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study of indican and had in many respects come to the same con- 
clusions as myself, I decided to publish my results, as it would be 
impossible to keep the matter a secret. 
It so happened that van Romsuren had also occupied himself 
with researches on indican and, without being acquainted with my 
work, had obtained results confirming my own. His results will, 
of course, be communicated in this paper. 
If the juice from the leaves of Indigofera leptostachya is collected 
in such a manner that there cannot be any question of enzyme- 
actions, for instance by treatment with boiling water or by using 
enzyme poisons (lime-water, solution of mercuric chloride), a liquid 
is obtained which, if not too acid, is perfectly stable, and contains 
a substance which is capable of yielding indigo. 
This may be demonstrated in four ways. 
1. By acidifying and carefully adding ferric chloride or any other 
suitable oxidizing agents. 
2. By the action of an enzyme contained in the indigo plant. 
3. By the action of enzymes not derived from the indigo plant. 
4. By the action of different bacteries. 
Enzyme 2 was prepared by repeatedly rubbing fresh indigo leaves 
with strong alcohol and removing the alcohol by squeezing the mass 
with the hands. The residue was then pressed between blotting 
paper in a copying press and allowed to dry in the sun. The dry 
product was then finely powdered and passed through a sieve. By 
keeping this powder under alcohol for a long time, it may be 
obtained perfectly white. 
The active principle is somewhat soluble in water, but much more 
so in a 10 per cent solution of common salt }). 
When I now allowed the powder or the solution prepared from 
it, to act on the liquid obtained by treating indigo leaves with boiling 
water, or on the solution obtained by crushing the leaves with milk 
of lime or lime water and neutralising the filtrate, I noticed the 
following effect : 
1. After a few minutes the liquid discoloured and yielded a 
little indigo. 
') Dr. Van RoMBureH paid me a visit at my laboratory when [ was engaged with these 
experiments and then informed me that he had also prepared active leaf powder 
in a slightly different manner. ‘This when in contact with sterilized infusion of indigo 
leaves caused the same effect as the usual fermentation principles. We, therefore, 
have come to the same result independently of each other, 
