1022 J. Sep. Sci. 2013, 36, 1022–1028 Zhi Yang 1 Xiaoman Liu 1 Kuiwu Wang 2 Xiaoji Cao 3 Shihua Wu 1 1 Research Center of Siyuan Natural Pharmacy and Biotoxicology, College of Life Sciences, Zhejiang University, Hangzhou, China 2 School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China 3 Research Center of Analysis and Measurement, Zhejiang University of Technology, Hangzhou, Zhejiang, China Received November 12, 2012 Revised December 29, 2012 Accepted December 30, 2012 Research Article Novel linear and step-gradient counter-current chromatography for bio-guided isolation and purification of cytotoxic podophyllotoxins from Dysosma versipellis (Hance) Dysosma versipellis (Hance) is a famous traditional Chinese medicine for the treat- ment of snakebite, weakness, condyloma accuminata, lymphadenopathy, and tumors for thousands of years. In this work, four podophyllotoxin-like lignans including 4 ′ - demethylpodophyllotoxin (1), -peltatin (2), podophyllotoxin (3), -peltatin (4) as major cytotoxic principles of D. versipellis were successfully isolated and purified by several novel linear and step gradient counter-current chromatography methods using the systems of hexane/ethyl acetate/methanol/water (4:6:3:7 and 4:6:4:6, v/v/v/v). Compared with isocratic elution, linear and step-gradient elution can provide better resolution and save more time for the separation of photophyllotoxin and its congeners. Their cytotoxicities were further evaluated and their structures were validated by high-resolution electrospray TOF MS and nuclear magnetic resonance spectra. All components showed potent anticancer activity against human hepatoma cells HepG2. Keywords: Counter-current chromatography / Dysosma versipellis (Hance) / Linear gradient / Natural Products / Step gradient / Traditional Chinese medicine DOI 10.1002/jssc.201201038 1 Introduction Dysosma versipellis (Hance), a well-known traditional Chinese medicine, has been used as a general remedy for the treat- ment of snakebite, weakness, condyloma accuminata, lym- phadenopathy, and tumors for thousands of years [1, 2]. Its major bioactive principles were found to be podophyllotoxin- like lignans that exhibit pronounced biological activities, such as antiviral [2–4], antitumor [5, 6], and insecticidal activity [7]. They are also used as important antiviral drugs for the treat- ment of Anogenital Warts [8, 9]. Although several compli- cated side effects such as nausea, vomiting damage of nor- mal tissues limit their clinical treatment of cancers [10, 11], podophyllotoxin, and its congeners are important precursors for the synthesis of their derivatives, which have better ac- tivities but much lower toxicity. Typically, etoposide (VP-16) and teniposide (VM-26), as two important podophyllotoxin derivatives have been used for the treatment of a variety of can- cers [2, 12, 13]. Thus an effective preparation of podophyllo- Correspondence: Dr. Shihua Wu, Research Center of Siyuan Natural Pharmacy and Biotoxicology, College of Life Sciences, Zhejiang University, Zijinggang Campus, Hangzhou 310058, P. R. China E-mail: drwushihua@zju.edu.cn Fax: +86-571-88206287 Abbreviations: CCC, counter-current chromatography; HEMWat, hexane/ethyl acetate/methanol/water toxin and its congeners is of great significance. Up to now, sev- eral chromatography assays including thin-layer chromatog- raphy [14], and HPLC [15] have been used to isolate these podophyllotoxin-like lignans. However, these methods are usually suffering from some complications, such as irre- versible adsorption losses on solid support matrix. In this work, we aim to establish several novel support- free counter-current chromatography (CCC) methods with linear and step-gradient elution for bio-guided isolation of podophyllotoxin and its congeners from D. versipellis (Hance). As well known, CCC is a unique liquid–liquid partition chro- matography without support matrix, eliminating irreversible adsorption of sample onto the solid support used in the con- ventional chromatography column [16]. In addition, CCC is an economic instrument with the capability of high mass loading and total recovery of injected sample [17]. In decades, theories, technical details, instruments, and practical applica- tions have been reported in numerous of publications [18–22]. New designs and elution modes have made it possible to iso- late compounds with a large range of polarities in a short processing time [23]. Therefore, CCC has been accepted as an efficient preparative and analytical technique to isolate various types of natural products, and even peptides and pro- teins [24–28]. Similar to HPLC [29], gradient elution [30] was found to be efficient in several CCC trials, such as pH-related gradient Colour Online: See the article online to view Figs. 2 and 3 in colour. C  2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com