TLC densitometric quantification of picrosides (picroside‐I and picroside‐II) in Picrorhiza kurroa and its substitute Picrorhiza scrophulariiflora and their antioxidant studies Shashi Shankar Tiwari, Madan Mohan Pandey, Sharad Srivastava and AKS Rawat* ABSTRACT: Picroside‐I and picroside‐II are known bioactive metabolites in Picrorhiza species. In the present study a simple, precise method has been established for the simultaneous determination of picrosides (picroside‐I and picroside‐II) in two different Picrorhiza species, P. kurroa and P. scrophulariiflora. This method was also validated for accuracy, precision, robustness, limit of detection and quantification, repeatability and recovery, according to International Conference of Harmonization guidelines. Separation and quantification was achieved by HPTLC using as the mobile phase chloroform– methanol (88:12, v/v) on precoated silica gel 60F 254 aluminum plates. Densitometric determination was carried out at wavelength λ max 254 nm in UV absorbance mode. Comparative study also revealed that picroside‐I and picroside‐II are higher in P. scrophulariiflora than P. kurroa. Picroside‐I content was found to be 1.258 and 1.611%, and picroside‐II was estimated as 0.481 and 0.613% in P. kurroa and P. scrophulariiflora, respectively. Antioxidant potential of these two Picrorhiza species was also studied using DPPH. At a concentration of 0.1 mg/mL the scavenging activities of P. kurroa and P. scrophulariiflora were found to 37.70 and 34.30%, respectively. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: HPTLC; picroside‐I; picroside‐II; Picrorhiza kurroa; Picrorhiza scrophulariiflora; antioxidant Introduction Picrorhiza kurroa Royle Ex Benth and Picrorhiza scrophulariiflora Pennel (Family: Scrophulariaceae) are extensively used in the traditional medical systems of India, China, Tibet, Nepal and Sri Lanka for various immune‐related diseases. Picrorhiza kurroa is predominant in the western Himalayas of northern India, while P. scrophulariiflora is mainly found in the Himalayan regions of Sikkim, Nepal and Tibet. Picrorhiza kurroa is used for the treatment of liver disorders, fever, asthma and jaundice (Bhandari et al., 2008; Smit et al., 2000; Sturm and Stuppner, 2001). It is also useful in gastrointestinal and urinary disorders, leukoderma, snake bite, scorpion sting and inflammatory affections (Joy and Kuttan 1999). It is reported to possess antiperiodic, cholagogue, stomachic, laxative, hepatoprotective and antiasthamatic activities (Sturm and Stuppner, 2000; Vaidya et al., 1996). Picrorhiza scrophulariiflora is used as a tonic, cathartic, stomachic and purgative. It is also widely used for the treatment of jaundice and the steaming of bones (Singh et al., 2005). Kutkin (a mixture of picroside‐I and picroside‐II) has significant hepatoprotective activity in hepatic damage caused by glactosamine in rats and plasmodium in berghei (mastomys) (Singh et al., 2005). There are several methods available for the chromatographic separation of picroside‐I and picroside‐II in P. kurroa (Bhandari et al., 2009; Dwivedi et al., 1997; Mishal and Sober 2005; Singh et al., 2005; Gaikwad et al., 2010). However, there is no detailed study available on the simultaneous determination of picrosides (picroside‐I and picroside‐II) in roots of P. scrophulariiflora. This prompted us to evaluate and compare both of these valuable species by simultaneously determining picroside‐I and picroside‐II and also their antioxidant activity. Therefore, in the present study we used HPTLC for the quanification of picrosides (picroside‐ I and picroside‐ II) in two species of Picrorrhiza, i.e. P. kurroa and P. scrophulariiflora, as well as comparing their antioxidant activities. Experimental Plant material, chemicals, and solutions Rhizome of two Picrorhiza species, P. kurroa and P. scrophulariiflora, were collected from Munsiyari region, Uttarakhand (India) in the month of * Correspondence to: AKS Rawat, Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute (CSIR), Lucknow‐226001, India. E‐mail: pharmacognosy1@rediffmail.com Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute (CSIR), Lucknow‐226001, India Abbreviations used: DPPH, 2, 2‐diphenyl‐2‐picryllhydrazyl. Biomed. Chromatogr. 2012; 26: 61–68 Copyright © 2011 John Wiley & Sons, Ltd. Research article Received 4 November 2010, Revised 3 February 2011, Accepted 3 February 2011 Published online in Wiley Online Library: 17 March 2011 (wileyonlinelibrary.com) DOI 10.1002/bmc.1626 61