Short communication A new megastigmane from Kalanchoe tubiflora (Harvey) Hamet Hui-Chi Huang a , Guan-Jhong Huang a , Chih-Chuang Liaw b , Chang-Syun Yang a , Chung-Ping Yang a , Chao-Lin Kuo a , Yen-Hsueh Tseng c , Sheng-Yang Wang c , Wen-Te Chang a, *, Yueh-Hsiung Kuo a,d, ** a Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan b Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung 804, Taiwan c Department of Forestry, National Chung Hsing University, Taichung 402, Taiwan d Tsuzuki Institute for Traditional Medicine, College of Pharmacy, China Medical University, Taichung 404, Taiwan 1. Introduction The plant Kalanchoe tubiflora (Harvey) Hamet (Crassulaceae) is a succulent plant native to Madagascar (Allorge-Boiteau, 1996), also known as Bryophyllum and Kitchingia (Chernetskyy, 2012). The plant of Kalanchoe genus has been applied medicinally for treatment of abscesses, bruises, coughs, fever, and stomachache in Southeast Asia (Kao, 1996). It is also reported to possess anti- leishmanial (Muzitano et al., 2006), anti-inflammatory (Costa et al., 2006), anti-ulcer (Pal and Nag Chaudhuri, 1991), anti-microbial (Akinpelu, 2000), cytotoxic (Wu et al., 2006), insecticidal (Suprat- man et al., 2000), lymphocyte-suppressive (Costa et al., 1994), and EBV-EA inhibitory activities (Supratman et al., 2001), respectively. In the previous studies, the Kalanchoe species has been found to contain bufadienolides (Wu et al., 2006; Supratman et al., 2000; Yamagishi et al., 1989), flavonoids (Muzitano et al., 2006; Akinpelu, 2000; Costa et al., 1994), triterpenoids (Gaind et al., 1976), fatty acids (Almeida et al., 2000), amino acids (Costa et al., 2006) and phenolic compounds (Gaind and Gupta, 1973). In addition, several bufadienolide compounds were revealed from the flowers of K. tubiflora (McKenzie et al., 1989; Capon et al., 1983, 1985). Since our interest was on the anti-inflammatory components discovery from plants, prompted us to investigate this whole plant. As a result, one new megastigmane, (6S,7R,8R,9S)-6-oxaspiro-7,8-dihy- droxymegastigman-4-en-3-one (1) (Fig. 1), three flavonoids (2–4), and six benzenoids (5–11) were isolated. The structure of the new compound was elucidated by extensive spectroscopic methods including 1D- and 2D-NMR experiments. The anti-inflammatory activity of selected isolated compounds was also evaluated as inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264.7 cell lines. 2. Results and discussion The EtOH extract of the whole plant of Kalanchoe tubiflora was suspended in water, and then partitioned with n-hexane, EtOAc, and n-BuOH by liquid–liquid extraction, successfully. The EtOAc fraction was successively subjected to repeated silica gel and Sephadex LH-20 column and then purified by RP-HPLC to yield 1–11. Phytochemistry Letters 6 (2013) 379–382 A R T I C L E I N F O Article history: Received 5 December 2012 Received in revised form 28 March 2013 Accepted 14 April 2013 Available online 3 May 2013 Keywords: Chinese herb Crassulaceae Kalanchoe tubiflora (Harvey) Hamet Megastigmane Tubiflorone Anti-inflammatory activity A B S T R A C T One new megastigmane, (6S,7R,8R,9S)-6-oxaspiro-7,8-dihydroxymegastigman-4-en-3-one (1) (tubi- florone, 1), and ten known compounds were isolated and characterized from the EtOH extract of Kalanchoe tubiflora (Harvey) Hamet. Structures of these isolates were assigned based on spectroscopic analyses that included 1D and 2D NMR techniques, such as HMQC, HMBC, and NOESY. The anti- inflammatory activities of selected isolated compounds (1–6 and 9–11) were evaluated as inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264.7 cell lines. Compounds 1–4, 6, 9, and 11 possessed nitric oxide inhibitory activity with IC 50 values ranging from 15.1 Æ 0.9 to 98.9 Æ 1.3 mM. ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe. * Corresponding author at: Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, No. 91, Hsueh-Shih Road, Taichung City 404, Taiwan. Tel.: +886 4 2205 3366x5203. ** Corresponding author at: Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, No. 91, Hsueh-Shih Road, Taichung City 404, Taiwan. Tel.: +886 4 2205 3366x5701. E-mail addresses: wtchang@mail.cmu.edu.tw (W.-T. Chang), kuoyh@mail.cmu.edu.tw (Y.-H. Kuo). 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 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe. http://dx.doi.org/10.1016/j.phytol.2013.04.002