Eremophilane-type sesquiterpenes from cultured lichen mycobionts of Sarcographa tricosa Duy Hoang Le a , Yukiko Takenaka a , Nobuo Hamada b , Takao Tanahashi a,⇑ a Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan b Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennouji-ku, Osaka 543-0026, Japan article info Article history: Available online 28 January 2012 This paper is dedicated to the memory of Prof. Dr. Meinhart H. Zenk. Keywords: Sarcographa tricosa Mycobiont Lichen Sesquiterpene Eremophilane abstract Spore-derived mycobionts of the crustose lichen Sarcographa tricosa were cultivated on a malt-yeast extract medium supplemented with 10% sucrose. Chemical investigation of the cultivated colonies led to isolation of three eremophilane-type sesquiterpenes, 3-epi-petasol (1), dihydropetasol (2) and sarcog- raphol (3), together with six known eremophilanes and ergosterol peroxide. These structures were elu- cidated by spectroscopic and chemical methods. This is the first report of eremophilane-type sesquiterpenes from the cultured mycobionts of lichen. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Lichens are symbiotic organisms of fungi (mycobionts) and photoautotrophic algal partners, namely, green algae and/or cya- nobacteria. About 18,500 different lichen taxa have been described worldwide. Lichens have adapted to extreme ecological conditions, being dominant at high altitudes and in Arctic boreal and tropical habitats. Vietnam has a tropical monsoon climate that is favorable for diverse tropical lichens, but previous studies on the Vietnamese lichens focused mainly on their taxonomy and not on their chem- ical constituents (Aptroot and Sparrius, 2006). Lichens produce many unique compounds, which are considered to have important biological and ecological functions, such as antimicrobial activity (Ahmadjian, 1993; Huneck, 1999, 2001). Most of these metabolites are produced by the fungal partner, in symbiosis or in the aposym- biotic state. Cultures of isolated lichen mycobionts, however, often exhibit the ability under osmotically stressed conditions to pro- duce substances that have never been detected in the lichenized state (Miyagawa et al., 1994; Tanahashi et al., 1997; Takenaka et al., 2003, 2010). From our interest in Vietnamese lichens and the metabolic ability of isolated lichen mycobionts, the spore- derived mycobionts of the crustose lichen Sarcographa tricosa col- lected in Vietnam were cultured and three new eremophilane-type sesquiterpenes together with seven known compounds were iso- lated. In this paper, the isolation and the structural determination of the new sesquiterpenes are described. Detailed spectroscopic data and further studies on the absolute stereochemistry of two known sesquiterpenes are also reported. 2. Results and discussion Specimens of S. tricosa (Ach.) Müll. Arg. were collected from tree bark in Dong Nai Province, Vietnam, in 2008. The polyspore- derived mycobionts were cultivated on a malt-yeast extract medium supplemented with 10% sucrose at 18 °C in the dark. After several months, the cultures were harvested and extracted with n-hexane and Et 2 O. These extracts were separated by chromato- graphic procedures to afford three new eremophilane-type sesqui- terpenes 1–3 together with six known eremophilanes 4–9 and ergosterol peroxide (10)(Fig. 1). Among the isolated compounds from the culture, petasol (4) and dihydrosporogen-AO 1 (5) were the major products. Petasol (4) was first isolated from Petasites fragrans in its free form (Sugama et al., 1983), although its esters such as petasin (11) and S-petasin (12) have since been isolated. Since alkaline hydroly- sis of the esters failed to yield petasol (4), but afforded isopetasol (9), the absolute stereostructure was investigated on its esters but not on petasol (4) itself (Aebi and Djerassi, 1959; Herbst and Djerassi, 1960; Neuenschwander et al., 1979). Dihydrosporogen- AO 1 (5) was isolated from the culture broth of fungus Alternaria citri. Its absolute configuration was determined previously by the CD spectrum of its dibenzoate derivative (Kono et al., 1989). As the 1 H NMR spectroscopic data of 4 and 5 have not been fully 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2012.01.009 ⇑ Corresponding author. Tel./fax: +81 78 441 7547. E-mail address: tanahash@kobepharma-u.ac.jp (T. Tanahashi). Phytochemistry 91 (2013) 242–248 Contents lists available at SciVerse ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem