Preparation method and application of ursodeoxycholic acid

Background and overview

Ursodeoxycholic acid, with the chemical name 3a,7β-dihydroxy-5β-cholestane-24-acid, is an organic compound that is odorless and has a bitter taste. This product is soluble in ethanol, insoluble in chloroform; soluble in glacial acetic acid, soluble in sodium hydroxide test solution. Ursodeoxycholic acid, as the main active component of the precious traditional Chinese medicine bear bile in my country, is widely used in clinical treatment of various hepatobiliary diseases, including gallstones, viral hepatitis, fatty liver, liver cirrhosis, etc. FDA-approved drug for the treatment of primary biliary cirrhosis.

Ursodeoxycholic acid is a chemical preparation of natural bile acid isolated from bear bile. It is an epistereoisomer of chenodeoxycholic acid. Short and small dose. It exists in bile combined with taurine in the body, is a hydrophilic bile acid, and is a cholesterol stone dissolving agent. It can reduce the secretion of cholesterol by the liver, reduce the saturation of cholesterol in bile, promote the secretion of bile acids, increase the solubility of cholesterol in bile, dissolve cholesterol stones or prevent the formation of stones. It can increase the secretion of bile, relax the sphincter of the bile duct orifice to produce a choleretic effect, which is conducive to the discharge of stones. This product does not dissolve other types of gallstones. Ursodeoxycholic acid is suitable for the treatment of cholesterol stones, hyperlipidemia, bile secretion disorders, primary biliary cirrhosis, chronic hepatitis, bile reflux gastritis and prevention of acute rejection and reaction of liver transplantation.

Preparation

Four enzymes, 7α-hydroxysteroid dehydrogenase, 7β-hydroxysteroid dehydrogenase, lactate dehydrogenase, and glucose dehydrogenase, were constructed on the same co-expression plasmid pRSFDuet-1, transformed into the BL21 expression host, and the constructed project The bacteria becomes an Escherichia coli, and the substrate is directly fermented and converted to chenodeoxycholic acid to generate ursodeoxycholic acid. The construction of the four enzyme co-expression plasmids. First, two dual-enzyme co-expression plasmids, pRSF-LDH-7α and pRSF-GDH-7β, were respectively constructed, and then the two gene fragments of LDH-7α and GDH-7β were spliced ​​together by overlap extension PCR. Together, the new gene fragment is ligated to the co-expression plasmid by restriction ligation. The correct four-enzyme co-expression plasmid pRSF-GDH-7β-LDH-7α was verified and transformed into the BL21 expression strain to obtain engineering bacteria and Escherichia coli transformed with chenodeoxycholic acid.

Whole-cell catalysis of chenodeoxycholic acid, firstly by fermenting, culturing and inducing expression to obtain a high-density fermentation broth of engineered bacteria, by adding exogenous conversion substrates chenodeoxycholic acid, sodium pyruvate and glucose, and continuing the induction culture , and finally obtain ursodeoxycholic acid with high conversion rate (>99%); the crude ursodeoxycholic acid fermentation broth, adjust the acid to collect all the ursodeoxycholic acid in the methanol extraction product after precipitation or directly use it in the fermentation broth Methanol is used to extract ursodeoxycholic acid, and after cooling and crystallization, crude ursodeoxycholic acid is obtained, and after two acetone-water refining, ursodeoxycholic acid >99.8% can be finally obtained.

Use

For the prevention and treatment of cholesterol gallstones. Gallstone-dissolving drugs are mainly used for cholesterol-type gallstones that are not suitable for surgical treatment, especially those with normal gallbladder function, stone diameter less than 15mm, X-ray transparent, and non-calcified floating cholesterol stones have a high cure rate. It also has a certain therapeutic effect on toxic hepatitis, cholecystitis, primary sclerosing cholangitis, and primary cholestatic liver cirrhosis. Improve the efficacy of alpha-interferon in the treatment of chronic hepatitis C. Diarrhea, rare constipation, allergic reaction, itching, headache, dizziness, stomach pain, pancreatitis and bradycardia. Complete biliary obstruction and severe liver dysfunction and pregnant women are hanged.

Can be used to prepare other chemicals

References

【1】CN114015712A