Pregnane-type steroidal glycosides from Gymnema grifﬁthii Craib. Suphongphan Srisurichan a , Songchan Puthong b , Surachai Pornpakakul a,⇑ a Research Centre for Bioorganic Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand b Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand article info Article history: Received 24 January 2014 Received in revised form 3 June 2014 Available online 19 July 2014 Keywords: Gymnema grifﬁthii Craib. Apocynaceae Ortho-acetate Pregnane-type steroidal glycoside abstract Eight pregnane-type steroidal glycosides substituted with ortho-acetate groups were isolated from the methanolic extract of the pericarp of Gymnema grifﬁthii fruits, and named gymnemogrifﬁthosides A–H. Their structures were determined by spectroscopic analysis (one and two dimensional nuclear magnetic resonance, high resolution electrospray ionization mass spectrometry and attenuated total reﬂectance- Fourier transformed infrared spectroscopy), while the absolute structure of the steroidal skeleton of one of these was additionally determined using Mosher’s method. All compounds were evaluated for their in vitro (i) cytotoxic effects against ﬁve human tumor cell lines (BT 474, Chago, Hep-G2, KATO-III and SW620) and (ii) a-glucosidase inhibitory activity. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Pregnane glycosides are C-21 steroidal glycosides that are widely distributed in the plant kingdom, especially in the subfam- ily Asclepiadoideae (family Apocynaceae; formerly Asclepiada- ceae) (Endress and Bruyns, 2000; Angiosperm Phylogeny Group, 2003). In these molecules, the sugar moiety is mostly linked at the C-3 position of the aglycone and generally occurs as a linear rather than a branched oligosaccharide chain (Panda et al., 2006). In recent years, several studies have reported that pregnane glyco- sides have considerable bioactivity, including compounds with antiproliferative (Ciofﬁ et al., 2006; Plaza et al., 2005), cytotoxic (Braca et al., 2002; De Leo et al., 2005; Chen et al., 2010; Waheeda et al., 2011; Al-Massarani et al., 2012), antifungal (Yoon et al., 2011) and antidepressant (Yang et al., 2011) activities. There are about 119 known species in the genera Gymnema (Apocynaceae: Asclepiadoideae), and they are distributed in tropi- cal or subtropical Asia, South Africa and Oceania (Gupta et al., 2012). Several species in the Gymnema genus are in the list of Indian and Chinese traditional medicine used for the treatment of diabetes, rheumatism, blood-vessel inﬂammation and snake- bites (Ping-tao et al., 1995; Khare, 2007). Additionally, extracts of various species have shown anti-sweetening (Suttisri et al., 1995), antidiabetic (Ahmed et al., 2010), hypoglycemic (Kang et al., 2012), anti-a-glucosidase and a-amylase (Ramkumar et al., 2010), antioxidant (Ramkumar et al., 2009) and cytotoxic (Gopiesh and Krishnan, 2009; Chakraborty et al., 2013; Ramkumar et al., 2013) activities. Previous phytochemical investi- gations of plants in the genus Gymnema have led to the isolation of steroidal glycosides (Yoshikawa et al., 1998, 1999) and triterpene saponins, especially the olenane type saponins (Liu et al., 1992; Sahu et al., 1996; Ye et al., 2000, 2001; Zhu et al., 2008). Gymnema grifﬁthii Craib. is widely distributed in the dry dipterocarp forests in the northern and southwestern part of Thailand. However, since no phytochemicals of G. grifﬁthii have been reported, herein reported are the isolation and structure elucidation of eight new C-21 steroidal glycosides (Fig. 1) from its fruits. 2. Results and discussion After extraction of the cut fresh pericarp of G. grifﬁthii fruits with methanol (MeOH), the residue was suspended in H 2 O and successively partitioned with dichloromethane (CH 2 Cl 2 ) and ethyl acetate (EtOAc). The CH 2 Cl 2 -soluble extract was fractionated by successive chromatographic techniques to give eight new steroidal glycosides 1–8. Gymnemogrifﬁthoside A (1) was obtained as a white amor- phous powder. Its molecular formula was established as C 46 H 72 O 19 , based on its high resolution electrospray ionization mass spectrometry (HRESIMS) data (m/z 951.4549 [M+Na] + , calcd 951.4560), suggesting eleven degrees of unsaturation. The attenu- ated total reﬂectance-Fourier transformed infrared (ATR-FTIR) spectrum of 1 showed absorption bands for hydroxy (3447 cm 1 ) and carbonyl (1731 cm 1 ) groups. Analysis of the 13 C NMR and HSQC spectra indicated that 1 contained eight methyl carbons, one methoxy carbon, eleven sp 3 methylene carbons (one oxygen- ated carbon at d C 60.2), eighteen sp 3 methine carbons (three http://dx.doi.org/10.1016/j.phytochem.2014.06.014 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +66 2 2187637; fax: +66 2 2187598. E-mail address: psuracha@chula.ac.th (S. Pornpakakul). Phytochemistry 106 (2014) 197–206 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem