Quantitative analysis of anti-inflammatory lignan derivatives in Ratanhiae radix and its tincture by HPLC-PDA and HPLC-MS

Root preparations of Krameria lappacea (Dombey) Burdet et Simpson are traditionally used against oropharyngeal inflammation. Besides antimicrobial and astringent procyanidines, lignan derivatives, including ratanhiaphenol I, II, III and (+)-conocarpan, contribute to the activity of Ratanhiae radix, exerting a significant topical anti-inflammatory activity in vivo, and in vitro by inhibiting NF-κB and the formation of inflammatory prostaglandins and leukotrienes. Besides gravimetrical analysis of the ratanhiaphenols I, II and III, the content of these compounds in the herbal drug has never been determined. The developed HPLC method enables the quantification of twelve active lignan derivatives in the roots, and is also suitable for the determination of the constituents in Tinctura Ratanhiae. Separation was achieved on a phenyl-hexyl column material using a solvent gradient consisting of 0.02% aqueous TFA and a mixture of acetonitrile/methanol (75:25, v/v). Sensitivity, accuracy (recovery rates were between 95% and 105.6%), repeatability (RSD ≤ 4.6%), and precision (intra-day precision ≤ 4.8%; inter-day precision ≤ 3.4%) of the method were determined. HPLC-MS experiments in positive and negative electrospray ionization mode confirmed identity and peak purity of analytes. The analysis of several root and tincture samples revealed that (+)-conocarpan and ratanhiaphenol II dominated with contents of 0.49-0.71% and 0.51-0.53% in the roots and 0.66-0.68 mg/ml and 0.70-0.71 mg/ml in the commercial tinctures, respectively.