The extraction of the isatan can also be effected with feebly acid alcohol, 

 both in the cold and at boiling temperature. Fresh leaves are then to be preferecl 

 to dried ones, because in drying there always gets lost some, at last all isatan. The 

 alcohol extract must be evaporated at low temperature and finally be neutralized 

 with chalk. After boiling a brownish, almost neutral and very rich isatan solution 

 is obtained, which can be purified with neutral lead acetate. 



For further concentration the isatan can be precipitated with basic lead acetate, 

 and the yellow precipitate be decomposed in the cold with oxalic acid. The lead 

 oxalate separates freely from the isatan solution, and the excess of oxalic acid 

 can be removed with chalk, the lead with sulphurated hydrogen. This solution 

 can be kept without decomposition for some time, but after a few weeks the 

 isatan vanishes. 



In the decoction method with oxalic acid, followed by lead precipitation, 

 the chlorophyll is removed from the very first and evaporation is excluded. More 

 plant slime will then precipitate with the lead than by alcohol extraction, but 

 on further purifying, this slime can be precipitated with ether-alcohol. I have as 

 yet not been able to prepare dry isatan, as a powder, from these extracts, such 

 as I before prepared the indican. 



The most characteristic difference between indican and isatan consists in 



their behaviour to alkalies: indican is constant in concentraded alkaline solutions, 



/% 



isatan is decomposed by very feeble alkalies, even in the cold. Concentrated 

 solutions of dinatrium phosphate, phosphoric salt and ammonium carbonate produce 

 indoxyl from isatan, already at room temperature. By acids, both indican and 

 isatan are decomposed, but indican with much more difficulty, which is especially 

 evident when using acid salts. So, isatan is already decomposed by boiling with 

 dilute kalium bioxalate, in which indican is constant. 



Both substances precipitate with basic lead acetate, producing yellow preci- 

 pitates, which colour is probably proper to the substances themselves, and not 

 to impurities. 



Isatase, the specific enzyme from woad, does not act on indican; isatan 

 on the other hand is not decomposed by the indigo-enzymes. 



Isatan is not directly splitted by the common microbes; indirectly it may, 

 of course, be decomposed by the alkali produced by microbes. Indican, on the 

 other hand, as I have formerly shown, is directly decomposed by many microbes, 

 either by ferment action of the protoplasm (katabolism), or by specific enzymes, 

 proper to the microbes. This difference between isatan and indican is probably 

 related to the nature of the substances set free in the decomposition beside the 

 indoxyl. So the glucose, from the indican, is an excellent nutrient for many 

 bacteria, whilst the very stability of the isatan in relation to microbes, seems to 

 indicate that the matter, which besides indoxyl originates from it, is no glucose, 

 perhaps no sugar at all. 



j. The isatase. 



The preparation of the woad-enzyme is effected in the same way as that of 

 the indigo-enzymes. The related parts of the plant are rubbed down in living 

 state under alcohol, and the alcohol is so often renewed until all the chlorophyll 



