A new cytotoxic apotirucallane from the roots of Walsura trichostemon Jirapast Sichaem a,d , Thammarat Aree a , Suttira Khumkratok b , Jonkolnee Jong-aramruang c , Santi Tip-pyang a, * a Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand b Walai Rukhavej Botanical Research Institute, Mahasarakham University, Mahasarakham 44000, Thailand c Department of Chemistry, Faculty of Science, Burapha University, Chonburi 20231, Thailand d Center for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Bangkok 10330, Thailand 1. Introduction The genus Walsura Roxb. (Meliaceae) is mainly distributed in India, China and Indonesia, and is comprised of between 30 and 40 species (Yunnan Institute of Botany, 1977). Previous chemical investigations showed that triterpenoids and tetranortriterpe- noids could be isolated from this genus (Luo et al., 2000; Purushothaman et al., 1985; Govindachari et al., 1995; Yin et al., 2007). Walsura trichostemon Miq. is naturally distributed in the evergreen forest throughout Southeast Asia, including countries such as Myanmar, Cambodia and Thailand (in North, Northeast and Southeastern). W. trichostemon is used in Thai traditional medicine to treat tendon disabilities, as a staunch, for cleaning wounds, and as a treatment for hemorrhoids. To the best of our knowledge, there have been no phytochemical investigations on this plant to date. Herein, we report the isolation and structure elucidation of 1, as well as the cytotoxicity of this compound against KB and HeLa cell lines. 2. Results and discussion Trichostemonate (1) was isolated as colorless crystals having the molecular formula of C 38 H 52 O 11 as established by HRESIMS. The UV absorption at 228 nm (log e 4.18) revealed the presence of an a,b-unsaturated ketone group (Omobuwajo et al., 1996). The 1 H NMR spectrum displayed resonances attributed to eleven methine protons, which included two coupled olefinic protons in ring A [d H 5.77 (d, J = 10.4 Hz) and 7.12 (d, J = 10.4 Hz)] and one olefinic proton in ring D [d H 5.33 (br, s)]. Signals attributable to four methylene protons, and eleven methyl groups which comprised of seven methyl protons [d H 1.04, 1.07, 1.13, 1.20, 1.24, 1.34 and 1.66] and four acetyl methyl protons [d H 1.95, 1.95, 2.10 and 2.17] were evident. The 13 C NMR spectrum showed 38 distinct carbon signals, comprising eleven sp carbons, four sp 2 carbons, eleven sp 3 carbons and twelve quaternary carbons as shown in Table 1. In the 1 H– 1 H COSY spectrum showed the homonuclear proton–proton spin system of CH-1-CH-2, CH-5-CH 2 -6-CH-7, CH-9-CH-11-CH 2 -12, CH-15-CH 2 -16-CH-17 and CH-21-CH-20-CH 2 -22 fragments. Four acetyl groups were located at C-7, C-11, C-21 and C-25, with these positions confirmed by the HMBC correlations from H-7, H-11, H- 21 and H-25 to the according acetyl carbonyl centers at d C 170.8, 170.0, 169.8 and 169.4, respectively. The information from the 1D and 2D NMR analysis, as well as comparisons with previous literature strongly suggested that 1 contained the characteristic tetracyclic system (rings A–D) of an apotirucallane-type triterpe- noid (Pan et al., 2009). The NMR data of 1 was closely related to that of the tetraacetate piscidinol C (Purushothaman et al., 1985), except for the presence of a tetrahydro-2H-3-pyranone ring (ring E) at C-17 in 1. This ring also was clearly verified by the HMBC cross-peaks of the H-24 methine proton (d H 5.15, br s) with C-23 (d C 203.2), C-25 (d C 81.3) and C-27 (d C 22.1), as well as the correlation of the H-21 methine proton (d H 6.20, br s) with C-20 Phytochemistry Letters 5 (2012) 665–667 A R T I C L E I N F O Article history: Received 30 March 2012 Received in revised form 2 July 2012 Accepted 3 July 2012 Available online 17 July 2012 Keywords: Walsura trichostemon Meliaceae Trichostemonate Cytotoxicity A B S T R A C T A new apotirucallane, trichostemonate (1), was isolated from the roots of Walsura trichostemon. The complete structural assignment of this new compound was elucidated from spectroscopic methods and the stereochemistry of 1 was confirmed by X-ray crystallographic analysis. Moreover, this new compound was evaluated for its cytotoxicity (KB and HeLa cells), and showed significant cytotoxicity against both KB and HeLa cell lines with IC 50 values of 3.28 and 0.93 mg/mL, respectively. ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +66 2 218 7625; fax: +66 2 218 7598. E-mail address: Santi.Ti@chula.ac.th (S. Tip-pyang). Contents lists available at SciVerse ScienceDirect Phytochemistry Letters jo u rn al h om ep ag e: ww w.els evier.c o m/lo c ate/p hyt ol 1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2012.07.001