Method for separating and purifying taurochenodeoxycholic acid from chicken bile

Abstract

The invention provides a method for separating and purifying taurochenodeoxycholic acid from chicken bile, which comprises the following steps: 1) extracting the chicken bile by using ethyl acetate, and collecting raffinate; (2) adding ethanol into the raffinate in the step (1), carrying out centrifugal treatment, collecting centrifuged supernate, evaporating to remove ethanol to obtain an extracting solution, and adjusting the pH value of the extracting solution to be acidic; (3) adding activated carbon into the extracting solution to perform adsorption treatment, then performing centrifugal treatment, and collecting centrifuged supernate to obtain a to-be-purified solution; and 4) carrying out chromatographic purification on the to-be-purified liquid to obtain a purified taurochenodeoxycholic acid product. According to the technical scheme, the pretreatment steps of the chicken bile are simple, after activated carbon treatment, filtering operation is easy, and adsorption resin used in the purification process is high in loading capacity and low in cost.

Description

Technical Field

[0001] The invention relates to the fields of pharmaceutical chemical industry and purification technology, and in particular to a method for separating and purifying taurochenodeoxycholic acid from chicken bile. Background Art

[0002] Taurochenodeoxycholic acid (TCDCA) is widely used in the medical field, such as for the treatment of biliary diseases, indigestion, hepatitis and other diseases. It has the functions of promoting bile secretion, enhancing bile excretion, and improving gallbladder function. It has certain antioxidant, anti-inflammatory, antiviral and other biological activities, and plays a positive role in improving human health and quality of life.

[0003] Chicken bile mainly contains bile pigments, cholesterol, mucin, inorganic salts and bile acids. Bile acids mainly include taurochenodeoxycholic acid, taurocholic acid, tauroallocholic acid and 3α-hydroxy-7-ketocholic acid, among which taurochenodeoxycholic acid has the highest content.

[0004] Taurochenodeoxycholic acid is an important raw material for the enzymatic synthesis of tauroursodeoxycholic acid. Therefore, it is of great significance to obtain high-purity, high-yield, and low-cost tauroursodeoxycholic acid from chicken bile for the preparation of tauroursodeoxycholic acid.

[0005] In the prior art, poultry bile is first extracted with water, and a modified superhydrophobic sponge is used to separate oil and water to obtain a decontaminated water extract; after the water extract is acidified, a modified reverse phase filler capped with trimethylchlorosilane is used for purification to obtain high-purity taurocholic acid and tauroursodeoxycholic acid. However, the C18 reverse phase filler used in this method has high pressure and is expensive. Summary of the invention

[0006] The present invention provides a method for separating and purifying taurochenodeoxycholic acid from chicken bile to solve the problems of complicated operation steps and high cost in the prior art. To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for separating and purifying taurochenodeoxycholic acid from chicken bile comprises the following steps:

[0008] Step 1) extracting chicken bile with ethyl acetate and collecting the raffinate;

[0009] Step 2) adding ethanol to the raffinate in step 1) and centrifuging the raffinate, collecting the supernatant after centrifugation and removing the ethanol to obtain an extract, and adjusting the pH of the extract to acidic;

[0010] Step 3) adding activated carbon to the extract for adsorption treatment, then centrifuging, and collecting the supernatant after centrifugation to obtain the liquid to be purified;

[0011] Step 4) The solution to be purified is subjected to chromatography purification to obtain a purified taurochenodeoxycholic acid product.

[0012] Optionally, the extraction in step 1) includes: mixing chicken bile and ethyl acetate in a volume ratio of 1:1-2, then standing for 40-120 minutes, and collecting the raffinate.

[0013] Optionally, in the step 2), the volume ratio of the raffinate to ethanol is 1:1-2.

[0014] Optionally, the centrifugation in step 2) is performed at 10000-12000 r / min for 15-20 min.

[0015] Optionally, in step 2), the pH of the extract is adjusted to 3-5.

[0016] Optionally, the amount of activated carbon added in step 3) is 0.5%-1.5% of the mass of the extract; the activated carbon is activated carbon particles of 200-500 mesh; the adsorption treatment time is 30-60min; and the centrifugal treatment after adsorption is centrifugation at 10000-12000r / min for 15-20min.

[0017] Optionally, in the step 4), an adsorption resin or a reverse phase filler is used as the stationary phase, and an aqueous solution of methanol is used as the mobile phase to perform chromatography purification on the liquid to be purified.

[0018] The adsorption resin adopts styrene-divinylbenzene copolymer microspheres with phenyl surface groups; the crosslinking degree is 50-80%, the average particle size of the polymer microspheres is 30-150um, the pore size is 50-300A, and the specific surface area is 300-700m 2 / g.

[0019] The reverse phase filler is a styrene-divinylbenzene copolymer microsphere with a surface group of C18 group; the crosslinking degree is 50-80%, the average particle size of the polymer microsphere is 30-150um, the pore size is 50-300A, and the specific surface area is 300-700m 2 / g.

[0020] Optionally, in step 4), an adsorption resin is used as the stationary phase; the purification step in step 4) comprises the following steps:

[0021] The adsorption resin is loaded into the column and balanced; then the sample is loaded at a loading capacity of 30-45 mg of pure product / ml of filler material, and the loading flow rate is not higher than 6 cm / min.

[0022] The impurities were washed with 20%-30% methanol aqueous solution for 12-15CV; then washed with pure water for 2-3CV; then eluted with 40%-60% methanol aqueous solution for 4-6CV, the elution flow rate was not higher than 6cm / min, and the peak was collected for 4-5CV.

[0023] Preferably, the adsorption resin column packing comprises: adding water to the adsorption resin to homogenize, pouring into a glass chromatographic column, allowing natural sedimentation, removing the upper water layer, and adding adsorption resin until the adsorption resin fills the chromatographic column, retaining a 2-4 cm water layer, and the height of the chromatographic column is 55-60 cm.

[0024] Preferably, the adsorption resin loaded in the column is equilibrated with sodium dihydrogen phosphate solution for 4-6CV, and the equilibrium flow rate is not higher than 6cm / min.

[0025] Optionally, in step 4), a reverse phase filler is used as the stationary phase; the purification step in step 4) comprises the following steps:

[0026] The reverse phase filler is loaded into the column and equilibrated; then the sample is loaded at a loading capacity of 40-55 mg of pure product / ml of filler, and the loading flow rate is not higher than 6 cm / min;

[0027] Use 20%-30% methanol aqueous solution to wash for 10-15CV; then wash with pure water for 2-3CV; then use 40%-60% methanol aqueous solution to elute for 8-10CV, the elution flow rate is not higher than 6cm / min, and collect 8-10CV after the peak appears.

[0028] Preferably, the filler is homogenized with 50% methanol, loaded into the column with water, switched with methanol after compaction, and then loaded into the column with water. Finally, the filler is stored in water, the column pressure is not less than 10 MPa, the height is 55-60 cm, and the flow rate is not higher than 6 cm / min.

[0029] Optionally, packing the reverse phase filler into the column includes: slurrying the filler with 50% methanol, then packing the column with water, switching with methanol after compaction, and then packing the column with water, and finally storing the filler in water, the compaction pressure of the column is not less than 10MPa, the height is not higher than 60cm, and the flow rate is not higher than 6cm / min.

[0030] Preferably, the adsorption resin loaded in the column is equilibrated with 20 mM sodium dihydrogen phosphate solution for 4-6 CV, and the equilibrium flow rate is not higher than 6 cm / min.

[0031] Optionally, after the chromatography treatment in step 4) is completed, the stationary phase is regenerated with 80% methanol for 3-6CV at a flow rate of no more than 6 cm / min.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The chicken bile of the present invention is purified by extraction, extraction, activated carbon adsorption, macroporous adsorption resin or reverse phase filler to obtain taurochenodeoxycholic acid with a purity of ≥95%. Compared with the prior art, the technical scheme of the present invention has simple pre-treatment steps for the chicken bile, and after the activated carbon treatment, it is easy to filter and operate. The adsorption resin used in the purification process has high loading capacity and low cost. Only methanol is used in the resin purification process, and the recovery is simple. No other toxic organic solvents are used in the entire purification process. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0035] Figure 1 This is a high performance liquid chromatogram of taurochenodeoxycholic acid before purification in Example 1 of the present invention;

[0036] Figure 2 This is a high performance liquid chromatogram of purified taurochenodeoxycholic acid according to Example 1 of the present invention;

[0037] Figure 3 is a high performance liquid chromatogram of purified taurochenodeoxycholic acid according to Example 2 of the present invention;

[0038] Figure 4 The HPLC chromatogram of taurochenodeoxycholic acid dry powder prepared in Example 2 of the present invention. DETAILED DESCRIPTION

[0039] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0040] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0041] The method of the present invention comprises the steps of:

[0042] Extraction: Cut the chicken bile, release the bile, add ethyl acetate for extraction, and collect the aqueous phase.

[0043] Extraction: Add ethanol to the aqueous phase collected in the previous step for extraction, collect the supernatant after centrifugation, remove the ethanol by rotary evaporation, and adjust the pH to acidic.

[0044] Activated carbon adsorption: Add a certain amount of activated carbon, centrifuge after adsorption, collect the supernatant and filter it for later use.

[0045] Chromatography: Purify the supernatant after filtration in the previous step with adsorption resin or reverse phase filler, collect and concentrate by rotary evaporation.

[0046] Furthermore, the specific process of the method of the present invention comprises the following steps:

[0047] Extraction: Fresh chicken bile is extracted with ethyl acetate, the ratio of chicken bile to ethyl acetate is 1:1-2, after shaking well, let stand for 40-120 minutes, and collect the aqueous phase.

[0048] Extraction: Add ethanol to the aqueous phase in the previous step, with the ratio of aqueous phase to ethanol being 1:1-2, stir evenly, leave for 2-4 hours, centrifuge at 10000r / min for 15 minutes, collect the supernatant, remove ethanol by rotary evaporation, and adjust the pH to 3-5.

[0049] Activated carbon adsorption: Add 0.5%-1.5% of the above acidic solution to activated carbon, the activated carbon is 200-500 mesh, the adsorption time is 30-60min, centrifuge at 10000r / min for 20min, and filter the supernatant for use.

[0050] Column Packing:

[0051] Adsorption resin column loading: Add water to the resin to make a slurry, pour it into a glass chromatographic column, let it settle naturally, remove the upper water layer, and add filler until the resin fills the chromatographic column, retaining a water layer of about 3 cm and a height of no more than 60 cm.

[0052] Reverse phase filler column loading: The filler is slurried with 50% methanol-water solution, loaded into the column with water, and after compaction, it is switched with methanol-water solution, and then loaded into the column with water. Finally, the filler is stored in water. The column loading pressure is not less than 10MPa, the height is not higher than 60cm, and the flow rate is not higher than 6cm / min.

[0053] purification:

[0054] Adsorption resin purification process flow: balance: 20mM sodium dihydrogen phosphate balance 4-6CV, flow rate not higher than 6cm / min, loading: take the filtered sample, load according to the loading capacity of 30-45mg (pure product) / ml (filler), flow rate not higher than 6cm / min, wash: 25% methanol aqueous solution wash 12-15CV, water wash: pure water wash 2-3CV, elution: 50% methanol aqueous solution wash 4-6CV, flow rate not higher than 6cm / min, collect 4-5CV after the peak, regeneration: 80% methanol aqueous solution regeneration 3-6CV, flow rate not higher than 6cm / min.

[0055] Reverse phase filler purification process flow: balance: 20mM sodium dihydrogen phosphate balance 4-6CV, flow rate not higher than 6cm / min, loading: take the sample after filtration, load according to the loading capacity of 40-55mg (pure product) / ml (filler), flow rate not higher than 6cm / min, wash: 25% methanol aqueous solution wash 10-15CV, water wash: pure water wash 2-3CV, elution: 40% methanol aqueous solution wash 8-10CV, flow rate not higher than 6cm / min, collect 8-10CV after the peak, regeneration: 80% methanol aqueous solution regeneration 3-6CV, flow rate not higher than 6cm / min.

[0056] Rotary evaporation: Rotary evaporate the eluate from the previous step at 40°C and dry to obtain dry powder.

[0057] More specifically, the adsorption resin refers to polymer microspheres formed by copolymerization of styrene and divinylbenzene, with a crosslinking degree of 50-80%, an average particle size of 30-150um, a pore size of 50-300A, and a specific surface area of ​​300-700m 2 / g, the surface group is phenyl.

[0058] The reverse phase filler is a polymer microsphere formed by copolymerization of styrene and divinylbenzene, with a crosslinking degree of 50-80%, an average particle size of 30-150um, a pore size of 50-300A, and a specific surface area of ​​300-700m 2 / g, and the surface group is C18.

[0059] The purity detection method of taurochenodeoxycholic acid in each embodiment is as follows:

[0060] Taurochenodeoxycholic acid is detected by liquid phase detection method, and the specific steps and parameters are as follows:

[0061] Chromatographic column: Shimadzu Shim-pack GIST C18 5um 4.6×250mm;

[0062] Column temperature: 40°C;

[0063] Wavelength: 210nm;

[0064] Flow rate: 0.8ml / min;

[0065] Mobile phase: methanol: mixed solution (the mixed solution is prepared by dissolving 2.34 g of sodium dihydrogen phosphate and 1.0 g of sodium heptane sulfonate in 350 ml of water) = 65:35.

[0066] Example 1

[0067] Step 1) Extraction: Cut fresh chicken bile, collect chicken bile, add ethyl acetate in a ratio of 1:1, shake well, let stand for 40 minutes, and collect the aqueous phase.

[0068] Step 2) Extraction: Add ethanol to the aqueous phase in the previous step, with the amount of aqueous phase to ethanol being 1:1, stir evenly, leave for 2 hours, centrifuge at 10000r / min for 15 minutes, collect the supernatant, remove ethanol by rotary evaporation, and adjust the pH to 3.5.

[0069] Step 3) activated carbon adsorption: add 0.5% activated carbon to the above solution with pH 3.5, the activated carbon is 200 mesh, the adsorption time is 60 min, centrifuge at 10000 r / min for 20 min, and filter the supernatant for use.

[0070] Step 4) Adsorption resin purification: 20SS resin (produced by Xi'an Lanxiao Technology New Materials Co., Ltd.) was loaded into the column, with an average particle size of 75um, a pore size of 64.6A, and a specific surface area of ​​557m 2 / g. Weigh 55g of resin, slurry with pure water, pour into a glass chromatographic column, settle naturally, add more resin, retain 3cm of water layer, resin filling height 57cm, diameter 1cm, column volume calculation according to height 60cm, one column volume equals 47ml. Balance: 20mM sodium dihydrogen phosphate balance 4CV, flow rate 6cm / min, sample loading: take the filtered sample, load according to the load 35mg (pure product) / ml (filler), flow rate 3cm / min, wash: 25% methanol aqueous solution wash 12CV, water wash: pure water wash 2CV, elution: 50% methanol aqueous solution wash 5CV, flow rate 6cm / min, collect 4CV after peak, regeneration: 80% methanol aqueous solution regeneration 3CV, flow rate 6cm / min. Test results: the purity of the sample solution is 69.499%, the purity after purification is 95.727%, and the yield is 78%. Figure 1 is a high performance liquid chromatogram of taurochenodeoxycholic acid before purification in Example 1 of the present invention, Figure 2 The HPLC chromatogram of purified taurochenodeoxycholic acid in Example 1 of the present invention is shown in FIG.

[0071] Step 5) Rotary evaporation: Rotary evaporate the eluate from the previous step at 40° C. and dry to obtain taurochenodeoxycholic acid dry powder.

[0072] Example 2

[0073] Step 1) Extraction: Cut fresh chicken bile, collect chicken bile, add ethyl acetate in a ratio of 1:1, shake well, let stand for 40 minutes, and collect the aqueous phase.

[0074] Step 2) Extraction: Add ethanol to the aqueous phase in the previous step, with the amount of aqueous phase to ethanol being 1:1, stir evenly, leave for 2 hours, centrifuge at 10000r / min for 15 minutes, collect the supernatant, remove ethanol by rotary evaporation, and adjust the pH to 4.

[0075] Step 3) activated carbon adsorption: add 0.5% activated carbon to the above pH 4 solution, the activated carbon is 200 mesh, the adsorption time is 60 minutes, centrifuge at 10000r / min for 20 minutes, and filter the supernatant for use.

[0076] Step 4) Reverse phase filler purification: 261-70 filler (produced by Xi'an Lanxiao Technology New Materials Co., Ltd.) column, filler average particle size 75um, pore size 96.7A, specific surface area 395m2 / g, filler homogenized with 50% methanol aqueous solution, water column, after compaction, switch with methanol aqueous solution, and then fill with water, the final filler is stored in water, column pressure 10MPa, height 60cm, flow rate 6cm / min. Balance: 20mM sodium dihydrogen phosphate balance 4CV, flow rate 6cm / min, sample loading: take the filtered sample, load according to the load 45mg (pure product) / ml (filler), flow rate 3cm / min, wash: 25% methanol aqueous solution wash 12CV, water wash: pure water wash 2CV, elution: 40% methanol aqueous solution wash 10CV, flow rate 6cm / min, collect 8.5CV after peak, regeneration: 80% methanol aqueous solution regeneration 6CV, flow rate 6cm / min.

[0077] Test results: the purity of the sample solution was 69.504%, the purity after purification was 95.824%, and the yield was 90%. Figure 3 is a high performance liquid chromatogram of purified taurochenodeoxycholic acid according to Example 2 of the present invention; Figure 4 The HPLC chromatogram of taurochenodeoxycholic acid dry powder prepared in Example 2 of the present invention.

[0078] Example 3

[0079] Step 1) Extraction: Cut the fresh chicken bile, collect the chicken bile, add ethyl acetate in a ratio of 1:2, shake well, let stand for 40 minutes, and collect the aqueous phase.

[0080] Step 2) Extraction: Add ethanol to the aqueous phase in the previous step, the amount of aqueous phase to ethanol is 1:2, stir evenly, let stand for 2 hours, centrifuge at 10000r / min for 15min, collect the supernatant, remove ethanol by rotary evaporation, and adjust the pH to 3.5.

[0081] Step 3) activated carbon adsorption: add 0.5% activated carbon to the above solution with pH 3.5, the activated carbon is 200 mesh, the adsorption time is 60 minutes, centrifuge at 10000r / min for 20 minutes, and filter the supernatant for use.

[0082] Step 4) Adsorption resin purification: 20SS resin (produced by Xi'an Lanxiao Technology New Materials Co., Ltd.) is loaded into the column, 55g of resin is weighed, pure water is homogenized, poured into a glass chromatographic column, and naturally settled. Resin is added, and a 3cm water layer is retained. The resin is loaded at a height of 57cm and a diameter of 1cm. The column volume is calculated according to a height of 60cm, and one column volume is equal to 47ml. Balance: 20mM sodium dihydrogen phosphate is balanced for 4CV, flow rate 6cm / min, sample loading: take the filtered sample, load it according to a load of 40mg (pure product) / ml (filler), flow rate 3cm / min, wash: 25% methanol aqueous solution for 12CV, water washing: pure water washing for 2CV, elution: 50% methanol aqueous solution for 5CV, flow rate 6cm / min, collect 4CV after the peak, regeneration: 80% methanol aqueous solution for 3CV, flow rate 6cm / min.

[0083] Test results: the purity of the sample solution was 69.499%, the purity after purification was 93.824%, and the yield was 70%.

[0084] Step 5) Rotary evaporation: Rotary evaporate the eluate from the previous step at 40° C. and dry to obtain taurochenodeoxycholic acid dry powder.

[0085] Comparative Example 1

[0086] Compared with Example 2, the first extraction step is not performed in this comparative example; the chicken bile is directly extracted with ethanol using the method of step 2). The other steps are consistent with Example 2, and the results are: subsequent filtration is difficult, the product has too many impurities, and the purity is low.

[0087] Test results: the purity of the sample solution was 69.302%, the purity after purification was 91.324%, and the yield was 70%.

[0088] Comparative Example 2

[0089] Compared with Example 3, the pH was not adjusted in this comparative example, and the supernatant with a pH of 8.5 was adsorbed and purified by the methods of step 3) and step 4). The other steps were consistent with Example 3, and the results showed that the activated carbon adsorption effect was poor and difficult to filter.

[0090] Test results: the purity of the sample solution was 69.832%, the purity after purification was 94.313%, and the yield was 85%.

[0091] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for separating and purifying taurochenodeoxycholic acid from chicken bile, characterized in that: The steps include: Step 1) extracting chicken bile with ethyl acetate and collecting the raffinate; Step 2) adding ethanol to the raffinate in step 1) and centrifuging the raffinate, collecting the supernatant after centrifugation and removing the ethanol to obtain an extract, and adjusting the pH of the extract to acidic; Step 3) adding activated carbon to the extract for adsorption treatment, then centrifuging, and collecting the supernatant after centrifugation to obtain the liquid to be purified; Step 4) The solution to be purified is subjected to chromatography purification to obtain a purified taurochenodeoxycholic acid product.

2. The method for separating and purifying taurochenodeoxycholic acid from chicken bile according to claim 1, characterized in that: The extraction in step 1) comprises: mixing chicken bile and ethyl acetate in a volume ratio of 1:1-2, then standing for 40-120 minutes, and collecting the raffinate.

3. The method for separating and purifying taurochenodeoxycholic acid from chicken bile according to claim 1, characterized in that: In the step 2), the volume ratio of the raffinate to ethanol is 1:1-2.

4. The method for separating and purifying taurochenodeoxycholic acid from chicken bile according to claim 1, characterized in that: The centrifugation in step 2) is performed at 10000-12000 r / min for 15-20 min.

5. The method for separating and purifying taurochenodeoxycholic acid from chicken bile according to claim 1, characterized in that: In the step 2), the pH of the extract is adjusted to 3-5.

6. The method for separating and purifying taurochenodeoxycholic acid from chicken bile according to claim 1, characterized in that: The amount of activated carbon added in the step 3) is 0.5%-1.5% of the mass of the extract; the activated carbon is activated carbon particles of 200-500 mesh; the adsorption treatment time is 30-60min; the centrifugal treatment after adsorption is 10000-12000r / min centrifugation for 15-20min.

7. The method for separating and purifying taurochenodeoxycholic acid from chicken bile according to claim 1, characterized in that: In the step 4), an adsorption resin or a reverse phase filler is used as the stationary phase, and an aqueous solution of methanol is used as the mobile phase to chromatographically purify the solution to be purified; The adsorption resin adopts styrene-divinylbenzene copolymer microspheres with phenyl surface groups; the crosslinking degree is 50-80%, the average particle size of the polymer microspheres is 30-150um, the pore size is 50-300A, and the specific surface area is 300-700m 2 / g; The reverse phase filler is a styrene-divinylbenzene copolymer microsphere with a surface group of C18 group; the crosslinking degree is 50-80%, the average particle size of the polymer microsphere is 30-150um, the pore size is 50-300A, and the specific surface area is 300-700m 2 / g.

8. The method for separating and purifying taurochenodeoxycholic acid from chicken bile according to claim 1, characterized in that: In the step 4), an adsorption resin is used as the stationary phase; the purification step in the step 4) comprises the following steps: The adsorption resin is loaded into the column and balanced; then the sample is loaded at a loading capacity of 30-45 mg of pure product / ml of filler, and the loading flow rate is not higher than 6 cm / min; Use 20%-30% methanol aqueous solution to wash for 12-15CV; then wash with pure water for 2-3CV; then use 40%-60% methanol aqueous solution to elute for 4-6CV, the elution flow rate is not higher than 6cm / min, and collect 4-5CV after the peak emerges; Preferably, the step of packing the adsorption resin into a column comprises: adding water to the adsorption resin to homogenize, pouring the mixture into a glass chromatographic column, allowing the mixture to settle naturally, removing the upper water layer, and adding more adsorption resin until the adsorption resin fills the chromatographic column, retaining a 2-4 cm water layer, and the height of the chromatographic column is 55-60 cm; Preferably, the adsorption resin loaded in the column is equilibrated with sodium dihydrogen phosphate solution for 4-6CV, and the equilibrium flow rate is not higher than 6cm / min.

9. The method for separating and purifying taurochenodeoxycholic acid from chicken bile according to claim 1, characterized in that: In the step 4), a reverse phase filler is used as the stationary phase; the purification step in the step 4) comprises the following steps: The reverse phase filler is loaded into the column and equilibrated; then the sample is loaded at a loading capacity of 40-55 mg of pure product / ml of filler, and the loading flow rate is not higher than 6 cm / min; Use 20%-30% methanol aqueous solution to wash for 10-15CV; then wash with pure water for 2-3CV; then use 40%-60% methanol aqueous solution to elute for 8-10CV, the elution flow rate is not higher than 6cm / min, and collect 8-10CV after the peak emerges; Preferably, the packing of the reverse phase filler into the column comprises: homogenizing the filler with 50% methanol, packing the column with water, switching with methanol after compaction, and then packing the column with water, and finally storing the filler in water, the packing pressure is not less than 10MPa, the height is 55-60cm, and the flow rate is not higher than 6cm / min; Preferably, the adsorption resin loaded in the column is equilibrated with 20 mM sodium dihydrogen phosphate solution for 4-6 CV, and the equilibrium flow rate is not higher than 6 cm / min.

10. The method for separating and purifying taurochenodeoxycholic acid from chicken bile according to claim 1, characterized in that: After the chromatography treatment in step 4) is completed, the stationary phase is regenerated with 80% methanol for 3-6CV at a flow rate of no more than 6 cm / min.

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