阅读说明：本技术 一种别熊去氧胆酸的合成方法 (Synthetic method of alloursodeoxycholic acid ) 是由 秦和平 张和平 祝国祥 林志锋 凌芬娜 于 2021-01-12 设计创作，主要内容包括：本发明公开了一种别熊去氧胆酸的合成方法,该方法以别鹅去氧胆酸为起始原料,加入磷酸缓冲液、烟酰胺腺嘌呤二核苷酸磷酸、乳酸脱氢酶、7-α类固醇脱氢酶、丙酮酸钠,进行反应,高温灭活酶后再降温；再加入L-苹果酸钠、苹果酸脱氢酶、7-β类固醇脱氢酶反应,高温灭活酶后再降至常温；再经过滤、洗涤、干燥等步骤得到别熊去氧胆酸。全过程包括别鹅去氧胆酸的氧化反应、还原反应步骤,合成别熊去氧胆酸。该方法整个反应过程在水溶液中进行,不使用有机溶剂,工艺简单、容易操作,得到的别熊去氧胆酸的纯度达到98.5％以上,可作药学研究机构进行毒理药理等药学性质样品使用,也可以作为熊去氧胆酸质量研究时的杂质对照品使用。(The invention discloses a synthetic method of alloursodeoxycholic acid, which takes allochenodeoxycholic acid as a starting material, adds phosphate buffer solution, nicotinamide adenine dinucleotide phosphate, lactate dehydrogenase, 7-alpha steroid dehydrogenase and sodium pyruvate for reaction, inactivates enzymes at high temperature and then cools; adding L-sodium malate, malate dehydrogenase and 7-beta steroid dehydrogenase for reaction, inactivating the enzymes at high temperature, and cooling to normal temperature; filtering, washing, drying, etc. to obtain the alloursodeoxycholic acid. The whole process comprises the steps of oxidation reaction and reduction reaction of allochenodeoxycholic acid to synthesize alloursodeoxycholic acid. The method has the advantages that the whole reaction process is carried out in aqueous solution, no organic solvent is used, the process is simple and easy to operate, the purity of the obtained alloursodeoxycholic acid reaches more than 98.5 percent, and the alloursodeoxycholic acid can be used as a sample of pharmacological properties such as toxicology and pharmacology of a pharmaceutical research institution and can also be used as an impurity reference substance in the quality research of the ursodeoxycholic acid.)

1. A synthetic method of alloursodeoxycholic acid is characterized in that the raw material used is allochenodeoxycholic acid, and the method comprises the following steps:

a. oxidation of allochenodeoxycholic acid

Putting allochenodeoxycholic acid into a reaction container, adding a phosphate buffer solution to adjust the pH value of the solution to 7.5-8.5, dissolving, adding nicotinamide adenine dinucleotide phosphate, lactate dehydrogenase, 7-alpha steroid dehydrogenase and sodium pyruvate, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 7.5-7.8, and reacting until the residual amount of the allochenodeoxycholic acid in the solution is less than 1%;

after the reaction is finished, heating, stirring and cooling;

b. reduction of allo7-ketolithocholic acid

Adding L-sodium malate, malate dehydrogenase and 7-beta steroid dehydrogenase into the reaction solution obtained in the step a, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 6.8-7.2, and reacting until the residual concentration of the allo-7-ketolithocholic acid in the solution is less than 0.1%;

after the reaction is finished, adding a sodium hydroxide solution to adjust the pH of the solution, heating, stirring, cooling to normal temperature, filtering to remove solid matters in the solution, adding acid into the filtrate to adjust the pH of the solution, filtering, washing and drying to obtain the alloursodeoxycholic acid.

2. The method as claimed in claim 1, wherein the concentration of phosphate buffer in step a is 50-100 mmol/L.

3. The method for synthesizing alloursodeoxycholic acid according to claim 1, wherein the mass-to-volume ratio of allochenodeoxycholic acid to the phosphate buffer in the step a is 1g:10 ml.

4. The method for synthesizing alloursodeoxycholic acid according to claim 1, wherein in the step a:

the addition amount of the nicotinamide adenine dinucleotide phosphate is 0.10-0.30% of the mass of the allochenodeoxycholic acid;

the addition amount of the lactate dehydrogenase is 0.10-0.30% of the mass of the allochenodeoxycholic acid;

the addition amount of the 7-alpha steroid dehydrogenase is 0.30-0.50% of the mass of the allochenodeoxycholic acid;

the addition amount of the sodium pyruvate is 30-50% of the mass of the allochenodeoxycholic acid.

5. The method for synthesizing alloursodeoxycholic acid according to claim 1, wherein after the reaction in step a is completed, the solution is heated to 40-80 ℃, stirred for 20-60 min to inactivate the lactate dehydrogenase and the 7-alpha steroid dehydrogenase, and then cooled to 25-30 ℃.

6. The method for synthesizing alloursodeoxycholic acid according to claim 1, wherein in the step b:

the adding amount of the L-sodium malate is 40-70% of the mass of the allochenodeoxycholic acid;

the addition amount of the malate dehydrogenase is 1.0-3.0% of the mass of the allochenodeoxycholic acid;

the addition amount of the 7-beta steroid dehydrogenase is 0.10-0.30% of the mass of the allochenodeoxycholic acid.

7. The method as claimed in claim 1, wherein the acid added to the filtrate in step b is an inorganic acid.

8. The method for synthesizing alloursodeoxycholic acid according to claim 1, wherein after the reaction in step b, 10% sodium hydroxide solution is added to adjust the pH of the solution to 8.0-8.5, the temperature is raised to 60-80 ℃, the solution is stirred for 1-3 hours to inactivate malate dehydrogenase and 7-beta steroid dehydrogenase, the temperature is lowered to normal temperature, solids in the solution are removed by filtration, the pH of the solution is adjusted to 2-3 by adding acid into the filtrate, and then the alloursodeoxycholic acid is obtained by filtering, washing and drying.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a synthetic method of alloursodeoxycholic acid.

Background

Ursodeoxycholic acid UDCA is a non-toxic hydrophilic cholic acid, can competitively inhibit the absorption of toxic endogenous cholic acid in ileum, and can increase cholestasis by activating a signal network consisting of calcium ions and protein kinase C and activating split-activity protein-based enzyme. Ursodeoxycholic acid can also competitively replace toxic bile acid molecules on cell break-in organelles, preventing hepatocytes and cholangiocytes from being damaged by more toxic bile acids. Clinically, ursodeoxycholic acid is mainly used for dissolving cholesterol gallstones, primary biliary cirrhosis PBC and chronic hepatitis C, and is also used for alcoholic liver diseases, non-alcoholic fatty liver, benign recurrent intrahepatic cholestasis and congenital intrabiliary cystic dilatation.

With the continuous and deep understanding of human beings on ursodeoxycholic acid, the usage amount of the ursodeoxycholic acid is greatly increased, and the yield of the ursodeoxycholic acid synthesized by taking chenodeoxycholic acid extracted from cholic acid and chicken bile as raw materials cannot meet the requirements of human beings. At present, some ursodeoxycholic acid which is synthesized by taking chenodeoxycholic acid extracted from duck bile as a raw material appears on the market. Chenodeoxycholic acid extracted from duck bile as raw material contains a small amount of allochenodeoxycholic acid, and the allochenodeoxycholic acid generates alloursodeoxycholic acid (3 alpha, 7 beta-dihydroxy-5 alpha-cholanic acid) through oxidation and reduction reactions in the synthesis process of the ursodeoxycholic acid. The alloursodeoxycholic acid can be used as an impurity reference substance for researching the properties of the alloursodeoxycholic acid by a pharmaceutical research institution and also can be used as an impurity reference substance for researching the ursodeoxycholic acid. Therefore, the preparation of high-purity alloursodeoxycholic acid and the research on the alloursodeoxycholic acid have certain significance.

Disclosure of Invention

The invention aims to: the alloursodeoxycholic acid synthesized by the method has high purity, can be used as a sample with pharmacological properties such as toxicology and pharmacology in a pharmaceutical research institution, and can also be used as an impurity reference substance in the research of the ursodeoxycholic acid.

In order to achieve the purpose, the invention adopts the following technical scheme:

a synthetic method of alloursodeoxycholic acid, which uses allochenodeoxycholic acid as a raw material, comprises the following steps:

a. oxidation of allochenodeoxycholic acid

Putting allochenodeoxycholic acid into a reaction container, adding a phosphate buffer solution to adjust the pH value of the solution to 7.5-8.5, dissolving, adding nicotinamide adenine dinucleotide phosphate, lactate dehydrogenase, 7-alpha steroid dehydrogenase and sodium pyruvate, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 7.5-7.8, and reacting until the residual amount of the allochenodeoxycholic acid in the solution is less than 1%;

after the reaction is finished, heating, stirring and cooling;

b. reduction of allo7-ketolithocholic acid

Adding L-sodium malate, malate dehydrogenase and 7-beta steroid dehydrogenase into the reaction solution obtained in the step a, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 6.8-7.2, and reacting until the residual concentration of the allo-7-ketolithocholic acid in the solution is less than 0.1%;

after the reaction is finished, adding a sodium hydroxide solution to adjust the pH of the solution, heating, stirring, cooling to normal temperature, filtering to remove solid matters in the solution, adding acid into the filtrate to adjust the pH of the solution, filtering, washing and drying to obtain the alloursodeoxycholic acid.

And the mass concentration of the phosphate buffer solution in the step a is 50-100 mmol/L.

In the step a, the mass-to-volume ratio of the allochenodeoxycholic acid to the phosphate buffer solution is 1g to 10 ml.

In the step a:

the addition amount of the nicotinamide adenine dinucleotide phosphate is 0.10-0.30% of the mass of the allochenodeoxycholic acid;

the addition amount of the lactate dehydrogenase is 0.10-0.30% of the mass of the allochenodeoxycholic acid;

the addition amount of the 7-alpha steroid dehydrogenase is 0.30-0.50% of the mass of the allochenodeoxycholic acid;

the addition amount of the sodium pyruvate is 30-50% of the mass of the allochenodeoxycholic acid.

And c, after the reaction in the step a is finished, heating the solution to 40-80 ℃, stirring for 20-60 min to inactivate the lactate dehydrogenase and the 7-alpha steroid dehydrogenase, and then cooling to 25-30 ℃ to facilitate the subsequent reaction.

In the step b:

the adding amount of the L-sodium malate is 40-70% of the mass of the allochenodeoxycholic acid;

the addition amount of the malate dehydrogenase is 1.0-3.0% of the mass of the allochenodeoxycholic acid;

the addition amount of the 7-beta steroid dehydrogenase is 0.10-0.30% of the mass of the allochenodeoxycholic acid.

The acid added to the filtrate in the step b is preferably inorganic acid.

And c, after the reaction in the step b is finished, adding a 10% sodium hydroxide solution to adjust the pH of the solution to 8.0-8.5, heating to 60-80 ℃, stirring for 1-3 hours to completely inactivate the malate dehydrogenase and the 7-beta steroid dehydrogenase, cooling to normal temperature, filtering to remove solid matters in the solution, adding acid into the filtrate to adjust the pH of the solution to 2-3, and filtering, washing and drying to obtain the alloursodeoxycholic acid.

And detecting the purity of the alloursodeoxycholic acid obtained by the reaction by using a High Performance Liquid Chromatograph (HPLC).

The invention has the following advantages and beneficial effects:

the invention provides a method for synthesizing high-purity alloursodeoxycholic acid by taking allochenodeoxycholic acid as a starting material. The purity of the obtained alloursodeoxycholic acid reaches more than 98.5 percent, the obtained alloursodeoxycholic acid can completely meet the requirements of research institutions on the property thereof, and the obtained alloursodeoxycholic acid can also be used as an impurity reference substance in the quality research of the ursodeoxycholic acid.

Drawings

FIG. 1 is a flow chart of the synthesis of alloursodeoxycholic acid.

Wherein: ACDCA-allochenodeoxycholic acid; A7-KLCA-allo 7-ketolithocholic acid; AUDCA-alloursodeoxycholic acid.

Detailed Description

The present invention will be further described with reference to the following examples, but is not limited thereto.

As shown in fig. 1, a method for synthesizing alloursodeoxycholic acid comprises the following steps:

a. oxidation of allochenodeoxycholic acid (ACDCA)

Putting allochenodeoxycholic acid into a reaction container, adding a phosphate buffer solution with the mass volume ratio of 1g to 10ml to the allochenodeoxycholic acid and the mass concentration of 50-100 mmol/L to adjust the pH value of the solution to 7.5-7.8, stirring and dissolving, adding Nicotinamide Adenine Dinucleotide Phosphate (NADP) accounting for 0.15% of the weight of the allochenodeoxycholic acid, Lactate Dehydrogenase (LDH) accounting for 0.15-0.18%, 7-alpha steroid dehydrogenase (7-alpha HSDH) accounting for 0.30-0.35% of the weight of the allochenodeoxycholic acid and sodium pyruvate accounting for 30-35%, controlling the temperature of the solution to 25-30 ℃ and the pH value to 7.5-8.0, and reacting until the residual amount of the allochenodeoxycholic acid in the solution is less than 1%;

after the reaction is finished, heating the solution to 50-55 ℃, stirring for 30min, and then cooling to 25-30 ℃;

b. reduction of allo7-ketolithocholic acid (A7-KLCA)

Adding L-sodium malate accounting for 45-50% of the weight of the allochenodeoxycholic acid, Malate Dehydrogenase (MDH) accounting for 1.8% of the weight of the allochenodeoxycholic acid and 7-beta steroid dehydrogenase (7-beta HSDH) accounting for 0.15-0.20% of the weight of the allochenodeoxycholic acid into the reaction solution obtained in the step a, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 6.8-7.2, and reacting until the residual concentration of the allochenodeoxycholic acid in the solution is less than 0.1%;

and after the reaction is finished, adding 10% sodium hydroxide solution to adjust the pH of the solution to 8.0-8.5, heating to 70-75 ℃, stirring for 2 hours, cooling to normal temperature, filtering to remove solid matters in the solution, adding acid into the filtrate to adjust the pH of the solution to 2-3, filtering, washing and drying to obtain alloursodeoxycholic acid (AUDCA).

After the experiment is completed, the purity of the obtained alloursodeoxycholic acid is detected by using a High Performance Liquid Chromatograph (HPLC), and corresponding data is read and recorded.

Example 1

Adding 10g of allochenodeoxycholic acid into a 500ml reaction bottle, adding 100ml of phosphate buffer solution with the substance amount concentration of 50mmol/L to adjust the pH value of the solution to 7.8, stirring for dissolution, adding 15mg of NADP, 15mg of LDH, 30mg of 7-alpha HSDH and 3.3g of sodium pyruvate, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 7.5-7.8, reacting for 5 hours, and sampling to measure that the residual amount of the allochenodeoxycholic acid in the solution is 0.73%. After the reaction is finished, heating to 55 ℃, stirring for 30min, and then cooling to 25-30 ℃.

Adding 4.5g L-sodium malate, 0.18g MDH and 15mg 7-beta HSDH into the reaction solution, controlling the temperature of the solution at 25-30 ℃ and the pH value to 6.8-7.2, reacting for 3 hours, and sampling to detect that the residue of the allo-7-ketolithocholic acid is less than 0.06 percent.

After the reaction is finished, adding sodium hydroxide with the concentration of 10% by weight to adjust the pH value of the solution to 8.2, heating to 75 ℃, stirring for 2 hours, then cooling to 30 ℃, filtering to remove solid matters in the solution, adding 1:1 hydrochloric acid into the filtrate to adjust the pH value of the solution to 2.59, filtering, washing and drying to obtain 9.6g of alloursodeoxycholic acid.

The purity of the alloursodeoxycholic acid is 98.761% by HPLC detection.

Example 2

Adding 10g of allochenodeoxycholic acid into a 500ml reaction bottle, adding 100ml of phosphate buffer solution with the substance amount concentration of 75mmol/L to adjust the pH value of the solution to 8.0, stirring for dissolution, adding 15mg of NADP, 18mg of LDH, 35mg of 7-alpha HSDH and 3.5g of sodium pyruvate, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 7.5-7.8, reacting for 5 hours, and sampling to measure that the residual amount of the allochenodeoxycholic acid in the solution is 0.66%. After the reaction is finished, heating to 50 ℃, stirring for 30min, and then cooling to 25-30 ℃.

Adding 4.6g L-sodium malate, 0.18g MDH and 16mg 7-beta HSDH into the reaction solution, controlling the temperature of the solution at 25-30 ℃ and the pH value to 6.8-7.2, reacting for 3 hours, and sampling to detect that the residue of the allo-7-ketolithocholic acid is less than 0.05 percent.

After the reaction is finished, adding a small amount of 10% sodium hydroxide to adjust the pH of the solution to 8.2, heating to 74 ℃, stirring for 2 hours, then cooling to 30 ℃, filtering to remove solid matters in the solution, adding 1:1 hydrochloric acid into the filtrate to adjust the pH of the solution to 2.65, filtering, washing and drying to obtain 9.6g of alloursodeoxycholic acid.

The purity of the alloursodeoxycholic acid is 98.700% by HPLC detection.

Example 3

Adding 10g of allochenodeoxycholic acid into a 500ml reaction bottle, adding 100ml of phosphate buffer solution with the substance amount concentration of 100mmol/L to adjust the pH value of the solution to 8.2, stirring for dissolution, adding 15mg of NADP, 18mg of LDH, 35mg of 7-alpha HSDH and 3.5g of sodium pyruvate, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 7.5-7.8, reacting for 5 hours, and sampling to measure that the residual amount of the allochenodeoxycholic acid in the solution is 0.3%. After the reaction is finished, heating to 55 ℃, stirring for 30min, and then cooling to 25-30 ℃.

Adding 4.8g L-sodium malate, 0.18g MDH and 18mg 7-beta HSDH into the reaction solution, controlling the temperature of the solution at 25-30 ℃ and the pH value to 6.5-7.5, reacting for 3 hours, and sampling to detect that the residue of the allo-7-ketolithocholic acid is less than 0.04%.

After the reaction is finished, adding a small amount of 10% sodium hydroxide to adjust the pH of the solution to 8.3, heating to 73 ℃, stirring for 2 hours, then cooling to 30 ℃, filtering to remove solid matters in the solution, adding 1:1 hydrochloric acid into the filtrate to adjust the pH of the solution to 2.74, filtering, washing and drying to obtain 9.7g of alloursodeoxycholic acid.

The purity of the alloursodeoxycholic acid is 98.650% by HPLC detection.

Example 4

Adding 10g of allochenodeoxycholic acid into a 500ml reaction bottle, adding 100ml of phosphate buffer solution with the substance amount concentration of 50mmol/L to adjust the pH value of the solution to 7.5, stirring for dissolution, adding 10mg of NADP, 30mg of LDH, 30mg of 7-alpha HSDH and 3.0g of sodium pyruvate, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 7.5-7.8, reacting for 5 hours, and sampling to measure that the residual amount of the allochenodeoxycholic acid in the solution is 0.84%. After the reaction is finished, heating to 40 ℃, stirring for 60min, and then cooling to 25-30 ℃.

Adding 4.0g L-sodium malate, 0.10g MDH and 15mg 7-beta HSDH into the reaction solution, controlling the temperature of the solution at 25-30 ℃ and the pH value to 6.8-7.2, reacting for 3 hours, and sampling to detect that the residue of the different 7-ketolithocholic acid is less than 0.08%.

After the reaction is finished, adding sodium hydroxide with the concentration of 10% by weight to adjust the pH value of the solution to 8.0, heating to 60 ℃, stirring for 3 hours, then cooling to 30 ℃, filtering to remove solid matters in the solution, adding 1:1 hydrochloric acid into the filtrate to adjust the pH value of the solution to 2.68, filtering, washing and drying to obtain 9.1g of alloursodeoxycholic acid.

The purity of the alloursodeoxycholic acid is 99.188% by HPLC detection.

Example 5

Adding 10g of allochenodeoxycholic acid into a 500ml reaction bottle, adding 100ml of phosphate buffer solution with the substance amount concentration of 75mmol/L to adjust the pH value of the solution to 8.0, stirring for dissolution, adding 20mg of NADP, 18mg of LDH, 40mg of 7-alpha HSDH and 4.0g of sodium pyruvate, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 7.5-7.8, reacting for 5 hours, and sampling to measure that the residual amount of the allochenodeoxycholic acid in the solution is 0.78%. After the reaction is finished, heating to 60 ℃, stirring for 40min, and then cooling to 25-30 ℃.

Adding 7.0g L-sodium malate, 0.3g MDH and 30mg 7-beta HSDH into the reaction solution, controlling the temperature of the solution at 25-30 ℃ and the pH value to 6.8-7.2, reacting for 3 hours, and sampling to detect that the residue of the different 7-ketolithocholic acid is less than 0.07 percent.

After the reaction is finished, adding a small amount of 10% sodium hydroxide to adjust the pH of the solution to 8.2, heating to 80 ℃, stirring for 1h, then cooling to 30 ℃, filtering to remove solid matters in the solution, adding 1:1 hydrochloric acid into the filtrate to adjust the pH of the solution to 2.02, filtering, washing and drying to obtain 9.2g of alloursodeoxycholic acid.

The purity of the alloursodeoxycholic acid is 99.273% by HPLC detection.

Example 6

Adding 10g of allochenodeoxycholic acid into a 500ml reaction bottle, adding 100ml of phosphate buffer solution with the substance amount concentration of 100mmol/L to adjust the pH value of the solution to 8.4, stirring for dissolution, adding 30mg of NADP, 10mg of LDH, 50mg of 7-alpha HSDH and 5.0g of sodium pyruvate, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 7.5-7.8, reacting for 5 hours, and sampling to measure that the residual amount of the allochenodeoxycholic acid in the solution is 0.68%.

After the reaction is finished, heating to 80 ℃, stirring for 20min, and then cooling to 25-30 ℃.

Adding 5.5g L-sodium malate, 0.2g MDH and 20mg 7-beta HSDH into the reaction solution, controlling the temperature of the solution at 25-30 ℃ and the pH value to 6.5-7.0, reacting for 3 hours, and sampling to detect that the residue of the different 7-ketolithocholic acid is less than 0.07 percent.

After the reaction is finished, adding a small amount of 10% sodium hydroxide to adjust the pH of the solution to 8.3, heating to 76 ℃, stirring for 2 hours, then cooling to 30 ℃, filtering to remove solid matters in the solution, adding 1:1 hydrochloric acid into the filtrate to adjust the pH of the solution to 2.78, filtering, washing and drying to obtain 9.3g of alloursodeoxycholic acid.

The purity of the alloursodeoxycholic acid is 99.167% by HPLC detection.

Example 7

Adding 10g of allochenodeoxycholic acid into a 500ml reaction bottle, adding 100ml of phosphate buffer solution with the substance amount concentration of 50mmol/L to adjust the pH value of the solution to 7.6, stirring for dissolution, adding 20mg of NADP, 10mg of LDH, 40mg of 7-alpha HSDH and 4.0g of sodium pyruvate, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 7.5-7.8, reacting for 5 hours, and sampling to measure that the residual amount of the allochenodeoxycholic acid in the solution is 0.87%. After the reaction is finished, heating to 80 ℃, stirring for 25min, and then cooling to 25-30 ℃.

Adding 7.0g L-sodium malate, 0.2g MDH and 20mg 7-beta HSDH into the reaction solution, controlling the temperature of the solution at 25-30 ℃ and the pH value to 6.8-7.2, reacting for 3 hours, and sampling to detect that the residue of the different 7-ketolithocholic acid is less than 0.08%.

After the reaction is finished, adding a small amount of 10% sodium hydroxide to adjust the pH of the solution to 8.2, heating to 80 ℃, stirring for 1h, then cooling to 30 ℃, filtering to remove solid matters in the solution, adding 1:1 hydrochloric acid into the filtrate to adjust the pH of the solution to 2.95, filtering, washing and drying to obtain 9.1g of alloursodeoxycholic acid.

The purity of the alloursodeoxycholic acid is 99.295% by HPLC detection.

Example 8

Adding 10g of allochenodeoxycholic acid into a 500ml reaction bottle, adding 100ml of phosphate buffer solution with the substance amount concentration of 75mmol/L to adjust the pH value of the solution to 8.2, stirring for dissolution, adding 30mg of NADP, 18mg of LDH, 30mg of 7-alpha HSDH and 5.0g of sodium pyruvate, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 7.5-7.8, reacting for 5 hours, and sampling to measure that the residual amount of the allochenodeoxycholic acid in the solution is 0.74%. After the reaction is finished, heating to 40 ℃, stirring for 60min, and then cooling to 25-30 ℃.

Adding 5.5g L-sodium malate, 0.10g MDH and 10mg 7-beta HSDH into the reaction solution, controlling the temperature of the solution at 25-30 ℃ and the pH value to 6.8-7.2, reacting for 3 hours, and sampling to detect that the residue of the allo-7-ketolithocholic acid is less than 0.06 percent.

After the reaction is finished, adding sodium hydroxide with the concentration of 10% by weight to adjust the pH value of the solution to 8.2, heating to 75 ℃, stirring for 2 hours, then cooling to 30 ℃, filtering to remove solid matters in the solution, adding 1:1 hydrochloric acid into the filtrate to adjust the pH value of the solution to 2.16, filtering, washing and drying to obtain 9.3g of alloursodeoxycholic acid.

The purity of the alloursodeoxycholic acid is 99.202% by HPLC detection.

Example 9

Adding 10g of allochenodeoxycholic acid into a 500ml reaction bottle, adding 100ml of phosphate buffer solution with the substance amount concentration of 100mmol/L to adjust the pH value of the solution to 8.5, stirring for dissolution, adding 10mg of NADP, 30mg of LDH, 50mg of 7-alpha HSDH and 3.0g of sodium pyruvate, controlling the temperature of the solution to be 25-30 ℃ and the pH value to be 7.5-7.8, reacting for 5 hours, and sampling to measure that the residual amount of the allochenodeoxycholic acid in the solution is 0.51%. After the reaction is finished, heating to 60 ℃, stirring for 40min, and then cooling to 25-30 ℃.

Adding 4.0g L-sodium malate, 0.3g MDH and 30mg 7-beta HSDH into the reaction solution, controlling the temperature of the solution at 25-30 ℃ and the pH value to 6.5-7.5, reacting for 3h, and sampling to detect that the residue of the allo-7-ketolithocholic acid is less than 0.05%.

After the reaction is finished, adding a small amount of 10% sodium hydroxide to adjust the pH of the solution to 8.3, heating to 60 ℃, stirring for 3 hours, then cooling to 30 ℃, filtering to remove solid matters in the solution, adding 1:1 hydrochloric acid into the filtrate to adjust the pH of the solution to 2.88, filtering, washing and drying to obtain 9.4g of alloursodeoxycholic acid.

The purity of the alloursodeoxycholic acid is 98.906% by HPLC detection.