Indigo naturalis is one of the TCM monographs from the Chinese Pharmacopoeia proposed
by the European Pharmacopoeia Commission to include in the Ph. Eur. [1]. It is prepared
from the leaves or stems of Baphicacanthus cusia (Nees) Bremek., Persicaria tinctoria (Aiton) Spach., Isatis tinctoria L. or Indigofera tinctoria L. after fermenting. Content: minimum 2.0 per cent of indigo (C16H10N2O2; Mr 262.3) and minimum 0.13 per cent of indirubin (C16H10N2O2; Mr 262.3) (dried drug). Several indigo samples, including very old samples from Java
(Indonesia) have been analysed by means of TLC and HPTLC and most proved to fulfil
the requirements. Proposed TLC: Test solution: To 0,5g of the powdered drug (355) add 25ml chloroform R. Sonicate during 30min
and filter. Reference solution: Dissolve 1mg of indigo R and 1mg of indirubin R in 5ml of chloroform R. Plate: TLC silica gel plate R (5–40µm [or TLC silica gel plate R 2–10µm]. Mobile phase: acetone R, chloroform R, light petroleum R (5:15: 30 V/V/V). Application: 20µl as bands of 10mm [or 10µl as bands of 7mm]. Development: over a path of 6cm [or 3cm]. Detection: immediately after development in daylight because of the instability of blue indigo
colour. HPLC: Test solution. To 25.00mg of the powdered drug add 25ml chloroform R and sonicate for about 10min
and add methanol R to 50.00ml and filter. Reference solution: Dissolve 1.000mg indigo CRS in 50.00ml chloroform R and sonicate for about 10min
and add methanol R to 100.00ml and filter. The same for indirubin CRS. Prepare the
solutions immediately before use. Column: – size: l =0.125m, Ø=4mm, – stationary phase: octadecylsilyl silica gel for chromatography R (5µm). Mobile phase. water R, methanol R (34:66 V/V). Flow rate: 1.0ml/min. Detection: spectrophotometer at 290 nm. Injection: 10µl.
Acknowledgements: Dr Riepl, TU München for the indirubin sample.
References: [1] Pharmeuropa 19.3 (2007)
