Steam distillation (Gerhard Vadopest 12-Kjedahl type
distillatory) was carried out for 7.3 min on the TCA extract. About
25 ml of filtrate were loaded into the distillation tube followed by
5 ml of 10% (w/v) NaOH. A beaker containing 10 ml of a 4% aqueous
boric acid solution (Merck 1.00165) and 0.04 ml of Mixed indicator
5 for ammonia titrations (Merck 1.06130) was placed at the end of
the condenser. The boric acid solution turned green when alkalinized
by the distilled TVB-N. The green alkaline distillate was titrated
using a digital burette (Schott type T80 /20) containing an
aqueous 0.1 N hydrochloric acid solution (Merck 1.09973). Complete
neutralization was obtained when the color turned pink on
the addition of a further drop of hydrochloric acid. This procedure
was repeated for triplicate analysis.