrysanthemum after harvesting, dry them in the sun, cut them into sections and crush them appropriately, and put them into an extraction tank. First, add 20 times the amount of distilled water and soak overnight, then heat and reflux for 2 hours. Take the residue and add 10 times and 8 times the amount of distilled water respectively for the second and third extractions, then heat and reflux for 1 hour and 0.5 hours respectively. Filter, combine the three extracts, and concentrate under reduced pressure to obtain an aqueous extract of chrysanthemum stems and leaves for later use.

[0063] (2) Take the water extract of chrysanthemum stems and leaves obtained in step (1), add anhydrous ethanol to make the alcohol content 50%, stir thoroughly, let stand overnight, and centrifuge to obtain the crude polysaccharide part (A) of chrysanthemum stems and leaves for later use.

[0064] (3) Take the remaining chrysanthemum stem and leaf residue after the extraction of chrysanthemum stem and leaf water extract in step (1), add 10 times the amount of 95% ethanol to soak overnight and percolate under normal pressure; add 8 times and 6 times the amount of 95% ethanol to percolate under normal pressure for the second and third times respectively, combine the percolates to obtain chrysanthemum stem and leaf ethanol extract for later use.

[0065] (4) Take the ethanol supernatant remaining from the preparation of crude polysaccharide of chrysanthemum stems and leaves in step (2), combine it with the ethanol extract of chrysanthemum stems and leaves in step (3), and concentrate it under reduced pressure to obtain chrysanthemum stem and leaf phenol ketone extract (B) for later use.

[0066] Using glucose as a reference standard, the absorbance of the prepared chrysanthemum stem and leaf crude polysaccharide fraction (A) was determined by UV-Vis spectrophotometry at 490 nm using the phenol-sulfuric acid method. The content of neutral polysaccharides was found to be 64.78%. Using glucuronic acid as a reference standard, the absorbance of the prepared chrysanthemum stem and leaf crude polysaccharide fraction (A) was determined by UV-Vis spectrophotometry at 512 nm using the carbazole-sulfuric acid method. The content of acidic polysaccharides was found to be 11.31%. The sum of the contents of neutral polysaccharides and total acidic polysaccharides in the prepared chrysanthemum stem and leaf crude polysaccharide fraction was greater than or equal to 76.09%.

[0067] Using rutin as a reference standard, the absorbance was measured at 510 nm using the aluminum nitrate-sodium nitrite colorimetric method and ultraviolet-visible spectrophotometry. The total flavonoid content in the prepared chrysanthemum stem and leaf phenolic extract (B) was 22.17%. Using gallic acid as a reference standard, the absorbance was measured at 760 nm using the Folin-phenol method. The total phenolic acid content in the prepared chrysanthemum stem and leaf phenolic extract (B) was 45.68%, and the total phenolic ketone content was greater than or equal to 67.85%.

[0068] Example 4

[0069] A method for preparing an active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish, comprising the following steps:

[0070] (1) Take the discarded stems and leaves of medicinal chrysanthemum after harvesting, dry them in the sun, cut them into sections and crush them appropriately, and put them into an extraction tank. First, add 20 times the amount of distilled water and soak overnight, then heat and reflux for 2 hours. Take the residue and add 10 times and 8 times the amount of distilled water respectively for the second and third extractions, then heat and reflux for 1 hour and 0.5 hours respectively. Filter, combine the three extracts, and concentrate under reduced pressure to obtain an aqueous extract of chrysanthemum stems and leaves for later use.

[0071] (2) Take the water extract of chrysanthemum stems and leaves obtained in step (1), add anhydrous ethanol to make the alcohol content 80%, stir thoroughly, let stand overnight, and centrifuge to obtain the crude polysaccharide part (A) of chrysanthemum stems and leaves for later use.

[0072] (3) Take the remaining chrysanthemum stem and leaf residue after the extraction of chrysanthemum stem and leaf water extract in step (1), add 10 times the amount of 75% ethanol to soak overnight and percolate under normal pressure; add 8 times and 6 times the amount of 75% ethanol to percolate under normal pressure for the second and third times respectively, combine the percolates to obtain chrysanthemum stem and leaf ethanol extract for later use.

[0073] (4) Take the ethanol supernatant remaining from the preparation of crude polysaccharide of chrysanthemum stems and leaves in step (2), combine it with the ethanol extract of chrysanthemum stems and leaves in step (3), and concentrate it under reduced pressure to obtain chrysanthemum stem and leaf phenol ketone extract (B) for later use.

[0074] Using glucose as a reference standard, the absorbance of the prepared chrysanthemum stem and leaf crude polysaccharide fraction (A) was determined by UV-Vis spectrophotometry at 490 nm using the phenol-sulfuric acid method. The content of neutral polysaccharides was found to be 65.19%. Using glucuronic acid as a reference standard, the absorbance of the prepared chrysanthemum stem and leaf crude polysaccharide fraction (A) was determined by UV-Vis spectrophotometry at 512 nm using the carbazole-sulfuric acid method. The content of acidic polysaccharides was found to be 11.94%. The sum of the contents of neutral polysaccharides and total acidic polysaccharides in the prepared chrysanthemum stem and leaf crude polysaccharide fraction was greater than or equal to 77.13%.

[0075] Using rutin as a reference standard, the absorbance was measured at 510 nm using the aluminum nitrate-sodium nitrite colorimetric method and ultraviolet-visible spectrophotometry. This method revealed that the total flavonoid content in the prepared chrysanthemum stem and leaf phenolic extract (B) was 25.93%. Using gallic acid as a reference standard, the absorbance was measured at 760 nm using the Folin-phenol method. This method revealed that the total phenolic acid content in the prepared chrysanthemum stem and leaf phenolic extract (B) was 47.27%, and the total phenolic ketone content was greater than or equal to 73.20%.

[0076] As can be seen from Examples 1 to 4, considering the content of total polysaccharide and total phenolic active ingredients in the crude polysaccharide part (A) of chrysanthemum stems and leaves and the phenolic extract (B) of chrysanthemum stems and leaves, the content of active ingredients is higher when extracted by the method in Example 4. Therefore, the following Examples 5 to 8 regarding the compounding of crude polysaccharide part (A) of chrysanthemum stems and leaves and the phenolic extract (B) of chrysanthemum stems and leaves are all based on Example 4.

[0077] Example 5

[0078] A method for preparing an active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish, comprising the following steps:

[0079] Steps 1 to 4 are the same as in Example 4.

[0080] (5) Take the crude polysaccharide part (A) of chrysanthemum stem and leaf from step (2) and the phenolic ketone part (B) of chrysanthemum stem and leaf from step (4) and mix them in a ratio of A:B = 2:1 to obtain the active extract of chrysanthemum stem and leaf.

[0081] Example 6

[0082] A method for preparing an active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish, comprising the following steps:

[0083] Steps 1 to 4 are the same as in Example 4.

[0084] (5) Take the crude polysaccharide part (A) of chrysanthemum stem and leaf from step (2) and the phenolic ketone part (B) of chrysanthemum stem and leaf from step (4) and mix them in a ratio of A:B = 1:1 to obtain the active extract of chrysanthemum stem and leaf.

[0085] Example 7

[0086] A method for preparing an active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish, comprising the following steps:

[0087] Steps 1 to 4 are the same as in Example 4.

[0088] (5) Take the crude polysaccharide part (A) of chrysanthemum stem and leaf from step (2) and the phenolic ketone part (B) of chrysanthemum stem and leaf from step (4) and mix them in a ratio of A:B = 1:2 to obtain the active extract of chrysanthemum stem and leaf.

[0089] Example 8

[0090] A method for preparing an active extract of chrysanthemum stems and leaves that can improve heavy metal poisoning in fish, comprising the following steps:

[0091] Steps 1 to 4 are the same as in Example 4.

[0092] (5) Take the crude polysaccharide part (A) of chrysanthemum stem and leaf from step (2) and the phenolic ketone part (B) of chrysanthemum stem and leaf from step (4) and mix them in a ratio of A:B = 1:3 to obtain the active extract of chrysanthemum stem and leaf.

[0093] Example 9

[0094] Pharmacodynamic experiments of active extracts from chrysanthemum stems and leaves on heavy metal poisoning in goldfish.

[0095] I. Experimental Materials and Drugs

[0096] 1. Drugs and reagents

[0097] Lead nitrate was purchased from Shanghai Aladdin (product number: L112113); sodium chloride injection was purchased from Henan Kelun Pharmaceutical Co., Ltd. (batch number: A19080101A); and reagent kits for alanine aminotransferase (ALT-GOT), aspartate aminotransferase (AST-GOT), blood urea nitrogen (BUN), and creatinine (CRE) were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0098] 2. Laboratory animals

[0099] Goldfish, weighing 15±3g, were purchased from the Confucius Temple Flower, Tree, Fish and Insect Market in Nanjing. They were housed in the goldfish breeding system of Nanjing University of Chinese Medicine at a temperature of (22±2)℃ and a relative humidity of (55±10)%, with 12h light / 12h darkness. They were fed standard fish food, and two-thirds of the water was changed half an hour after feeding each day. The animal experimental protocol was reviewed and approved by the Animal Ethics Committee of the Drug Safety Evaluation Research Center of Nanjing University of Chinese Medicine, and all operations complied with relevant regulations on experimental animal ethics.

[0100] 3. Experimental Apparatus

[0101] OLYMPUS microscopes (OLYMPUS Corporation, Japan); BT125 electronic balance (Sartorius Scientific Instruments Co., Ltd.); ELISA reader (Bio-Tek Corporation, USA); Leica ASP300S fully automatic dehydrator, Leica RM2255 semi-automatic microtome, Leica TS5015 fully automatic staining machine, and Olympus BX51 optical microscope.

[0102] 4. Test drug and treatment method

[0103] The treatment group was given the active extracts of chrysanthemum stems and leaves from Examples 5-8, with a concentration of 10 mg / L water based on the amount of dried chrysanthemum stems and leaves.

[0104] II. Experimental Methods

[0105] After 7 days of acclimatization, goldfish were randomly divided into 6 groups: blank control group (NC), model group (MO), Example 5 (SS5), Example 6 (SS6), Example 7 (SS7), and Example 8 (SS8), with 10 fish in each group. Except for the blank control group, a lead poisoning model was prepared by adding 10 μM lead nitrate to the fish tank for one week. After the modeling was completed, the treatment group was given the drug for one week. After the last administration, blood was collected by puncture with a disposable sterile syringe. After blood collection, the organs of the goldfish were dissected. Some of the tissues were fixed in 10% formalin and then dehydrated, cleared, paraffin-embedded, embedded, and sectioned in stages. Routine H&E staining was performed, and the histological changes were observed under a light microscope. The remaining tissues were used for subsequent metabolomics experiments.

[0106] III. Experimental Results

[0107] 1. Serum biochemical indicators

[0108] Serum biochemical results showed that, compared with the blank control group, the model group goldfish had significantly increased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE), indicating that lead nitrate caused significant liver and kidney damage in the model group goldfish (p<0.001, p<0.001, p<0.001, p<0.001). Compared with the model group, Examples 5-8 all significantly reduced serum alanine aminotransferase (ALT). Figure 1 A, p<0.001, p<0.001, p<0.001, p<0.001), aspartate aminotransferase (AST) Figure 1 B, p<0.001, p<0.001, p<0.001, p<0.001), blood urea nitrogen (BUN) Figure 1 C, p<0.001, p<0.001, p<0.001, p<0.001), creatinine CRE ( Figure 1The levels (D, p<0.001, p<0.001, p<0.001, p<0.001) indicate that the active extracts of chrysanthemum stems and leaves in the above examples can improve the liver and kidney function of goldfish caused by lead nitrate, especially the effect of Example 7 is the most significant. Figure 1 ).

[0109] 2. Histopathological observation

[0110] Histopathological results showed that: (1) Heart: In the blank control group (NC), the heart tissue structure was normal and intact, the myocardial fibers were neatly arranged, and no degeneration or necrosis was observed. No pathological changes such as congestion, edema, or inflammatory cell infiltration were observed in the interstitium. In the model group (MO), the heart tissue structure of the goldfish was basically intact, with focal granulomas observed. Except for the case of example 6 (SS6) where mild focal granulomas were observed, no obvious pathological changes were observed in the hearts of the goldfish in the other treatment groups (SS5, SS7, SS8). Figure 2 ). This indicates that the active extract of chrysanthemum stems and leaves can improve cardiac damage caused by lead poisoning to a certain extent, especially in Examples 5, 7, and 8. (2) Liver: The liver tissue structure of the blank control group (NC) was normal and intact, the hepatocytes were neatly arranged, and no pathological changes such as degeneration or necrosis were observed. No pathological changes such as dilation or congestion of the hepatic sinusoids were observed. The liver of the goldfish in the model group (MO) showed moderate multifocal granulomas. No obvious pathological changes were observed in Examples 5 to 8 (SS5, SS6, SS7, SS8) of the active extract of chrysanthemum stems and leaves. Figure 3 ). This indicates that the active extract of chrysanthemum stems and leaves can improve liver damage caused by lead poisoning, and the effects of Examples 5 to 8 are comparable. (3) Kidney: The blank control group (NC) showed normal and intact kidney tissue structure, normal glomerular and tubular structures, no degeneration or necrosis, and no pathological changes such as congestion or edema in the interstitium. The model group (MO) showed moderate tubular casts in the kidneys of goldfish, with dark blue calcium salt-like substances in the lumen, and moderate pigment deposition in the renal interstitium. In the chrysanthemum stem and leaf active extract treatment group, except for Example 5 (SS5), there was slight pigment deposition and granuloma in the renal interstitium, and in Examples 6 (SS6) and 8 (SS8), there was slight pigment deposition in the renal interstitium, and in Example 7 (SS7), no obvious pathological changes were observed. Figure 4 ). This indicates that the active extract of chrysanthemum stems and leaves can improve kidney damage caused by lead poisoning to a certain extent, especially the best effect in Example 7. (4) Brain: The brain tissue structure of blank control group (NC), model group (MO), and chrysanthemum stem and leaf active extract treatment groups (Examples 5-8, SS5, SS6, SS7, SS8) was normal and intact, no neuronal degeneration or necrosis was observed, and no pathological changes such as congestion, edema, and inflammatory cell infiltration were observed in the interstitium. Figure 5(5) Intestine: The blank control group (NC) had a normal and intact intestinal tissue structure. The intestinal wall included the mucosa, submucosa, muscularis propria, and serosa. The mucosa included the mucosal epithelium and lamina propria, which protruded into the lumen to form villi. No mucosal epithelial cell shedding or necrosis was observed. No congestion or edema was observed in the submucosa. The muscularis propria and serosa were normal. The model group (MO) showed significant intestinal dilation, reduced intestinal villi, and thinning of the intestinal wall. The intestinal wall tissue structure of the chrysanthemum stem and leaf active extract administration groups (Examples 5, 6, and 8, SS5, SS6, and SS8) was normal and intact, with mild intestinal dilation observed. No obvious pathological changes were observed in Example 7 (SS7) of the chrysanthemum stem and leaf active extract administration group. Figure 6 ). This indicates that the active extract of chrysanthemum stems and leaves can improve intestinal damage caused by lead poisoning to a certain extent, especially the best effect of Example 7. (6) Gills: The blank control group (NC) has a normal and intact gill tissue structure, with gill rakers, gill arches and gill filaments. The gill rakers are long and conical, and the gill filaments are arranged in a comb-like manner on the gill arches. The gill lamellae are evenly arranged on both sides of the gill filaments. The gill lamellae are mainly composed of respiratory epithelium, column cells and capillary network. No gill lamellae or epithelial cell shedding or necrosis was observed. The model group (MO) has a disordered gill tissue structure, with severe gill lamellae shedding, necrosis and shedding of epithelial cells covering some of the gill lamellae that have not shed, and exposure of the capillary network. The active extract of chrysanthemum stems and leaves in Example 5 (SS5) and Example 6 (SS8) have disordered gill tissue structure and moderate gill lamellae shedding; Example 7 (SS7) has disordered gill tissue structure and mild gill lamellae shedding; Example 8 (SS8) has a basically intact gill tissue structure and slight gill lamellae shedding. Figure 7 The results indicate that the active extract of chrysanthemum stems and leaves can improve gill damage caused by lead poisoning to a certain extent, especially in Examples 7 and 8. In summary, the active extract of chrysanthemum stems and leaves provided in Example 7 has the best overall effect on the damage to various tissues and organs of goldfish caused by lead poisoning.

[0111] Example 10

[0112] Metabolomics experiment on heavy metal poisoning in goldfish using active extracts of chrysanthemum stems and leaves

[0113] I. Experimental Materials and Drugs

[0114] 1. Drugs and reagents

[0115] Merck chromatographic acetonitrile was purchased from Merck GmbH, Germany; heavy water (D2O, brand: Sigma, batch number: MKBV6574VS) and tris(trimethylsilane) phosphate (TMSP, brand: CIL Cambridge Isotope Laboratory, batch number: I1-12486) were purchased from Qingdao Tenglong Microwave Technology Co., Ltd.

[0116] 2. Laboratory animals

[0117] Same as Example 9.

[0118] 3. Experimental Apparatus

[0119] LABCONCO FreeZone freeze dryer (LABCONCO, USA); BT125 electronic balance (Sartorius Scientific Instruments Ltd.); Bruker nuclear magnetic resonance spectrometer (Bruker GmbH, Germany).

[0120] 4. Test drug and treatment method

[0121] Same as Example 9.

[0122] II. Experimental Methods

[0123] Take 0.2g of goldfish liver from each group, add 5ml of pre-cooled acetonitrile-water solution (vol / vol = 1:1) to each 1g of tissue, grind into a homogenate, centrifuge at 12000rpm for 10min at 4℃, collect the supernatant, concentrate using a nitrogen blower, and freeze-dry. Reconstitute the freeze-dried liver extract with 550μL of phosphate buffer (0.2M Na2HPO4 / NaH2PO4, pH 7.0) prepared with heavy water (D2O). Vortex the sample, then centrifuge at 12000rpm for 10min at 4℃ to remove insoluble matter. Add 50μL of TMSP solution (1mg / ml) prepared with heavy water to the supernatant, vortex to mix, and transfer to a 5mm NMR tube for 1H NMR data acquisition. Spectral acquisition was performed using a NOESYPR pulse sequence: relaxation delay -90° - t1 - 90° - tm - 90° - signal acquisition - free induction decay signal. Noise suppression was applied during the cyclic delay and NOESY mixing time to suppress water peaks. 128 free induction decay signals generated 32K data points, resulting in a spectral width of 10kHz. Each scan had an acquisition time of 2.54s, with a cyclic delay and mixing time (tm) of 2s and 100ms, respectively. Topspin 3.0 software was used for automatic phase and baseline correction of all spectra, and the TSP signal was corrected to 0.00ppm.

[0124] After removing the water peak and its adjacent signals (4.3–5.45 ppm), all spectra were shift-corrected using an R-language program. Binning was performed on the 0.7–9.4 ppm region using the PROcess software package; the adaptive binning method was based on code implemented in Matlab (version 7.3, MathWorks, Natick, MA) as described in the literature. Before multivariate statistical analysis, the binned data underwent probabilistic quotient normalization, centering, and Pareto scaling using R software. Small molecule metabolites were assigned by comparing with references, consulting the Human Metabolome Database (HMDB, www.hmdb.ca), and using the NMR data identification software Chenomx NMRSuite, version 4.0 (Chenomx Inc., Edmonton, Canada).

[0125] III. Experimental Results

[0126] 1. Identification of small molecule metabolites in the liver

[0127] By comparing with references, consulting relevant databases such as HMDB, MMCD, and KEGG, and using the nuclear magnetic resonance data identification software Chenomx NMR Suite, a total of 53 small molecule metabolites were identified from goldfish livers. The order is: 1.Isoleucine, 2.Leucine, 3.Valine, 4.3-Hydroxybutyrate, 5.Lactate, 6.Alanine, 7.Lysine, 8.Acetate, 9.Proline, 10.Glutamate, 11.Methionine, 12.Glutamine, 13.Glutathione, 14.2- Aminoadipate, 15.Succinate, 16.Methylamine, 17.Aspartate, 18.Sarcosine, 19.Asparagine, 20.Trimethylamine, 21.Creatine, 22.Choline, 23.Betaine, 24.Taurine, 25.TMAO / trimethylamine N-oxide, 26.Methanol, 27.Glucose, 28.Maltose, 29.Uridine, 30.GTP, 31.UDP-glucose / UDPG, 32.Inosine, 33.AMP, 34.Fumarate, 35.Tyrosine, 36. Tryptophan, 37.Phenylalanine, 38.Nicotinurate, 39.Guanosine, 40.NAD+, 41.Hypoxanthine, 42.Formate, 43.Mannose, 44.Pyridoxine, 45.Flavin Mononucleotide / FMN, 46.1,7-Dimethylxanthine / Paraxanthine, 47.dGTP, 48.Uridine 5'-monophosphate / UMP, 49.Carnosine, 50.Guanosine diphosphate / GDP, 51.Adenosine monophosphate / AMP, 52.Imidazoleacetic acid, 53.NADP+.

[0128] 2. Regulatory effect of active extracts from chrysanthemum stems and leaves on liver metabolic disorders in goldfish poisoned by lead.

[0129] The supervised multivariate analysis method OPLS-DA was used to process the NMR metabolomics data of goldfish livers in each group. The score plot showed that the blank group (NC, ▼) and the model group (MO, ▲) were completely separated, located in the positive and negative regions of the OPLS-DA score plot, respectively. The treatment groups of chrysanthemum stem and leaf active extract (Examples 5-8, +, ×, ○, ◇) were located between the two, especially SS7, which was closest to NC and farthest from MO. This indicates that lead nitrate caused significant metabolic disorders in goldfish livers, and the treatment groups of Examples 5-8 could correct the above metabolic disorders to a certain extent, especially the regulatory effect of Example 7 was the most significant. Figure 8 A). The S-Plot of OPLS-DA showed that, compared with the control group, the levels of Aspartate, Fumarate, and Maltose in the liver of goldfish in the model group were significantly increased, while the levels of Valine, Lactate, Alanine, Acetate, Methionine, Succinate, Sarcosine, Asparagine, Creatine, Betaine, Taurine, Methanol, Glucose, UDPG, dGTP, and UMP were significantly decreased. Each drug-treated group showed a certain corrective effect on the above-mentioned metabolic disorders, especially SS7.

[0130] 3. Effects of active extracts from chrysanthemum stems and leaves on the hepatic metabolic pathways of goldfish poisoned by lead.

[0131] Metabolic pathway analysis of the above-mentioned significantly different metabolites revealed the following main metabolic disorders in goldfish liver caused by lead nitrate: taurine and hypotaurine metabolism disorders, alanine, aspartic acid, and glutamate metabolism abnormalities, glycine, serine, and threonine metabolism disorders, pyruvate metabolism abnormalities, tricarboxylic acid cycle disorders, and impaired glycolysis and gluconeogenesis. The active extracts from chrysanthemum stems and leaves can play a corrective and restorative role by regulating these disordered metabolic pathways.

[0132] The above experimental results show that the active extract of chrysanthemum stems and leaves provided by the present invention can improve the pathological changes in fish caused by heavy metal poisoning, such as cardiac and hepatic granulomas, renal tubular casts, intestinal dilatation, and gill lamellae shedding. At the same time, it can improve metabolic dysfunction caused by heavy metal poisoning, such as taurine and hypoturine metabolism disorders, pyruvate metabolism abnormalities, tricarboxylic acid cycle disorders, glycolysis and gluconeogenesis disorders.

[0133] The activity of the chrysanthemum stem and leaf active extracts prepared by different processes varies slightly. Through comparison, it can be seen that the chrysanthemum stem and leaf active extract prepared in Example 7 has the strongest activity and has achieved very good and unexpected technical effects.

[0134] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A chrysanthemum stem leaf active extract having improved fish heavy metal poisoning, characterized in that, It is prepared by the following preparation method: (1) Take the waste chrysanthemum stems and leaves after the medicinal chrysanthemum is harvested, dry them, cut them into sections and moderately crush them, then add 20 times, 10 times and 8 times the amount of distilled water, heat them to reflux and extract them for 3 times, each time for 0.5-2 hours, filter them, combine the three times of extract filtrates, and reduce the pressure to concentrate them to obtain the chrysanthemum stem and leaf water extract extract, which is ready for use; (2) Take the chrysanthemum stem and leaf water extract extract obtained in step (1), add anhydrous ethanol to make the alcohol content 50%, 65%, 80% or 95%, stand still overnight, centrifugalize to obtain the chrysanthemum stem and leaf crude polysaccharide part (A), which is ready for use; (3) Take the chrysanthemum stem and leaf residue after the chrysanthemum stem and leaf water extract is extracted in step (1), add 55%, 65%, 75%, 85% or 95% ethanol, percolate and extract 1-3 times, filter and combine them to obtain the chrysanthemum stem and leaf ethanol extract, which is ready for use; (4) Take the ethanol supernatant remaining in the preparation process of the chrysanthemum stem and leaf crude polysaccharide in step (2), combine it with the chrysanthemum stem and leaf ethanol extract in step (3), and reduce the pressure to concentrate them to obtain the chrysanthemum stem and leaf phenolic ketone extract (B), which is ready for use; (5) Take the chrysanthemum stem and leaf crude polysaccharide part (A) in step (2) and the chrysanthemum stem and leaf phenolic ketone part (B) in step (4), and compound them in the ratio of 2:1, 1:1, 1:2 or 1:3 to obtain the chrysanthemum stem and leaf active extract.

2. An active extract of Chrysanthemum stem and leaves having improved fish heavy metal poisoning, characterized in that, It is prepared by the following preparation method: (1) Take the waste chrysanthemum stems and leaves after the medicinal chrysanthemum is harvested, dry them, cut them into sections and moderately crush them, then add 20 times, 10 times and 8 times the amount of distilled water, heat them to reflux and extract them for 3 times, each time for 0.5-2 hours, filter them, combine the three times of extract filtrates, and reduce the pressure to concentrate them to obtain the chrysanthemum stem and leaf water extract extract, which is ready for use; (2) Take the chrysanthemum stem and leaf water extract extract obtained in step (1), add anhydrous ethanol to make the alcohol content 50%, 65%, 80% or 95%, stand still overnight, centrifugalize to obtain the chrysanthemum stem and leaf crude polysaccharide part (A), which is ready for use; (3) Take the chrysanthemum stem and leaf residue after the chrysanthemum stem and leaf water extract is extracted in step (1), add 55%, 65%, 75%, 85% or 95% ethanol, percolate and extract 1-3 times, filter and combine them to obtain the chrysanthemum stem and leaf ethanol extract, which is ready for use; (4) Take the ethanol supernatant remaining in the preparation process of the chrysanthemum stem and leaf crude polysaccharide in step (2), combine it with the chrysanthemum stem and leaf ethanol extract in step (3), and reduce the pressure to concentrate them to obtain the chrysanthemum stem and leaf phenolic ketone extract (B), which is ready for use; (5) Take the chrysanthemum stem and leaf crude polysaccharide part (A) in step (2) and the chrysanthemum stem and leaf phenolic ketone part (B) in step (4), and compound them in the ratio of 2:1, 1:1, 1:2 or 1:3 to obtain the chrysanthemum stem and leaf active extract.

3. The chrysanthemum stem and leaf active extract for improving fish heavy metal poisoning according to claim 1 or 2 is used for preparing fish medicine or functional feed for improving heavy metal poisoning.

4. Use according to claim 3, characterized in that, The chrysanthemum stem and leaf active extract is prepared into a drug or functional feed with a pharmaceutically or food acceptable carrier.

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