Development of a validated LC-PDA method for the quantification of anti-inflammatory secondary metabolites from Ratanhiae radix

Roots of Krameria lappaceae (Dombey) Burdet et Simpson, listed in many pharmacopoeias (ÖAB90, Ph. Eur. 6.0) are traditionally used against oropharyngeal inflammation due to their high tannin content. We could show in previous studies that lignan derivatives contribute strongly to the anti-inflammatory properties of Ratanhiae radix. All eleven isolated constituents [1], including ratanhiaphenol I, II and conocarpan, exerted a pronounced topical anti-inflammatory potential in vivo (ID₅₀ values 0.3–0.6µM/cm²) and could also inhibit the NF-κB activation in a dose-dependent manner [2]. Besides gravimetrical analysis of the ratanhiaphenols I, II and III the content of these compounds in the drug has never been determined. Therefore, we developed a validated LC-PDA method (according to the ICH-guidelines) for the quantification of the eleven active lignan derivatives in the roots (ASE extraction). The method is also suitable to determine the lignan derivatives in the tincture (pre-purification over polyamide). Separation was achieved on a phenyl-hexyl column using a solvent gradient consisting of 0.02% aqueous TFA and a mixture of acetonitrile/methanol (v/v; 75/25). The two major compounds were identified as conocarpan (1) and ratanhiaphenol II (2) with contents of 0.61%±0.02% and 0.56%±0.02% in the roots and 0.16mg/ml (RSD 1.1%) and 0.17mg/ml (RSD 0.4%) in the tincture, respectively. The recovery rates, determined in two different concentrations for both sample types, ranged from 95.5 to 104.8% for 1 and 2. The outcome of this study confirms that the isolated lignan derivatives are present in concentrations relevant for the previously determined anti-inflammatory activities.

Acknowledgements: This work was granted by the Austrian Science Foundation (NFN: DNTI, S10703-B03)

References: 1. Baumgartner L et al. (2010) Book of Abstracts; International Symposium Drugs from Nature Targeting Inflammation, SL6.

2. Baumgartner L et al. (2009) Planta Med 75:PA20.