A Proposed Biosynthetic Pathway of Picrosides Linked through the Detection of Biochemical Intermediates in the Endangered Medicinal Herb Picrorhiza kurroa Varun Kumar, a Hemant Sood, b Manu Sharma a and Rajinder Singh Chauhan a * ABSTRACT: Introduction – Picrorhiza kurroa Royle ex Benth is an important medicinal herb used in the preparation of several herbal drug formulations due to the presence of picroside-I (P-I) and picroside-II (P-II) along with other iridoid-glucosides derivatives. Objective – The endangered status of P. kurroa coupled with lack of information on biosynthesis of P-I and P-II necessitate deciphering the biosynthetic pathway for picrosides. Methods – LC with electrospray ionisation (ESI) and quadrupole time of flight combined with MS/MS was used to detect intermediates and assemble the picrosides biosynthetic pathway in P. kurroa. Results – The presence of catalpol and aucubin, the major backbone structures of picrosides, along with intermediate metabolites boschnaloside, bartsioside and mussaenosidic acid, was confirmed in ESI negative mode with pseudomolecular ion peaks, that is, m/z 361, m/z 343, m/z 345, m/z 329 and m/z 375 ions and their fragmentation patterns. Conclusion – The picrosides biosynthetic pathway is expected to provide a reliable platform towards understanding the molecular components (genes/enzymes) of P-I and P-II biosynthesis in P. kurroa for their eventual utilisation in various applications. Copyright © 2013 John Wiley & Sons, Ltd. Supporting information can be found in the online version of this article. Keywords: LC–MS; biosynthetic pathway; aucubin; bartsioside; picrosides; Picrorhiza kurroa Introduction Picrorhiza kurroa Royle ex Benth is a medicinal herb belonging to the family of Scrophulariaceae and mainly found in the Himalayan regions of India at altitudes of 3000–4300 m (Sood and Chauhan, 2010). It is widely used as a hepatoprotective (Saraswat et al., 1999) in various formulations such as Picroliv (Ansari et al., 1991), Katuki, Arogya, Livomap and Kutaki (Bhandari et al., 2009), and also possesses other pharmacological activities such as anti-carcinogenic (Joy et al., 2000), anti-oxidant (Rajkumar et al., 2011), immunomodulatory (Gupta et al., 2006), anti-allergic, anti-asthmatic (Dorsch et al., 1991), superoxide scavenging (Chander et al., 1992) and anti-diabetic (Joy and Kuttan, 1999) properties. Picrorhiza kurroa contains two major medicinal components, picroside-I (P-I) and picroside-II (P-II) along with kutkoside (Singh and Rastogi, 1972), picroside-III (P-III) (Weinges et al., 1972), picroside-IV (P-IV), verminoside, specioside (Li et al., 1998) and other iridoid-glucosides (Table 1) (Mondal et al., 2012). The increasing demand, limited cultiva- tion and reckless collection from the wild have rendered P. kurroa a critically endangered plant species (Rai et al., 2000; Mehra et al., 2011; Sood and Chauhan, 2011). Despite the several medicinal properties attributed to P-I and P-II, their biosynthetic pathway is unknown in P. kurroa. Picrosides are biosynthesised by the isoprenoid biosynthesis pathway through the precursor geranyl diphosphate (GPP) (Gahlan et al., 2012) and no information is available as of today on the formation of picrosides from GPP. The biosynthesis of P-I and P-II has been found to occur differentially in P. kurroa, the former in shoots and the latter in stolons or roots (Sood and Chauhan, 2010; Pandit et al., 2012). Whether both the mevalonate (MVA) and non-mevalonate (i.e. methylerythritol phosphate, MEP) pathways or either of them contribute to the formation of GPP, was undertaken through the expression analysis of pathway genes vis-à-vis differential P-I and P-II biosynthesis in different tissues/organs of P. kurroa (Pandit et al., 2012). Four genes (DXPS, ISPD, ISPE, MECPS) of the MEP pathway and one gene (PMK) of the MVA pathway had shown elevated levels of expression in shoots, thereby, correlating with the biosynthesis of P-I in P. kurroa and two genes of the MEP pathway showed higher expression in roots containing P-II. The expression analysis showed that both the MVA and MEP * Correspondence to: R. S. Chauhan, Department of Pharmacy, Jaypee University of Information Technology, Waknaghat, Solan-173234, Himachal Pradesh, India. Email: rajinder.chauhan@juit.ac.in a Department of Pharmacy, Jaypee University of Information Technology, Waknaghat, Solan-173234, Himachal Pradesh, India b Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan-173234, Himachal Pradesh, India Phytochem. Anal. 2013 Copyright © 2013 John Wiley & Sons, Ltd. Research Article Received: 26 December 2012, Revised: 25 February 2013, Accepted: 28 February 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/pca.2437