Isolation and Quantitation of Ecologically Important Phloroglucinols and Other Compounds from Eucalyptus jensenii † Jasmeen Sidana, a William J. Foley b and Inder Pal Singh a * ABSTRACT: Introduction – Eucalyptus jensenii has not been explored for its phytoconstituents except for its essential oil although a formylated phloroglucinol compound jensenone has been the focus of several ecological studies. Objective – i) To optimise the extraction conditions for preparative scale isolation of jensenone and other secondary metabolites from leaves. (ii) To develop an RP-HPLC method for simultaneous determination of phloroglucinols and other compounds in Eucalyptus leaves. Methodology – Jensenone and other compounds were isolated from acetone extract using VLC over TLC grade silica. HPLC quantitation of isolated compounds was undertaken on a C 18 -column using acetonitrile–water (2% formic acid) gradient elution. Results – Extraction conditions for isolation of jensenone were optimised and more than 99% pure jensenone was obtained in a yield of 2.1% from the dried leaves. Ten phloroglucinol compounds, including a new nuclear methylated phloroglucinol named 4-O-demethyl miniatone, and two triterpenoids were also isolated. A RP-HPLC method was developed for simultaneous quantitation of phloroglucinols and other compounds in different extracts of E. jensenii leaves. A total of 19 compounds reported from different species of Eucalyptus was separated by this method. Conclusion – The method described for isolation of jensenone is a simple, rapid and low-cost procedure amenable to the preparative scale. A new monomeric phloroglucinol compound was isolated and characterised from the acetone extract of E. jensenii leaves. The HPLC method developed can be applied successfully to different eucalypt matrices for precise and accurate determination of 19 secondary metabolites belonging to different chemical classes. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: Anti-feedant; 4-O-demethyl miniatone; formylated phloroglucinols; Jensenal; Eucalyptus jensenii INTRODUCTION Eucalyptus jensenii Maiden is a small ironbark tree of northern Australia. Although not known to be an important part of the diet of either mammals or insect herbivores, it is of interest because of the high concentration of a potent mammalian anti-feedant called jensenone (Boland et al., 1992; Lawler et al., 1998). Jensenone is a formylated phloroglucinol compound similar to the sideroxylonals that are key determinants of feeding by koalas and other marsupials and also many species of Christmas beetles (Anoplognathus spp). Other pharmacological activities of jensenone include anti-fungal and anti-leishmanial activity. It is effective against Candida albicans with a half-maximal inhibition concentration (IC 50 ) of 5.5 mg/mL. It has shown moderate anti-leishmanial activity in vitro (IC 50 of 19 mg/mL against Leishmania donovanii promastigotes (Bharate et al., 2007). Jensenone has been isolated by acid base extraction of a crude extract of E. jensenii prepared by soxhlet extraction of dried and powdered leaves with 20% acetone in petroleum ether (Lawler et al ., 1998). For further ecological and pharmacological studies, it was nec- essary to obtain reasonable quantities of very pure compound. In the present article, we report the optimisation of extraction con- ditions, HPLC quantitation and a method for rapid isolation of jensenone from the dried leaves of E. jensenii. The acetone extract prepared for the large-scale isolation of jensenone was further used for the phytochemical investigations of this plant and a new nuclear methylated phloroglucinol compound named 4-O-demethyl minia- tone (1), structurally related to torquatone and miniatone, was iso- lated along with other known compounds; namely jensenone (2) (Boland et al., 1992), miniatone (3) (Ireland et al., 2004), euglobal G1 (4), euglobal G2 (5), euglobal G3 (6), euglobal G4 (7) (Takasaki et al., 1990), grandinol (8) (Crow et al., 1977), a dimer of jensenone (9) (Mitaine-Offer et al., 2003), jensenal (10) (Moore et al., 2004, 2005), maslinic acid (11) (Kim et al., 2005) and corosolic acid (12) (Lee et al., 2009). Also, a RP-HPLC method for the simultaneous determination of 19 chemical compounds of Eucalyptus has been established and validated. This method was applied successfully to quantify eight phloroglucinols and two flavonoids in the extracts of E. jensenii leaves and will facilitate further ecological studies. The structures of these compounds are shown in Fig. 1. * Correspondence to: I. P. Singh, Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab-160062, India. E-mail: ipsingh@niper.ac.in; ipsingh67@yahoo.com a Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab-160062, India b Research School of Biology, The Australian National University, Canberra 0200, Australia † NIPER Communication Number 486 Phytochem. Anal. 2012 Copyright © 2012 John Wiley & Sons, Ltd. Research Article Received: 12 September 2011; Revised: 17 November 2011; Accepted: 23 November 2011 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/pca.2345