Sesquiterpenoids from Artemisia vestita and Their Antifeedant and Antifungal Activities

Four new sesquiterpenoids, named artemivestinolide D–G (1–4) and three known sesquiterpenoids (5–7), were isolated from Artemisia vestita. The structures of these new compounds were determined based on extensive spectroscopic data analyses. Furthermore, the electronic circular dichroism data determined the absolute configurations of the new compounds. The antifeedant and antifungal activities of the isolates were evaluated against third-instar larvae of Plutella xylostella and three plant pathogenic fungi. Compounds 1–7 showed moderate antifeedant activities and compounds 1–4 and 6–7 exhibited antifungal activities.


Introduction
Artemisia (Compositae) species are widespread throughout China and receive much attention due to their remarkable biological activities and structural diversities [1][2][3]. A. vestita is distributed at wasteland and river beaches of China and it has been widely used in traditional Tibetan and Chinese Traditional Medicine for treating various inflammatory diseases [4]. Modern pharmacological studies indicated that ethanol extracts of A. vestita exhibited anti-inflammatory activities [5]. The isolated compounds from A. vestita showed inhibitory effects against NO production induced by LPS [6,7]. Furthermore, A. vestita was found to possess strong insecticidal activity against Sitophilus zeamais and Haemonchus contortus [8,9]. A number of sesquiterpenoids have been isolated from A. vestita and have shown biological activities [4][5][6]. Sesquiterpenoids have important roles in plants' defense against pests and plant pathogenic fungi so that they can be utilized as natural insecticides and antifungal agents [10,11]. In our continuing research to explore insecticidal and antifungal compounds from plant resources and to extend the knowledge towards the sesquiterpenoids molecules, the whole plants of A. vestita were collected and the extracts were carefully investigated. From the results of our studies on this species, we report herein the isolation, structural determination, and bioactivity of four new sesquiterpenoids, artemivestinolide D-G (1-4), along with three known metabolites, 1α-acetoxyeudesm-4-en-6β,11βH-12,6-olide (5) [6], dehydrocostus lactone (6) [12], and dihydroestafiatone (7) [13] (Figure 1). Herein, we reported the isolation and structural elucidation of the undescribed sesquiterpenoids by extensive spectroscopic techniques (Supplementary Materials) and chemical means. The CD exciton chirality method and calculated ECD spectra were used to determine the absolute configurations of the new compounds. The antifeedant activities against 3rd instar larvae of Plutella xylostella and antifungal activities for plant pathogenic fungi of the isolated compounds were evaluated.   (Table 1) indicated 19 carbon resonances, including two ester carbonyls at δ C 178.7 (C-12) and 176.6 (C-16), two olefinic carbons at δ C 126.4 (C-4) and 128.6 (C-5), and two oxygenated carbons at δ C 78.9 (C-1) and 82.6 (C-6). The above NMR spectroscopic data and the degrees of unsaturation suggested that this compound was a sesquiterpene lactone containing three rings. Considering its biological source, it should be an eudesm-12,6-olide derivative [6]. In the HMBC spectrum ( Figure 2), correlations were observed from H-1 to C-16 and from H-17 to C-1, indicating that an isobutyrate group was attached to C-1. The HMBC correlations of H 3 -15 to C-3, C-4, and C-5 allowed the assignment of the olefinic carbons at C-4 and C-5. The HMBC correlations of H-6 to C-5, C-7, and C-8 confirmed the 12,6-olide. The relative stereochemistry of 1 was established by the NOESY spectrum ( Figure 3). The NOESY correlations between H 3 -14 with H-6, and between H-7 with H-1 and H 3 -13 indicated that H 3 -14 and H-6 were β-oriented, and H-1, H-7, and H 3 -13 were α-oriented ( Figure 2). The absolute configuration at C-6 of 1 was determined to be S from the CD spectrum using empirical rules of π-π * transition of α,β-unsaturated γ-lactones [6,14], in which a negative Cotton effect was observed at 212 nm. Furthermore, the absolute configuration of 1 was assigned as (1R,6S,7S,10R,11S) based on the fact that the calculated ECD curve for (1R,6S,7S,10R,11S)-1 agreed well with the experimental spectrum for 1 ( Figure 4). Thus, compound 1 was elucidated as (1R,6S,7S,10R,11S)-1-isobutyryloxy-eudesma-4(5)-en-12,6-olide, named artemivestinolide D (1).     were α-oriented. The absolute configuration at C-6 of 2 was determined as S based on the negative Cotton effect at 211 nm compared with compound 1 in the CD experimental spectrum [6,14]. Thus, compound 2 was elucidated as (1R, 6S, 7S, 10R, 11S)-1-methacryloyloxy-eudesma-4(5)-en-12,6-olide, named artemivestinolide E (2).
The isolated sesquiterpenoids were preliminarily investigated for their antifeedant activities against third-instar larvae of P. xylostella and antifungal activities against three pathogenic fungi (Pyricularia oryzae, Botrytis cinerea, and Fusarium oxysporum). As shown in Table 2, all compounds were found to have potential deterrence against the P. xylostella, with EC 50 values ranging from 25.3-42.1 µg/cm 2 . Furthermore, some sesquiterpenoids showed antifungal activities against pathogenic fungi. For P. oryzae, compounds 1 and 6 displayed antifungal activities (MIC of 128 mg/L). For B. cinerea, compounds 1, 2, 4, and 7 showed antifungal effects (MIC of 256 mg/L). For F. oxysporum, compounds 3 and 6 displayed antifungal activities (MIC of 256 mg/L). These results may reveal a way to search for natural product inspired synthetic insecticides and antifungal agents.

Antifeedant Bioassay
The antifeeding effect was estimated through a no-choice assay using leaf disc method [11]. Leaf discs (1 cm × 1 cm) of cabbage were cut and immersed in acetone solution with compounds for 5 s, and air dried at room temperature. Five treated leaf discs and 10 larvae were placed together in one 90 mm diameter Petri dish. Residual leaf area of the control and the treatment were measured separately by graph paper at 24 h post treatment and the percentage antifeedant index was then determined using the following equation: antifeedant index (%) = [(CK−T)/CK] × 100, where CK refers to the leaf area consumed in the control disc while T indicates the leaf area consumed in the treatment.

Antifungal Bioassay
The test phytopathogenic fungi used in this study were P. oryzae, B. cinerea, and F. oxysporum. The microdilution method, with 96-well microliter plates using a potato dextrose (PD) medium, was used to evaluate antifungal activity of the compounds [18]. After incubation for 48 h at 28 ± 0.5 • C, minimum inhibitory concentration (MIC) was taken as the lowest concentration of the test compounds in 96-well plate, in which no microbial growth could be observed.

Conclusions
In this study, seven sesquiterpenoids, including four new compounds, were isolated from A. vestita, and their structures were primarily elucidated on the basis of NMR and MS studies. The absolute configuration of the new compounds was determined by CD exciton chirality and calculated ECD methods. The isolated compounds were tested for antifeedant activities against third-instar larvae of P. xylostella and antifungal activities against three pathogenic fungi, with all compounds showing moderate antifeedant activities and some compounds exhibiting antifungal activities.