MICROBIOLOGICAL PROCESS FOR THE PREPARATION OF URSOCHOLIC ACID

Abstract

A microbiological process is provided for the preparation of ursocholic acid, which includes the biotransformation of β-sitosterol in the presence of specific microorganisms.

Claims

1. A microbiological process for the preparation of ursocholic acid of formula (I) ##STR00004## comprising the biotransformation of β-sitosterol of formula (II) ##STR00005## in the presence of Pleurotus incarnatus.

2. The process according to claim 1, wherein Pleurotus incarnatus is Pleurotus incarnatus CBS 498.76.

3. The process according to claim 1, wherein the biotransformation occurs in an adequate culture broth.

4. The process according to claim 1, wherein the Pleurotus incarnatus concentration is comprised between 1 mg/l and 100 mg/l.

5. The process according to claim 1, wherein the β-sitosterol concentration is comprised between 1 g/l and 10 g/l.

6. The process according to claim 1, wherein the biotransformation occurs in a time comprised between 120 hours and 360 hours.

7. A process for the preparation of a cholic derivative comprising the process for the preparation of ursocholic acid according to claim 1.

8. The process for the preparation of a cholic derivative according to claim 7, wherein the cholic derivative is selected from ursodeoxycholic acid, chenodeoxycholic acid, obeticholic acid, lithocholic acid and cholic acid.

9. The process according to claim 6, wherein the biotransformation occurs in a time of about 240 hours.

10. The process according to claim 5, wherein the β-sitosterol concentration is about 2 g/l.

11. The process according to claim 4, wherein the Pleurotus incarnatus concentration is comprised between 10 mg/l and 50 mg/l.

12. The process according to claim 3, wherein the culture broth is selected from Donova, SAWADA, and Bushnell-Haas Broth.

13. The process according to claim 2, wherein the biotransformation occurs in an adequate culture broth.

14. The process according to claim 13, wherein the culture broth is selected from Donova, SAWADA, and Bushnell-Haas Broth.

15. The process according to claim 13, wherein a concentration of Pleurotus incarnatus is between 1 mg/l and 100 mg/l.

16. The process according to claim 13, wherein the Pleurotus incarnatus concentration is comprised between 10 mg/l and 50 mg/l.

17. The process according to claim 15, wherein a concentration of β-sitosterol is between 1 g/l and 10 g/l.

18. The process according to claim 17, wherein the β-sitosterol concentration is about 2 g/l.

19. The process according to claim 17, wherein the biotransformation occurs in a time comprised between 120 hours and 360 hours.

20. The process according to claim 19, wherein the biotransformation occurs in a time of about 240 hours.

Description

EXAMPLES

Example 1

[0040] 5.0 g of magnesium sulfate, 5.0 g of monopotassium phosphate, 50.0 g of sucrose, 20.0 mL of Cornsteep liquor and water are loaded into a 2 L fermenter together with the Pleorotus incarnatus strain. It is left under stirring for 72 hours.

[0041] 2 g of β-sitosterol are subsequently added to the mixture and it is left under stirring (160 rpm) for 240 hours at 28° C.

[0042] At the end the mass is isolated and purified by centrifugation of the culture broth. The solid is separated from the liquid phase, the pH of the solution is adjusted to about 2-3 with acetic acid and extracted with ethyl acetate. The organic phase is washed with water and evaporated to give 1.8 g of ursocholic acid with HPLC purity of 90%.

[0043] mp 145-146° C.; [α] D +68.8° (c 0.5, EtOH); 1H-NMR δ 3.80 (1 H, t, J=2.8 Hz, H-12β), 3.33 (2 H, m, H-3βand H-7α), 0.94 (3 H, d, J=7 Hz, C-21 Me), 0.86 (3 H, s, C-19 Me), 0.63 (3 H, s, C-18 Me).

Example 2

[0044] 5.0 g of magnesium sulfate, 5.0 g of monopotassium phosphate, 50.0 g of sucrose, 20.0 mL of Cornsteep liquor and water are loaded into a 2 L fermenter together with the Pleorotus incarnatus strain. It is left under stirring for 72 hours.

[0045] 2 g of β-sitosterol are subsequently added to the mixture and it is left under stirring (160 rpm) for 240 hours at 28° C.

[0046] At the end the mass is isolated and purified by centrifugation of the culture broth. The solid is separated from the liquid phase, the pH of the solution is adjusted to about 2-3 with acetic acid and extracted with ethyl acetate. The organic phase is washed with water and evaporated to give 1.8 g of ursocholic acid with HPLC purity of 90%.

[0047] mp 145-146° C.; [α] D+68.8° (c 0.5, EtOH); 1H-NMR δ 3.80 (1 H, t, J=2.8 Hz, H-12β), 3.33 (2 H, m, H-3β and H-7α), 0.94 (3 H, d, J=7 Hz, C-21 Me), 0.86 (3 H, s, C-19 Me), 0.63 (3 H, s, C-18 Me).