Insect Antifeedant Components of Senecio fistulosus var. fistulosus—Hualtata

From a bioactive methanolic extract of Senecio fistulosus, the antifeedant effects of the alkaloidal and non-alkaloidal fractions were tested against the insects Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi, with the non-alkaloidal fraction being antifeedant. The phytochemical study of the non-alkaloidal fraction of S. fistulosus, resulted in the isolation of four compounds, two 9-oxo-furanoeremophilanes (1, 2), an eremophilanolide, 1β,10β-epoxy-6-acetoxy-8α-hydroxy-eremofil-7(11)-en-8β,12-olide (3) and a maaliol derivative (4). The alkaloidal fraction yielded two known pyrrolizidine alkaloids (5, 6). Compounds 1, 3 and 4 are new natural products. Furanoeremophilane 2 was a strong antifeedant against S. littoralis and maaliane 4 inhibited the settling of M. persicae.


Introduction
The genus, Senecio (Asteraceae), is distributed worldwide and contains pyrrolizidine alkaloids (PAs). PAs are toxic to mammals and feeding deterrents for insect herbivores [1]. Compounds present in the non-alkaloidal fraction of Senecio spp have been described as part of their defense [1][2][3]. The most frequent chemical groups found in the non-alkaloidal fraction of Senecio are eremophilane-type sesquiterpenes of the furanoeremophilane and eremophilanolide type [1]. Some of these compounds have insect antifeedant, acaricidal, fungicidal, cytotoxic, phytotoxic, antioxidant, anti-inflammatory, and antimicrobial effects [1-6] and have been proposed as being an important part of Senecio defense [1][2][3][4][5].
In Chile, the genus Senecio is abundant (~210 species) [7]. There are several reports on eremophilane sesquiterpenes from Chilean Senecio species with defensive properties [1 -3]. The species, Senecio fistulosus, grows from the western area of Patagonia to central Chile and it is used in folk medicine for its effects on the heart [8,9]. A previous study on the phytochemistry of S. fistulosus, from the central region of Chile, reported the presence of furanoeremophilanes 4α-hydroxy-6β-angeloxy-10βacetoxy-9-oxo-furanoeremophilane and 4α-hydroxy-6β-angeloxy-9-oxo-furanoeremophilane [10], but there are no reports on the defensive chemistry of this species.
In this work, the authors studied the chemical defenses of S. fistulosus var. fistulosus from the Magallanic region, containing a large number of the Chilean Senecio species and subspecies distributed in the Patagonic Cordillera and the coastal areas [7].
The structural elucidation was carried out based on their 1 H and 13 C NMR spectra including (1D) and (2D) (COSY, HSQC, HMBC and NOESY) experiments, X-ray diffraction, as well as its physical, spectrometric (EIMS and HREIMS) and comparison with the chemical bibliography reported for similar compounds. Compounds 1, 3 and 4 are described here for the first time as natural products. A previous study on S. fistulosus reported the presence of furanoeremophilanes 4α-hydroxy-6β-angeloxy-10βacetoxy-9-oxo-furanoeremophilane and 4α-hydroxy-6β-angeloxy-9-oxo-furanoeremophilane [10]. The difference in furanoeremophilane composition could be related to the different origin of the plant populations studied (Magallanes versus the central region of Chile).
The structural elucidation was carried out based on their 1 H and 13 C NMR spectra including (1D) and (2D) (COSY, HSQC, HMBC and NOESY) experiments, X-ray diffraction, as well as its physical, spectrometric (EIMS and HREIMS) and comparison with the chemical bibliography reported for similar compounds.
The antifeedant effects of compounds 1, 2 and 4 are shown in Table 4. Furanoeremophilane 2 was a strong antifeedant to S. littoralis (EC 50 = 0.64 µg/cm 2 ) while the maaliane 4 affected M. persicae (EC 50 = 0.97 µg/cm 2 ). Antifeedant furanoeremophilanes have been described in Senecio species such as S. magellanicus (against M. persicae and S. littoralis) [2] and S. otites (against M. persicae and R. padi) [11,18]. Furanoeremophilanes are less abundant in Senecio than eremophilanolides. Therefore, the studies on their structure-activity relationships (SAR) are limited. Table 5 shows a compilation of the available information on the SAR of these structures, including the results presented in this work. The active compounds against the aphid M. persicae are characterized by the absence of substituents in C-1, C-3 and C-10, regardless of the substituent in C-6 (8 -10, 11, 12). The presence of β-OH/C-1 and the α-OAng/C-3 group (compound 2) resulted in an important antifeedant activity against S. littoralis. In addition, the C-6 substitution pattern together with the C-1/C-10 unsaturation determined post-ingestion effects on S. littoralis [11]. Maalianes have been isolated from a range of organisms, such as liverworts, marine sponges, soft corals and bacteria, however, they are not abundant in nature. A small amount of biological activity has been reported and includes fish toxicity, in vitro antimalarial activity, cytotoxicity and antimicrobial [19]. This is the first report on the insect antifeedant effects of a maaliane sesquiterpene.
PAs with unsaturated retronecines are potentially more toxic than rosmarinecine and petasinecine type (1,2-saturated base) PAs [21]. For example, rosmarinine with a petasinecine, did not form hepatotoxic reactive pyrrole intermediates [22,23] and cytotoxic assays have demonstrated a higher toxicity of retronecine and otonecine PAs compared with platynecine PAs [24]. Therefore, PAs 5 and 6 have a low risk of associated toxicity.

General
For column chromatography (CC), Si-gel (107734, 107741, and 107749, Merck) and Sephadex LH-20 (Sigma-Aldrich) were used. For TLC chromatography, Si-gel (105554 and 105715; Merck) plates were used and visualized with óleum solution (sesquiterpenes) and Dragendorff's reagent (alkaloids). The prep. HPLC chromatography was carried out on a Beckman 125P system equipped with an Ultrasphere semiprep column (10 × 250 mm) and a UV/visible diode array detector 168. Optical rotations were determined at 20 • C on a Perkin-Elmer 343 Plus polarimeter. IR Spectra were recorded in CHCl 3 on a Perkin Elmer 1600 spectrophotometer. NMR spectra were recorded on a pulsed-field gradient Bruker Advance II-500 MHz spectrometer (solvent as internal standard CDCl 3 , at δ H 7.26 and δ C 77.0) and the Bruker software was used for DEPT, 1 H, 1 H-COSY (Homonuclear correlation spectroscopy), NOESY (Nuclear Overhauser Effect Spectroscopy), HSQC (Heteronuclear single quantum coherence spectroscopy) and HMBC (Heteronuclear Multiple Bond Correlation). EI and HR-EI-MS spectra were recorded in m/z on a Micromass Autospec spectrometer.

Extraction and Isolation
Aerial parts of S. fistulosus (Asteraceae), identified by Orlando Dollenz, were collected in Sierra Baguales (March 2009, Punta Arenas, Magallanes, Chile,) during the flowering period. A voucher specimen (# 7569) has been deposited in the Herbarium of the Patagonian Institute, Magallanes University (UMAG), Punta Arenas, Chile.
Crystallographic data (excluding structure factor tables) has been deposited with de Cambridge Crystallographic Data Center as supplementary publications no. CCDC1455588. Copies of the data can be obtained free of charge on application to The Director, CCDC, 12 Union Road, Cambridge CB1EZ, UK ((Fax: Int. + (1223) 336 033); e-mail: deposit@ccdc.cam.ac.uk)). Antifeedant bioassays: The upper surface of C. anuum and H. vulgare leaf disks or fragments (1.0 cm 2 ) were treated with 10 µl of the test substance. The crude extracts and products were tested at an initial dose of 100 or 50 µg/cm 2 respectively. Five Petri dishes (9 cm diam.) or twenty ventilated plastic boxes (2 × 2 cm) with two newly molted S. littoralis L6 larvae (≤24 h) or ten apterous aphid adults (24-48 h old) each were allowed to feed at room temperature for S. littoralis (<2 h) or in a growth chamber for the aphids (24 h, environmental conditions as above). Each experiment was repeated 2-3 times (SE < 10%) and terminated when the consumption of the control disks reached 65-75% for S. littoralis or after 24 h for aphids. The leaf disk area consumed was measured on their digitalized images (Image J, http://imagej.nih.gov/ij). Settling was measured by counting the number of aphids settled on each leaf fragment. Feeding or settling inhibition (%FI or %SI) was calculated as % FI/%SI = [1 − (T/C) × 100], where T and C are the consumption/settling of treated and control leaf disks, respectively. The antifeedant effects (% FI/SI) were analyzed for significance by the nonparametric Wilcoxon signed-rank test. Extracts and compounds with an FI/SI ≤ 75% were further tested in a dose-response experiment (3-4 serial dilutions) to calculate their relative potency (EC 50 , the effective dose to give a 50% feeding/settling reduction) from a linear regression analysis (% FI/SI on Log-dose) [33].

Conclusions
Senecio fistulosus is characterized by their content in sesquiterpenes (furanoeremophilanes, eremophilanolides and maaliane type) and pyrrolizidine alkaloids. The antifeedant properties of ethanolic, non-alkaloidal, alkaloidal extracts and compounds have been studied. Most of the insect antifeedant effects were found in the ethanolic and non-alkaloidal extracts, containing mainly sesquiterpenes with low amounts of PAs. The isolated furanoeremophilanes sesquiterpenes type had structure-dependent antifeedant effects. In addition to their antifeedant action, these sesquiterpenes could play a role in insect-plant interactions. Funding: This work has been supported by grants CTQ2015-64049-C3-1-R, (MINECO/FEDER), UMAG 027103-026703 (Dirección de Investigación, Chile) and a JAEPRE-DOC-CSIC predoctoral fellowship to L.R.V.

Conflicts of Interest:
The authors declare no conflicts of interest.