Method for extracting bilirubin, cholic acid and chenodeoxycholic acid from chicken bile

Abstract

The invention discloses a method for extracting bilirubin, cholic acid and chenodeoxycholic acid from chicken bile. The method comprises the steps of pretreatment, chicken bile treatment, hydrolysis reduction, chromatographic separation and the like. The method comprises the following steps: slicing frozen chicken gallbladder, heating, unfreezing and melting to denature protein into solid, then cooling, performing three-phase centrifugal degreasing and deproteinization, and adding three enzymes including biliverdin reductase, coenzyme I and glucose dehydrogenase to reduce free biliverdin and combined biliverdin, so as to obtain the biliverdin-free biliverdin-glucose dehydrogenase-free biliverdin-glucose dehydrogenase-free biliverdin-glucose dehydrogenase. The method comprises the following steps: adding cholic acid and chenodeoxycholic acid into a container, adding gluconic acid hydrolase and bile salt hydrolase to hydrolyze biliverdin and bile acid, adding trypsin to hydrolyze impure protein, performing chromatography by using macroporous silica gel, and separating bilirubin, cholic acid and chenodeoxycholic acid by using different molecular space structures to obtain high-purity bilirubin, cholic acid and chenodeoxycholic acid. The method has the advantages of simple process, few types of used solvents, low toxicity, and facilitation of environmental protection and industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of chicken bile processing, and in particular to a method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile. Background Art

[0002] Bilirubin has multiple drug activities such as antipyretic, expectorant, sedative, anticonvulsant, antihypertensive, promoting heme regeneration, inhibiting Japanese encephalitis virus, etc. It is also an important raw material for artificial bezoar. At present, the main way to obtain bilirubin is to extract it from the bile of pigs, cattle, sheep and other animals. Due to the shortage of raw material resources and backward production technology, the supply of bilirubin is seriously insufficient and the price is very expensive.

[0003] Fresh chicken bile is mainly composed of water (about 97%), bile acid (about 2.5%) and biliverdin (up to about 0.03%), in addition to phospholipids, cholesterol, sodium, potassium, calcium, phosphate, carbonate, chloride and a small amount of protein. However, the current extraction of chicken bile generally only involves the extraction of bile acid or chenodeoxycholic acid, which is easy to cause waste of raw materials, and the use of flammable and explosive reagents in the extraction process has safety risks and cannot meet the requirements of safety, energy saving and green environmental protection development. Therefore, we propose a method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile.

[0005] To achieve the above object, the present invention is implemented by the following technical scheme: a method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile, comprising the following process steps: (1) Preprocessing The antioxidant, glacial acetic acid and water are mixed in a ratio of 1:1:1000 to prepare a composite acid solution A, wherein the antioxidant is one of sodium pyrosulfite, sodium thiosulfate or vitamin C; the sodium pyrosulfite, sodium thiosulfate, sodium hydroxide and water are mixed in a ratio of 1:1:1:1000 to prepare a composite acid solution B; (2) Chicken bile processing Cut the frozen chicken gall into appropriate sizes, put it into a container for thawing, and add 0.1% sodium sulfite or sodium thiosulfate for protection. The thawed material is heated to 60°C-70°C to fully denature the protein in the chicken gall into a solid. Then, nitrogen is filled in the container for protection. After the material in the container is cooled to room temperature, it is centrifuged in a three-phase centrifuge to remove solid impurities and grease, and the water phase is collected to obtain the chicken gall. (3) Hydrolysis reduction The chicken bile collected in step (2) is put into a reactor, stirred, nitrogen-filled, and then glucose, coenzyme I, biliverdin reductase, glucuronide dehydrogenase and glucuronide hydrolase are added, wherein the amount of the above enzymes added is 1 / 10,000 to 5 / 10,000 of the chicken bile, and then the composite alkaline solution B is added to adjust the pH value to 6.5±0.5, and the temperature is controlled to 37°C, and the reaction is carried out for 30 minutes, and then 5 / 10,000 of the chicken bile ... (4) Chromatographic separation S1. Chromatography loading: Pre-treat the chromatography column, filter the chicken bile that has passed the hydrolysis test through a 5um titanium rod barrel, and load 4 times the column volume of chicken bile; S2. Separation of bilirubin: Use composite alkaline solution B for chromatography for 3 times of the volume of the chromatography column, when the column is washed for 1-2 times of the volume of the chromatography column, the bilirubin pigment band begins to flow out, and when the column is washed for 3 times of the volume of the chromatography column, the bilirubin pigment band is completely washed out, collect the pigment band aqueous phase, add dichloromethane with a volume of 0.2 times the amount of chicken bile, add composite acid solution A to adjust the pH to 5±0.2, extract bilirubin, and then use 90%-95% ethanol for crystallization, filter, and dry to obtain a bilirubin product with a content of more than 95%, wherein the amount of ethanol added is 0.1 times the amount of chicken bile; S3. Separation of high-purity bile acid: After washing the bilirubin with water, the chicken bile is further washed with 20% alcohol methanol water for 3 times the volume of the column. Under this condition, chenodeoxycholic acid is almost not washed out, while bile acid begins to wash out at 1-2 times the volume of the column. The bile acid with a high content of about 1 times the volume of the column is collected, hydrochloric acid is added to adjust the pH value to 5-6, stirred for 30 minutes for crystallization, filtered and dried to obtain bile acid with a purity of more than 98%; S4. Collect high-purity chenodeoxycholic acid Add 50% alcohol methanol to the chicken bile from which bilirubin and bile acid have been separated, collect 1 volume of the chromatographic column of the bile acid and chenodeoxycholic acid mixture, then collect and wash out 2 volumes of the chromatographic column of high-purity chenodeoxycholic acid solution to obtain a 98% pure chenodeoxycholic acid solution, recover the solvent, add hydrochloric acid to adjust the pH value to 3-4 for crystallization, and obtain chenodeoxycholic acid with a purity of more than 98%.

[0006] The method comprises the following steps: slicing frozen chicken gall, heating and thawing the slices to denature the protein into a solid, cooling the slices, performing three-phase centrifugal defatting and protein removal, adding three enzymes, namely, biliverdin reductase, coenzyme I (NAD+) and glucose dehydrogenase (GDH), to reduce free biliverdin and bound biliverdin, adding gluconate hydrolase and bile salt hydrolase to hydrolyze biliverdin and bile acid, adding trypsin to hydrolyze the miscellaneous protein, and finally using macroporous silica gel for chromatography to separate bilirubin, bile acid and chenodeoxycholic acid by utilizing different molecular spatial structures, so as to obtain high-purity bilirubin, bile acid and chenodeoxycholic acid; wherein, the bilirubin extraction rate is 0.02-0.03% and the content is more than 95%; the bile acid yield is about 0.3-0.4% and the purity is more than 98%; the chenodeoxycholic acid yield is 2-3% and the purity is more than 98%.

[0007] Compared with the prior art, the present invention has the following beneficial effects: 1. The use of abundant chicken bile to extract bilirubin solves the problem of severe shortage of bilirubin supply caused by shortage of animal resources such as pigs, cattle and sheep; 2. The method of the present invention can obtain bile acid and chenodeoxycholic acid while extracting bilirubin, thus solving the problems of raw material waste and backward production technology. The process steps are simple, fewer types of solvents are used, and the toxicity is low. The method can meet the production requirements of safety, energy saving, and green environmental protection, and is conducive to large-scale industrial production. DETAILED DESCRIPTION

[0008] The technical solution in the method of the present invention is clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0009] A method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile in this embodiment comprises the following specific steps: (1) Preprocessing Prepare composite acid solution A by mixing one of sodium metabisulfite, sodium thiosulfate and vitamin C with glacial acetic acid and water in a ratio of 1:1:1000, and prepare composite acid solution B by mixing sodium metabisulfite, sodium thiosulfate, sodium hydroxide and water in a ratio of 1:1:1:1000; (2) Chicken bile processing Cut the frozen chicken gall into appropriate sizes, put it into a container for thawing, and add 0.1% sodium sulfite or sodium thiosulfate for protection. The thawed material is heated to 60°C-70°C to fully denature the protein in the chicken gall into a solid. Then, nitrogen is filled in the container for protection. After the material in the container is cooled to room temperature, it is centrifuged in a three-phase centrifuge to remove solid impurities and grease, and the water phase is collected to obtain the chicken gall. (3) Hydrolysis reduction The chicken bile collected in step (2) is put into a reactor, stirred, nitrogen-filled, and then glucose, biliverdin reductase, coenzyme I, glucuronide dehydrogenase and glucuronide hydrolase are added, wherein the amount of the above enzymes added is 1 / 10,000 to 5 / 10,000 of the amount of the chicken bile; then composite alkaline solution B is added to adjust the pH value to 6.5±0.5, and the temperature is controlled to 37°C, and the reaction is carried out for 30 minutes, and then 5 / 10,000 of the amount of the chicken bile bile bile hydrolase is added, and the stirring reaction is continued for 60 minutes, and then 1 / 10,000 of the amount of the chicken bile trypsin is added, and the temperature is raised to 50°C-55°C, and the reaction is carried out for 30 minutes to hydrolyze the impurities; (4) Chromatographic separation Chromatography loading: According to the requirements of the chromatography silica gel manufacturer, use 0.1 mol / L hydrochloric acid, tap water and 0.1 mol / L sodium hydroxide solution, each 2 times the volume of the chromatography column, to activate the chromatography column, then use tap water to wash it to neutrality, then filter the chicken bile that has passed the hydrolysis test through a 5um titanium rod, and load 4 times the volume of the chromatography column with chicken bile; (5) Separation of bilirubin Use composite alkaline solution B to wash the column for 3 times the volume of the chromatography column, the bilirubin pigment band begins to flow out at 1-2 times the volume of the chromatography column, and the bilirubin pigment band is completely washed out at 3 times the volume of the chromatography column, collect the pigment band water phase, add dichloromethane with a volume of 0.2 times the amount of chicken bile, add composite acid solution A to adjust the pH to 5.0±0.2 to extract bilirubin, and then use 90%-95% methanol or ethanol for crystallization, filter, and dry to obtain a bilirubin product with a content of more than 95%, and the amount of ethanol added is 0.2 times the amount of chicken bile; (6) Separation of high-purity bile acid The chicken bile after washing the bilirubin is further chromatographed using 3 times the volume of the chromatography column with 20% alcohol methanol water. Under this condition, chenodeoxycholic acid is almost not eluted, while bile acid is eluted at 1-2 times the volume of the chromatography column. The bile acid with a high content of 1 times the chromatography volume is collected, hydrochloric acid is added to adjust the pH value to 5-6, stirred for 30 minutes for crystallization, filtered and dried to obtain bile acid with a purity of more than 98%; (7) Collecting high-purity chenodeoxycholic acid Add 50% alcohol methanol to the chicken bile from which bilirubin and bile acid have been separated, collect 1 volume of the chromatographic column mixed solution of bile acid and chenodeoxycholic acid, recover and dissolve it, and then repeat the process, then continue to collect 2 volumes of the chromatographic column eluate of chenodeoxycholic acid to obtain a 98% pure chenodeoxycholic acid solution, recover the solvent, add hydrochloric acid to adjust the pH to 3-4 for crystallization, and filter and dry to obtain chenodeoxycholic acid with a purity of more than 98%. Embodiment 1

[0010] 500 g of frozen fresh chicken gallbladder was cut into suitable sizes, put into a container for thawing, and 0.5 g of sodium sulfite was added for protection. The thawed material was heated to 60° C. to fully denature the protein in the chicken gallbladder into a solid. Then, nitrogen was filled in the container for protection. After the material in the container was cooled to room temperature, it was centrifuged in a three-phase centrifuge to remove solid impurities and grease, and 410 g of chicken gallbladder was collected; 410g of chicken bile was put into a reactor, and after stirring and nitrogen filling, 20g of glucose, 0.1g of biliverdin reductase, 50mg of coenzyme-1, 0.1g of glucuronic acid dehydrogenase, and 0.2g of glucuronic acid hydrolase were added, and the pre-configured composite alkali solution B was added to adjust the pH value to 5.8, and the temperature was controlled to 37°C, and the reaction was carried out for 30 minutes, and then 0.2g of bile salt hydrolase was added, and the stirring reaction was continued for 60 minutes, and then 0.05g of trypsin was added, and the temperature was raised to 50°C, and the reaction was carried out for 30 minutes to hydrolyze the impurities; 100g of 5um macroporous chromatography silica gel was filled into the column, 100ml of 0.1mol / L hydrochloric acid was used for column washing, and then tap water was used to wash the column. When the pH value was 6-7, 200ml of 0.1mol / L sodium hydroxide solution was used for activation, and then tap water was used to wash the chromatography column until the pH value was 7-8. Chicken bile that passed the hydrolysis test was filtered through a 5um pore size filter membrane, 400g of chicken bile hydrolyzate was weighed and loaded, and after loading, The column was washed with 300 ml of composite alkali solution B. The bilirubin pigment band began to flow out at about 130 ml of the column. The bilirubin pigment band was washed out at about 300 ml. 170 g of the pigment band aqueous phase was collected, 80 g of dichloromethane was added, and the composite acid solution A was added to adjust the pH to 5.05 to extract bilirubin. The mixture was recovered under reduced pressure into a paste, and then refined with 40 g of 90% ethanol to obtain 116 mg of bilirubin, with a detection content of 96% and a yield of 0.232‰. In the chicken bile after washing the bilirubin with water, 300 ml of 20% alcohol methanol was used to continue washing the column. Under this condition, chenodeoxycholic acid was almost not washed out, while bile acid began to wash out at around 140 ml. 100 g of high-content bile acid was collected, hydrochloric acid was added to adjust the pH value to 5, crystallized, stirred for 30 minutes, filtered, and dried to obtain 2.1 g of bile acid, HPLC purity 98.1%, yield 0.22%; In the chicken bile from which bilirubin and bile acid have been separated, 300 g of methanol with 50% alcohol content is added, and after collecting 100 ml of mixed bile acid, the solvent is recovered in industrial production and then applied, and then 200 ml of the eluate is collected to obtain 200 g of chenodeoxycholic acid solution with a purity of 98%. After the solvent is recovered, hydrochloric acid is added to adjust the pH to 3.2, and chenodeoxycholic acid will precipitate white crystals, which are filtered and dried to obtain 12.3 g of chenodeoxycholic acid, and the purity tested by HPLC is: 98.2%. Embodiment 2

[0011] 500 g of frozen fresh chicken gallbladder was cut into suitable sizes, put into a container for thawing, and 0.5 g of sodium thiosulfate was added for protection. The thawed material was heated to 65° C. to fully denature the protein in the chicken gallbladder into a solid, and then nitrogen was filled in the container for protection. After the material in the container was cooled to room temperature, it was centrifuged in a three-phase centrifuge to remove solid impurities and grease, and 430 g of chicken gallbladder was collected; 430g of chicken bile was put into a reactor, and after stirring and nitrogen filling, 20g of glucose, 0.1g of biliverdin reductase, 50mg of coenzyme-1, 0.1g of glucuronic acid dehydrogenase, and 0.2g of glucuronic acid hydrolase were added, and the pre-configured composite alkali solution B was added to adjust the pH value to 5.8, and the temperature was controlled to 37°C, and the reaction was carried out for 30 minutes, and then 0.2g of bile salt hydrolase was added, and the stirring reaction was continued for 60 minutes, and then 0.05g of trypsin was added, and the temperature was raised to 50°C, and the reaction was carried out for 30 minutes to hydrolyze the impurities; 100g of 5um macroporous chromatography silica gel was filled into the column, 100ml of 0.1mol / L hydrochloric acid was used for column washing, and then 200ml of tap water was used to wash the column. When the pH value was 6-7, 200ml of 0.1mol / L sodium hydroxide solution was used for activation, and then the column was washed with tap water until the pH value was 7-8. The chicken bile that passed the hydrolysis test was filtered through a 5um pore size filter membrane, and 400g of the chicken bile hydrolyzate was weighed and loaded. After the loading is completed, 300g of composite alkali solution B is used to wash the column. The bilirubin pigment band begins to flow out at about 130ml of the column washing, and the bilirubin pigment band is washed out at about 300ml. 170g of the pigment band aqueous phase is collected, 80g of dichloromethane is added, and the composite acid solution A is added to adjust the pH to 4.95 to extract bilirubin, and the reduced pressure is recovered into a paste, and then 40g of 90% ethanol is used for purification to obtain 126mg of bilirubin, and the detection content is 95.8%; In the chicken bile after washing the bilirubin with water, 300 ml of 20% alcohol methanol was used to continue washing the column. Under this condition, chenodeoxycholic acid was almost not washed out, while bile acid began to wash out at around 140 ml. 100 g of high-content bile acid was collected, hydrochloric acid was added to adjust the pH value to 5, crystallization was performed, and after stirring for 30 minutes, filtration was performed and dried to obtain 1.98 g of bile acid with a HPLC purity of 98.2%. In the chicken bile from which bilirubin and bile acid have been separated, 300 g of methanol with 50% alcohol content is added, and after collecting 100 ml of mixed bile acid, the solvent is recovered in industrial production and then applied, and then 200 ml of the eluate is collected to obtain 200 g of chenodeoxycholic acid solution with a purity of 98%. After the solvent is recovered, hydrochloric acid is added to adjust the pH to 3.2, and chenodeoxycholic acid will precipitate white crystals, which are filtered and dried to obtain 11.5 g of chenodeoxycholic acid, and the purity tested by HPLC is: 98.2%. Embodiment 3

[0012] 500 g of frozen fresh chicken gall was cut into suitable sizes, put into a container for thawing, and 0.1 g of sodium thiosulfate was added for protection. The thawed material was heated to 70° C. to fully denature the protein in the chicken gall into a solid, and then nitrogen was filled in the container for protection. After the material in the container was cooled to room temperature, it was centrifuged in a three-phase centrifuge to remove solid impurities and grease, and 415 g of chicken gall was collected; 415g of chicken bile was put into a reactor, and after stirring and nitrogen filling, 20g of glucose, 0.1g of biliverdin reductase, 50mg of coenzyme-1, 0.1g of glucuronic acid dehydrogenase, and 0.2g of glucuronic acid hydrolase were added, and the pre-configured composite alkali solution B was added to adjust the pH value to 5.8, and the temperature was controlled to 37°C, and the reaction was carried out for 30 minutes, and then 0.2g of bile salt hydrolase was added, and the stirring reaction was continued for 60 minutes, and then 0.05g of trypsin was added, and the temperature was raised to 50°C, and the reaction was carried out for 30 minutes to hydrolyze the impurities; 100g of 5um macroporous chromatography silica gel was filled into the column, 100ml of 0.1mol / L hydrochloric acid was used for column washing, and then tap water was used to wash the column. When the pH value was 6-7, 200ml of 0.1mol / L sodium hydroxide solution was used for activation, and then tap water was used to wash the chromatography column until the pH value was 7-8. Chicken bile that passed the hydrolysis test was filtered through a filter membrane with a pore size of 5um, 400g of chicken bile hydrolyzate was weighed for sample loading, and after the sample loading was completed, 300ml of composite alkali solution B was used for column washing. The bilirubin pigment band began to flow out after about 130ml of column washing, and the bilirubin pigment band was washed out after about 300ml. 170g of pigment band aqueous phase was collected, 80g of dichloromethane was added, and composite acid solution A was added to adjust the pH value to 4.95, bilirubin was extracted, and the mixture was recovered under reduced pressure into a paste, and then 40g of 90% ethanol was used for purification to obtain 123mg of bilirubin, and the detection content was 96.3%; In the chicken bile after washing the bilirubin with water, 300 ml of 20% alcohol methanol was used to continue washing the column. Under this condition, chenodeoxycholic acid was almost not washed out, while bile acid began to wash out at around 140 ml. 100 g of high-content bile acid was collected, hydrochloric acid was added to adjust the pH value to 5, crystallization was performed, and after stirring for 30 minutes, filtration was performed and dried to obtain 1.9 g of bile acid with a HPLC purity of 98.3%; In the chicken bile from which bilirubin and bile acid have been separated, 300 g of methanol with 50% alcohol content is added, and after collecting 100 ml of mixed bile acid, the solvent is recovered in industrial production and then applied, and then 200 ml of the eluate is collected to obtain 200 g of chenodeoxycholic acid solution with a purity of 98%. After the solvent is recovered, hydrochloric acid is added to adjust the pH to 3.2, and chenodeoxycholic acid will precipitate white crystals, which are filtered and dried to obtain 10.6 g of chenodeoxycholic acid, and the purity tested by HPLC is: 98.2%.

[0013] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile, characterized in that: The process steps include: (1) Preprocessing The composite acid solution A is prepared by using an antioxidant, glacial acetic acid and water, and the composite acid solution B is prepared by using sodium pyrosulfite, sodium thiosulfate, sodium hydroxide and water; (2) Chicken bile processing Cut the frozen chicken gall into appropriate sizes, thaw and heat, cool to room temperature and centrifuge continuously by three-phase centrifugation, collect the water phase, which is the chicken bile; (3) Hydrolysis reduction Add glucose, coenzyme I, biliverdin reductase, glucuronide dehydrogenase and glucuronide hydrolase to the chicken bile in step (2), then add composite alkaline solution B to adjust the pH value to 6.5±0.5, add bile salt hydrolase and trypsin to hydrolyze the impurities in the chicken bile; (4) Chromatographic separation Chicken bile was chromatographed on macroporous silica gel to separate bilirubin, bile acid and chenodeoxycholic acid.

2. A method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 1, characterized in that: The configuration ratio of the antioxidant, glacial acetic acid and water in the composite acid solution A is 1:1:1000, and the antioxidant is one of sodium pyrosulfite, sodium thiosulfate and vitamin C; the configuration ratio of sodium pyrosulfite, sodium thiosulfate, sodium hydroxide and water in the composite acid solution B is 1:1:1:1000.

3. A method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 2, characterized in that: When the frozen sliced ​​chicken bile in step (2) is put into a container for thawing, 0.1% sodium sulfite or sodium thiosulfate is added for protection, and the thawed material is heated to 60° C.-70° C. to fully denature the protein in the chicken bile into a solid. Then, nitrogen is filled in the container for protection. After the material in the container is cooled to room temperature, it is centrifuged using a three-phase centrifuge to remove solid impurities and grease, and the water phase collected is the chicken bile.

4. A method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 3, characterized in that: The hydrolysis reduction is to put the chicken bile collected in step (2) into a reactor, stir, fill with nitrogen, then add glucose, coenzyme I, biliverdin reductase, glucuronide dehydrogenase and glucuronide hydrolase, the amount of the above enzymes added is 1 / 10,000 to 5 / 10,000 of the chicken bile, then add composite alkaline solution B to adjust the pH value to 6.5±0.5, and control the temperature to 37°C, react for 30 minutes, then add 5 / 10,000 of the chicken bile bile bile hydrolase, continue stirring and reacting for 60 minutes, then add 1 / 10,000 of the chicken bile bile trypsin, raise the temperature to 50°C-55°C, react for 30 minutes, and hydrolyze the impurities.

5. The method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 1, characterized in that: The chromatographic separation in step (4) comprises the following specific steps: S1. Chromatography loading: Pre-treat the chromatography column, filter the chicken bile that has passed the hydrolysis test through a 5um titanium rod barrel, and load the chicken bile in an amount of 3-4 times the volume of the chromatography column; S2. Separation of bilirubin: Use composite alkaline solution B for chromatography for 3 times of the volume of the chromatography column, when the column is washed for 1-2 times of the volume of the chromatography column, the bilirubin pigment band begins to flow out, and when the column is washed for 3 times of the volume of the chromatography column, the bilirubin pigment band is completely washed out, collect the pigment band aqueous phase, add dichloromethane with a volume of 0.2 times the amount of chicken bile, add composite acid solution A to adjust the pH to 5±0.2 to extract bilirubin, recover the solvent, and then use 90%-95% ethanol for crystallization, filter, and dry to obtain a bilirubin product with a content of more than 95%, wherein the amount of ethanol added is 0.1 times the amount of chicken bile; S3. Separation of high-purity bile acid: The chicken bile after washing the bilirubin is further washed with 20% alcohol methanol water for 3 times the volume of the chromatography column, bile acid begins to be washed out at 1-2 times the volume of the chromatography column, 1-2 times the volume of the chromatography column bile acid aqueous solution is collected, hydrochloric acid is added to adjust the pH value to 5-6, stirred for 30 minutes for crystallization, filtered and dried to obtain bile acid with a purity of more than 98%; S4. Collect high-purity chenodeoxycholic acid Continue to extract 3 times the volume of the chromatography column from the chicken bile from which bilirubin and bile acid have been separated, add 50% alcohol methanol water for chromatography elution, collect 1 times the volume of the chromatography column eluate, which is a mixed solution of bile acid and chenodeoxycholic acid, and recover the solvent before using it for the next batch; continue to collect 2 times the volume of the chromatography column eluate to obtain a chenodeoxycholic acid solution with a purity of 98%. After recovering the solvent, add hydrochloric acid to adjust the pH value to 3±0.3 for crystallization, filter and dry to obtain chenodeoxycholic acid with a purity of more than 98%.

6. A method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 5, characterized in that: In the chromatographic separation of step (4), the extraction rate of bilirubin is 0.02-0.03%.

7. The method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 5, characterized in that: In the chromatographic separation of step (4), the yield of bile acid is 0.3-0.4%.

8. The method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 5, characterized in that: In the chromatographic separation of step (4), the yield of chenodeoxycholic acid is 2-3%.