Hypaphorine, an indole alkaloid from Erythrina velutina, induced sleep on normal mice Masaaki Ozawa, Kazuki Honda, Izumi Nakai, Akio Kishida, Ayumi Ohsaki * Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10, Kanda Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan article info Article history: Received 22 April 2008 Revised 30 May 2008 Accepted 3 June 2008 Available online 6 June 2008 Keywords: Hypaphorine Indole alkaloid Sleep induced Non-REM Medicinal plant Erythrina velutina abstract An indole alkaloid (hypaphorine (1)) was isolated from Brazilian medicinal plant, Erythrina velutina (Leguminosae). This compound was investigated for sleep promoting effects in mice, and the results showed that it significantly increased non-rapid eye movement (NREM) sleep time during the first hour after its administration. The NREM sleep time was enhanced by 33% in the experimental mice when com- pared to that of the controls. This study therefore confirmed its sleep promoting property. Ó 2008 Elsevier Ltd. All rights reserved. Problems with sleeping in the general population are associated with a year to year increase in the aging rate, graveyard shift, and mental disorders including depression, neurosis, and dementia. The current clinical approach employs different varieties of benzo- diazepine derivatives that are used as hypnotic agents for those having difficulties in falling asleep and maintaining sleep. How- ever, these agents are often associated with adverse effects such as dependence, amnesia, nightmares, and prolonged sleepiness. 1 In addition, drug developments of the substitutes for benzodiaze- pines are required for discontinuation of chronic users. In our search for biologically active compounds from tropical plants, 2 we have studied the seeds of Erythrina velutina. This plant is commonly called ‘Mulungu’ in Brazil, and its bark is used for sedation, hypnogenesis, control of convulsions, and nervous coughs in the northern region in Brazil. 3 We study herein the alka- loidal constituents of the seed part of E. velutina. In this report, we describe the isolation and characterization of an indole derivative, hypaphorine (1) from Brazilian medicinal plant, E. velutina Willd (Leguminosae), 4 and the evaluation of its potency as a potential hypnotic agent. The seeds of E. velutina (954 g) were crushed and then extracted with MeOH (19.0 g). The MeOH-soluble materials were succes- sively partitioned between petroleum ether, EtOAc, and 3% aque- ous tartaric acid. Water-soluble materials were adjusted to pH 10 with Na 2 CO 3 and successively partitioned between CHCl 3 (6.07 g) and n-BuOH (3.48 g) to obtain the alkaloidal portions. The CHCl 3 -soluble materials (3.11 g) were subjected to silica gel column (CHCl 3 -MeOH, 100:0 ? 0:100) 5 and the last fraction was eluted with pure MeOH (349 mg) followed by a silica gel column [MeOH/(CH 3 ) 2 CO, 99:1] to isolate hypaphorine (1, 44 mg) 6 (Fig. 1) and glycoerysodine (2, 5 mg). 7 The data of hypaphorine (1) were analyzed with 1 H and 13 C NMR including 2D-techniques and FABMS spectra. Hypaphorine (1) was identified by comparison with the data in the literature, and was also determined as the 9S configuration from its optical rotation value. 8 The hypnotic activity of hypaphorine (1) was evaluated by the electroencephalographic analysis of the sleep–wake cycle in freely behaving mice through the analysis of rapid eye movement (REM) sleep, non-REM sleep (NREM), wakefulness, and total sleeping times, respectively. Male mice (C57BL, 16–21-week, Crea Japan Inc., Tokyo, Japan) were obtained and kept under controlled condi- tions at 24 ± 1 °C with relative humidity of 56 ± 6%, and a light– 0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2008.06.002 * Corresponding author. Tel./fax: +81 3 5280 8153. E-mail address: a-ohsaki.fm@tmd.ac.jp (A. Ohsaki). Figure 1. Chemical structure of hypaphorine (1). Bioorganic & Medicinal Chemistry Letters 18 (2008) 3992–3994 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl