A new sesquiterpene lactone from yacon leaves

Abstract The chemical constituents of 60% EtOH extract of yacon leaves were separated to yield a new compound, together with four known compounds, which were isolated for the first time from yacon. The new compound was characterised and named as chlorodalin (1) on the basis of NMR (1D and 2D), HR-MS and other spectral methods. The cytotoxic activities of 1–5 were evaluated on two human tumour cell lines and the new compound showed significant cytotoxic activity. Graphical Abstract


Introduction
Yacon, Smallanthus sonchifolius (Peoepp. & Endl.) H. Robinson belonging to family Asteraceae (Kakuta et al. 1992) is an indigenous plant of the Andes of South America (Lachman et al. 2003). It was firstly introduced into China from Japan as an anti-diabetic herbal tea (Genta et al. 2010) in the early 1990s. Currently, anti-diabetes, antioxidant and antitumour actions of yacon leaves were evaluated (Valentova et al. 2003;Genta et al. 2010;Raga et al. 2010;de Moura et al. 2012;Russo et al. 2015). Up to now, ent-kaurenic acids, phenolic acids, diterpenes, lignans and sesquiterpene lactones were isolated from yacon leaves (Kakuta et al. 1992;Jirovský et al. 2003;Dou et al. 2008Dou et al. , 2010Mercado et al. 2010;Xiang et al. 2010a;Lv et al. 2012;Oliveira et al. 2013). Previously, the active constituents of yacon leaves were explored for anti-diabetic effect and a series of smaditerpenic acids possessing α-glucosidase inhibitory action were discovered (Dou et al. 2008;Xiang et al. 2010b). The constituents of yacon leaves were further investigated to furnish a new compound 1, together with four known compounds 2, 3, 4 and 5, which were obtained for the first time from this plant ( Figure 1). This paper deals with the structure elucidation of the new compound and their cytotoxic activity.

Results and discussion
Compounds 1-5 were isolated from the 60% ethanol extract of yacon leaves using silica gel and ODS column chromatography and purified by preparative HPLC. The chemical structures were elucidated on the basis of spectral data analysis.
Compound 1 is the uvedalin chloride resulted in the opening of epoxy group of uvedalin and most possibly, it was transformed from uvedalin in yacon leaves during the isolation process with chlorinated solvents or by hydrolysis in storage over one year, although the leaves were stored in a dry and dark place. So fresh leaves were adopted and immediately extracted ultrasonically with methanol for UPLC/TOF-MS analysis, indicating that compound 1 naturally occurred in the fresh yacon leaves.
Evaluating the cytotoxic activity of the five compounds, compound 1 showed significant cytotoxicity against two human cancer cell lines (Table 1).

Instruments and chemicals
Optical rotations were recorded on a JASCO P-2000 (Japan) digital polarimeter. CD was measured on MOS-450 (France). HR-MS was obtained on a Waters Xevo Q-TOF (USA). NMR spectra were recorded on a Bruker ARS 500 NMR spectrometer (Germany). Preparative HPLC separations were performed on HITACHI 7100 equipped with a YMC-Pack ODS-A, 10 × 250 mm column (Japan), using a flow rate of 2.0 mL/min at a column temperature of 25 °C, and

Cytotoxicity
The in vitro cytotoxicity of the five compounds against two human cancer cell lines (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) was investigated by the MTT colorimetric method. 5-Fu (5-fluorouracil), a known anticancer agent, was used as positive control. The human cancer cell lines were grown in HyClone's modified 1640 medium containing 10% foetal bovine serum and cultivated in humidified incubators (SANYO, Japan). Afterwards, the cells were seeded in 96-well plates and cultivated for 12 h, and then treated with compounds of five concentrations (12.5, 25, 50, 100 and200 μM) for 48 h. Then, 10 μL of MTT (5 mg/mL) was added to each well, and the cells were incubated for additional 4 h. Then, DMSO (100 μL per well) was added to dissolve the formazan crystals. Absorbance was measured at 492 nm by enzyme immunoassay instrument (Caretium, KC-100, Shenzhen, China). [2R-(1R*, 2α, 4α)]4-(2,4-dihydroxy-2,6,6-trimethylcyclohexylidene)-3-buten.  3H, s, H-12). This is the first time to report the data of 13 C NMR and 1 H NMR of compound 2 and its CD showed opposite to that of grasshopper keton (Shinde et al. 2007). The NMR data of C/H 2,3,4 are the only difference compared with grasshopper keton (Baumeler et al. 1990;Shinde et al. 2007).
Adenine ( The NMR spectra data of compound 3 were agreed with the data of Adenine in the reference (Lim et al. 2006).

Supplementary material
Supplementary material relating to this article is available online, alongside the CD and HR-MS of compound 1 and 2, and the NMR data of compounds 1-5.

Disclosure statement
None of the authors declare any conflict of interests in this study.