Ursolic acid analogues: non-phenolic functional food components in Jamaican raspberry fruits Camille S. Bowen-Forbes a,b , Vanisree Mulabagal b , Yunbao Liu b , Muraleedharan G. Nair b, * a Department of Chemistry, The University of the West Indies, Mona, Kingston, Jamaica b Bioactive Natural Products and Phytoceuticals, Department of Horticulture, 173 National Food Safety and Toxicology Center, East Lansing, Michigan 48824, USA article info Article history: Received 7 November 2008 Received in revised form 14 January 2009 Accepted 25 February 2009 Keywords: Rubus rosifolius Antioxidant Anti-inflammatory Tumour cell proliferation inhibition Terpenoids abstract The Rubus genus produces numerous species that are known for their medicinal properties. Rubus rosifo- lius, called the red raspberry, grows wild in elevated regions in Jamaica. Phytochemical examination of the ethyl acetate extract of the fruit yielded eight compounds of the 19-a-hydroxyursane type: euscaphic acid (1), 1-b-hydroxyeuscaphic acid (2), hyptatic acid B (3), 19a-hydroxyasiatic acid (4), trachelosperog- enin (5), 4-epi-nigaichigoside F1 (6), nigaichigoside F1 (7), and trachelosperoside B-1 (8), as confirmed by NMR spectroscopy. Inhibition of cell proliferation by these compounds were determined by using MCF-7 (breast), SF-268 (CNS), NCI H460 (lung), HCT-116 (colon) and AGS (gastric) human tumour cells. Among the human tumour cell lines assayed, only compounds 3 and 6 displayed significant growth inhibition and was specific to colon tumour cells by 56% and 40%, respectively. These ursolic acid analogues were also tested for anti-inflammatory activity using in vitro cycloxegenase-1 (COX-1) and cycloxegenase-2 (COX-2) enzyme inhibitory assays. Compounds 1, 2 and 3 showed selective COX-1 enzyme inhibitory activity (13%, 25% and 35%) at 25 lg/ml. In the lipid peroxidation (LPO) inhibitory assays, compounds 2, 4, 7 and 6 inhibited LPO by 62%, 60%, 53% and 68%, respectively, at 25 lg/ml. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Fruits and vegetables are an excellent source of diverse non- nutrient phytochemicals, many of which possess favourable quali- ties such as the ability to act as cellular antioxidants or as anti- inflammatory agents (Deighton, Brennan, Finn, & Davies, 2000; Seeram, Momin, Nair & Bourquin, 2001). Rubus is a diverse plant genus comprised of 250 species. The plants have been traditionally employed in the treatment of several diseases, particularly diabe- tes (Kanegusuku et al., 2007). The berries are rich in vitamins A, B and C, while the leaves and roots have been used in various medicinal applications (Byamukama, Kiremire, Andersen, & Stei- gen, 2005; Deighton et al., 2000). Rubus rosifolius (Rosaceae) is a red raspberry that is native to Eastern China and is distributed in the Caribbean, Hawaii, Australia and Asia (Adams, 1972). Also called the West Indian raspberry, it is one of the many fruit-bearing plant species in Jamaica that is under utilized, being known and ea- ten by only a minority of the populace. Much of the work done on the Rubus genus to date has been focused on either the anthocya- nin content of the fruits or the phytochemistry of the aerial parts (Byamukama et al., 2005; Deighton et al., 2000; Ohtani et al., 1990; Zhou, 1992). This is in contrast to the little research that has been undertaken on the phytochemistry of the fruits. Of the compounds isolated from this genus, triterpenoids of the ursane and oleanane types are among those reported (Ohtani et al., 1990; Zhou, 1992). The methanolic extract of the aerial parts of R. rosifolius was shown to possess strong analgesic properties, the active principle being identified as 28-methoxytormentic acid (Kanegusuku et al., 2007). Several sesquiterpenoids including b- caryophyllene, humulene, bicyclygermacrene, and rosifoliol, the latter of which was first isolated from R. rosifolius, were isolated from the essential oil Southwell, 1978; Southwell & Tucker, 1996). In the present study, we conducted phytochemical research of the fruits of the plant. Herein we report the isolation of eight 19a-hydroxyursolic acid analogues and their COX-1 and COX-2 en- zyme, lipid peroxidation and tumour cell proliferation inhibitory activities. This is the first report on the terpenoidal constituents in R. rosifolius fruits, and of their bioactivity. 2. Materials and methods 2.1. General experimental procedures 1 H and 13 C NMR spectra were recorded on an INOVA Varian VRX 500 MHz instrument using CD 3 OD. Chemical shifts are in d (parts per million) relative to d 3.30 and 49.0 for 1 H and 13 C NMR, respec- tively. Merck Silica gel 60 (35–70 lm particle size) was used for medium pressure liquid chromatography (MPLC). The Preparative 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.02.075 * Corresponding author. Tel.: +1 517 432 3100x141; fax: +1 517 432 2310. E-mail address: nairm@msu.edu (M.G. Nair). Food Chemistry 116 (2009) 633–637 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem