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World J Microbiol Biotechnol (2012) 28:835840 DOI 10.1007/s11274-011-0878-0
ORIGINAL PAPER
Preparative separation of helvolic acid from the endophytic fungus Pichia guilliermondii Ppf9 by high-speed counter-current chromatography
Jianglin Zhao Yan Mou Tijiang Shan
Yan Li Shiqiong Lu Ligang Zhou
Received: 10 July 2011 / Accepted: 31 August 2011 / Published online: 9 September 2011 Springer Science+Business Media B.V. 2011
Abstract High-speed counter-current chromatography (HSCCC) was applied for preparative separation of helvolic acid from the crude extract of the endophytic fungus Pichia guilliermondii Ppf9, associated with the medicinal plant Paris polyphylla var. yunnanensis for the rst time. The two-phase solvent system consisted of n-hexaneethyl acetatemethanolwater (4.5:4.5:5.0:5.0, v/v) appending with phosphoric acid (0.2%, v/v) was employed. The revolution speed of the separation column, ow rate of the mobile phase and separation temperature of the apparatus were 800 rpm, 3 ml min-1 and 25C, respectively. About6.8 mg of helvolic acid was successfully obtained from 450 mg of the crude extract by HSCCC within 4 h separation procedure, and its purity reached to 93.2% according to the HPLC analysis. The product was further characterized by MS, 1H-NMR and 13C-NMR spectra.
Keywords Preparative separation Helvolic acid
Endophytic fungus Pichia guilliermondii Ppf9
High-speed counter-current chromatography
Introduction
Plant endophytic fungi are an important and novel resource of natural bioactive compounds with great potential
applications in agriculture, medicine and food industry (Strobel et al. 2004; Gunatilaka 2006; Verma et al. 2009; Zhao et al. 2011). During the past two decades, many valuable bioactive compounds with antimicrobial, insecticidal, cytotoxic and anticancer activities have been obtained from the endophytic fungi (Zhang et al. 2006; Aly et al. 2010; Yu et al. 2010; Zhou et al. 2010; Kharwar et al. 2011). In our previous study, one antimicrobial nordammarane triterpenoid (helvolic acid, Fig. 1) was successfully obtained by bioassay-guided fractionation from the endophytic fungus Pichia guilliermondii Ppf9 derived from the medicinal plant Paris polyphylla var. yunnanensis (Zhao et al. 2010). Helvolic acid, a pronounced antibiotic, has been successfully obtained from many fungal microorganisms such as rice fungal pathogen Sarocladium oryzae (Sakthivel et al. 2002), endophytic Aspergillus sp. CY725 from Cynodon dactylon (Li et al. 2005), marine-derived fungus Aspergillus sydowi (Zhang et al. 2008), entomopathogenic fungus Metarhizium anisopliae (Lee et al. 2008), and endophytic Alternaria sp. FL25 from...