Natural indigo dye is prepared by fermentation of indigo leaves, the decay of the cell-walls liberating the enzymes in the tissues, which bring about the chemical changes illustrated in the above equations.
THE CYANOPHORE GLUCOSIDES
Several glucosides which yield hydrocyanic acid as one of the products of their hydrolysis are of common occurrence in plants. These are generally spoken of as the "cyanogenetic" glucosides; but as they do not actually produce cyanogen compounds, but only liberate them when hydrolyzed, the recently suggested term "cyanophore" undoubtedly more correctly indicates their properties.
The best known and most widely distributed of these is amygdalin. Amygdalin was first discovered in 1830, and was one of the first substances to be recognized as a glucoside. It is found in large quantities in bitter almonds and in the kernels of apricots, peaches, and plums; also in the seeds of apples, etc., in fact in practically all the seeds of plants of the Rose family. It is the mother substance for "oil of bitter almonds," which is widely used as a flavoring extract.
Amygdalin has been the object of very extensive studies, and even yet the exact nature of the linkage between its constituent groups is not certainly known. When completely hydrolyzed, it yields two molecules of glucose and one each of benzaldehyde and hydrocyanic acid. Recent studies indicate that the two sugar molecules are separately united to the other constituents, rather than united with each other in the disaccharide relationship. In other words, amygdalin is a true glucoside rather than a maltoside. This is indicated by the fact that when submitted to the action of all known hydrolyzing agents which affect it, it has never been found to yield maltose as one of the products of hydrolysis. Furthermore, the rate of hydrolysis of amygdalin is not affected by the presence of maltose; and the segregation of the two glucose molecules is accomplished by enzymes other than maltase, which is the only enzyme which is known to break up a maltose molecule. Since the exact nature of the linkage is not known, it is customary and convenient to indicate the unit groups as linked together in the following order:
| C6H11O5— | O—C6H10O4— | O—C6H5·CH | —C | ≡N |
| (1) | (2) | (3) | (4) |
A study of the hydrolysis reactions of amygdalin shows that there are three different linkages in the molecule which may be broken by the simple interpolation of a single molecule of water and a fourth which may be split by a different type of hydrolysis, namely, the C≡N linkage. These are indicated by the numbers below the corresponding portion of the formula above. Most hydrolyzing agents break the molecule first at (1), yielding one molecule of glucose and one of mandelo nitrile glucoside (see [page 77]). The next step usually breaks the latter at the point indicated by (2), yielding glucose and benzaldehyde cyanhydrin, or mandelo nitrile. The latter in turn breaks down at (3) into benzaldehyde and HCN. But when amygdalin is boiled with concentrated hydrochloric acid, the first change is the splitting off at (4) of the nitrogen in the form of ammonia and the consequent conversion of the CN group into a COOH group, producing amygdalinic acid. On further hydrolysis, this breaks up in the same order as before. Similarly, it is possible to convert mandelo nitrile into mandelic acid by splitting off the nitrogen to form a COOH group, instead of splitting off the HCN group leaving benzaldehyde.
The mandelo nitrile glucoside contains an asymmetric carbon atom which is wholly outside its glucose group, thus C6H10O5—O—C6H5·CH·CN. Hence, it may exist in dextro, levo, and racemic forms. In the amygdalin molecule, it exists in the dextro form, which has been named "prunasin." The levo form, known as "sambunigrin," has been obtained by hydrolysis of a compound isomeric with amygdalin, whose composition has not been definitely worked out; while the racemic form, known as "prulaurasin," has been prepared from isoamygdalin, by the action of alkalies. Hence, all the possible compounds indicated by the presence of the asymmetric carbon have been found and identified.
The crude enzyme preparation which is obtained from almond seeds, known as "emulsin," contains two enzymes, amygdalase, which breaks the amygdalin molecule at linkage (1), and prunase, which breaks it at (2). The action of amygdalase must always precede that of prunase. In other words, it is never possible to break off a disaccharide sugar from the molecule, either by the action of prunase alone, or by means of any other hydrolytic agent.