Indigo naturalis: Qingdai, a TCM monograph proposed for the European Pharmacopoeia (Ph. Eur.)

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; M_r 262.3) and minimum 0.13 per cent of indirubin (C₁₆H₁₀N₂O₂; M_r 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)