Isolation of Sodium Hyaluronate

Abstract

DURING recent investigations for the development of new methods for the isolation and purification of hyaluronic acid from human umbilical cords, the following interesting observation was made. The cords, stored in acetone, were extracted with hot water (65°) and the aqueous extract allowed to ‘settle’ overnight. The clear supernatant liquid was kept under toluene for fourteen days, when it deposited a precipitate. This was removed, and the clear liquid was acidified with glacial acetic acid. Half the resulting mucin clot was stored in acetone for several weeks. Upon attempting to dissolve it at pH 8.0, it was found to be incompletely soluble, whereas the original clot was soluble. The ‘acetone-clot’ was extracted with water at pH. 8.0, the extract giving only a very faint cloudiness upon acidification to pH 4.0. It was poured into 3 vol. of alcohol and the precipitate re-extracted with water. Finally, a preparation of sodium hyaluronate was obtained having only 4.5 per cent nitrogen (on free acid) and 7.7 per cent ash (3.3 per cent sodium approximately); whereas the mucin clot, after reprecipitation, had 15 per cent nitrogen and negligible ash content. In fact, hyaluronic acid, containing only 6–10 per cent protein, had been extracted at pH 8.0 from the mucin clot after acetone treatment.