Extract From Thymus Gland

In the preparation of the vitamin fraction from the thymus gland of the hog, the following changes were found to be necessary in the above method. Thymus glands could not be ground and dried in the usual way on account of the difficulty presented by the large amount of connective tissue. The glands were therefore cut up with scissors into small pieces and allowed to stand for several days in 05 per cent alcohol; for 6,000 grams of glands six liters of alcohol were used. The alcoholic extract was then filtered off, the residue placed in a filter press and the remainder of the liquid removed in this manner. The pressed-out thymus glands were then minced in a meat chopper and again extracted with 8 liters of 95 per cent alcohol. The filtrate from the first extraction was evaporated in vacuo. On account of the tendency to foam, the filtrate was slowly dropped into the flask by means of a long-necked separatory funnel. The weight of the sirupy residue obtained from the first extraction amounted to 120 grams. The second alcoholic extract was treated in the same manner. These fractions were then extracted with ether in a separatory funnel. The lower layer, containing the vitamin fraction, was then run off and the ether evaporated in vacuo.

The greater part of the vitamin is extracted by the first treatment with alcohol. The two fractions were united and weighed 181 grams.

9 British Patent 133,183, Oct. 15, 1918.

9a Synthetic fats, prepared by combining higher fatty acids with glycerine, may have vitamins added thereto. (O. Schicht Akt. Ges., British Patent 160,840, Mar. 31, 1921).

10 Voegtlin, Neill and Hunter, Hyg. Lab. Bull. 116, 1920.

These extracts were, however, considerably less efficient than either activated Lloyd's reagent or Funk's fraction from rice polishings.

Punk's method of separation by means of phosphotungstic acid is described as follows: 11

Since only relatively small quantities of vitamins are contained in the precipitate due to phosphotungstic acid, the separation of these vitamins according to this method is accomplished only with extreme difficulty. Funk finds by treating the phosphotungstic acid precipitate with acetone, the major portion of the precipitate, containing the inactive ingredients, may be dissolved and eliminated, leaving almost all of the vitamins in the insoluble residue of the precipitate. Thus the percentage of vitamins in the precipitate may be so increased that Punk claims these substances may be isolated, generally in crystalline form, after decomposing the precipitate and evaporating or concentrating the solution. Neutral lead acetate is an effective agent for decomposing the precipitate produced by phosphotungstic acid. The method may be employed for the treatment of organic extracts! such for example, as extract from the thyroid gland (scutiform glandule), to separate the active substances.

For example, 350 grams of a moist precipitate obtained from yeast extract by means of phosphotungstic acid is ground up in a mortar, together with one-half a liter of acetone, causing most of the precipitate to go into solution. The portion insoluble in acetone is separated by filtration and this residue then well washed with acetone. The residue when dried weighs about 20 grams and hence represents only a minor portion of the entire precipitate produced with the phosphotungstic acid. This insoluble residue is further treated by well mixing and grinding with a solution of neutral lead acetate and then placed on the filter, the filtrate being freed from excess of lead by treatment with hydrogen sulphide. After filtering to separate the sulphide of lead formed, the filtrate is evaporated in vacuo. The resultant product is a white substance crystallising in the form of needles and weighing from 0.2 to 0.3 grams"12

11 U. S. Patent 1,162,908.

12 See also Eddy, J. Biol. Ctam. 27,113,1016.

Gams13 claims to have obtained much more active preparations by eliminating the phosphotungstic acid and treating directly with lead acetate in the following manner: The vitamin-containing material is extracted with dilute alcohol and the extract freed from alcohol by distillation in vacuo. The aqueous extract thus obtained is treated successively in an acid state by lead acetate and in a neutral state by basic lead acetate. The treatment with lead acetate in an acid state is claimed to produce only the precipitation of the most of the inactive ingredients, the active substance remaining in solution. By the subsequent treatment with basic lead acetate in a neutral solution, according to Gams, only further impurities are precipitated without appreciable loss of active substances. The lead is separated and by evaporating the solution in vacuo Gams secures a highly active preparation which is easily soluble in water to a clear solution and which he claims may be preserved indefinitely and adapted to be administered per se.

The food products or other substances of vegetable and animal origin employed as parent materials may be subjected previously to a mechanical, physical or chemical treatment making their extraction with alcohol more easy, for instance a comminution with quarts sand, a plasmolysis, a hydrolysis with dilute acids or alkalis, or a digestion with pepsin and hydrochloric acid.

The process is illustrated by the following examples:

Rice bran in a finely-divided state is extracted, in the cold, with dilute alcohol and the solution obtained is freed from alcohol by evaporating in vacuo. The aqueous milky liquid is separated by decantation from the oil and grease floating on its surface, is acidified with acetic acid and a solution of lead acetate is added until no further precipitate is produced. The clear solution, separated by filtering off the lead acetate precipitates is accurately neutralised and a neutralised solution of basic lead acetate is added until precipitation is complete. The solution separated from the precipitate by filtering is freed from lead by means of hydrogen sulphide or sulphuric acid. In the first case, the solution obtained by filtering is directly evaporated to dryness in vacuo and in the second case the excess of sulphuric acid is removed by the addition of barium hydroxid and the solution, showing still a feeble acid reaction with Congo indicator, is concentrated in vacuo. In each case a light yellow crystalline powder is obtained, which is easily soluble in water to a clear solution and contains most of the vitamin present in the parent material.