CS269835B1 - Method of isolation of [23R / 3ώ, 7úC, 23-trihydroxy-5-cholan-24-one (β) -phocaecholic acid] - Google Patents

Abstract

i<ySeiina /23R/ 3ώ , 7 «6 , 23-tri-hydroxy-. -5/4 -cholan-24-ova is prepared from available raw materials. From a mixture of crude bile acids obtained by alkaline hydrolysis of duck bile and purified by recrystallization, after acetylation and esterification by chromatography-; ky, pure /23R/ 3ιο , 7 ¢6 , * ZJ-trihydroxy-S/J -cholan-24-ova (/j-phoca- ; echolic) acid is separated.

Description

The invention relates to a method for isolating /23R/ 3.0, 7 oc, 23-trihydroxy-5/3-cholan-24-ic acid (β-phocaecholic acid) from duck bile.

A method for isolating the acid from the bile of marine mammals such as sea lions (pinnipedia) is known. The disadvantage of isolating the acid from the bile of these animals is the difficult availability of the raw material. From the bile of these animals, (b - phocaecholic acid was isolated either by precipitation as a barium salt or by chromatography of its esters on alumina, elution being carried out with benzene, ether and ethanol (1, 2).

The above-mentioned disadvantage is eliminated by the method of the invention, the essence of which lies in the fact that crude bile acids obtained by alkaline hydrolysis of duck bile, especially bile of domesticated ducks, are purified by recrystallization after acetylation and esterification, and from the obtained mixture of bile acids, pure acid /23R/ 3 «6 , 7, 23- ” is isolated by chromatography.

-trihydroxy-5/4-cholan-24-ova ( /3-phocaechol).

The use of β-phocaecholic acid is expected either for pharmaceutical purposes, or as a preparation for scientific work related to the study of bile acid and steroid metabolism and its disorders.

Duck bile is mixed with 3 parts by weight of denatured alcohol, the resulting precipitate is filtered off and the solution is filtered through a layer of alumina, possibly shaken with a sufficient amount of this sorbent. In this way, a light yellow solution is obtained from the original dark green to black bile. This solution is evaporated to 1/6 of the volume and enough solid KOH (or NaOH) is added to it so that the resulting concentration is 10% (weight/volume). The solution is then refluxed for 12 hours or heated in an autoclave at 120 °C for 2 hours.

The cooled solution is acidified with 7N HCl while cooling until a precipitate forms, which is isolated and dried at a temperature of max. 60 °C in a stream of air. The yield is about 40 g from 1 l of bile.

This mixture of bile acids is esterified by dissolving in methanol containing 10% concentrated sulfuric acid (or HCl). 500 ml of the esterification mixture is taken per 100 g of bile acids. The resulting solution is left for 12 to 24 hours at room temperature and then diluted with excess distilled water. The precipitate formed is isolated and dried. It is then dissolved in a 5-fold volume of the acetylation mixture acetic anhydride/pyridine (volume/volume) 1/2 and heated at reflux for 2 hours at 100 °C. The resulting methyl acetate esters are isolated as a precipitate after precipitation with excess distilled water under vigorous cooling and recrystallized from methanol. The white crystals are dried, the yield is about 70%.

This crystalline mass of bile acid mixture is then hydrolyzed by boiling with 5% (w/v) methanolic KOH at reflux. After 2 hours of boiling, the solution is diluted with 2 times its volume of water and the methanol is evaporated. The resulting precipitate (after acidification) is isolated and dried. This precipitate consists mainly of chenodeoxycholic acid and /b-phocaecholic acid. ,

From this mixture, /J-phocaecholic acid is isolated in two ways: 1. The mixture is converted into methyl esters and chromatographed on Silpearl or neutral *

A^O-j. For 1 part of the separated mixture, 20 parts by weight of the sorbent are taken and chromatographed with a mixture of ethyl acetate/heptane (or toluene) in the ratio 2/1 (volume/volume). /5-phocaecholic acid flows out after chemodeoxycholic acid. The yield of the pure fraction is about 20%.

2. The acid mixture is not esterified, but chromatographed on the same sorbents as the free acids using the petroleum ether/acetone system 2/1 (v/v), 1/1 (v/v), acetone alone. /^-phocaecholic acid flows out in the last (second) fraction. The yield is about 30%.

Claims (1)

SUBJECT OF THE INVENTION A method for isolating /J-phocaecholic acid, characterized in that from a mixture of crude bile acids obtained by alkaline hydrolysis of duck bile, with subsequent acidification and recrystallization of the product, the diacetate or triacetate of methyl esters (the resulting product) formed after esterification, acetylation and recrystallization from methanol is subjected to hydrolysis and acidification, the resulting mixture of bile acids - in particular chenodeoxycholic and /j-phocaecholic acids - is subjected to chromatographic separation after possible conversion to methyl esters.