Diterpenoid Alkaloids from Two Aconitum Species ... - ACS Publications

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Diterpenoid Alkaloids from Two Aconitum Species with Antifeedant Activity against Spodoptera exigua Ji-Fa Zhang,†,‡ Lin Chen,†,§ Shuai Huang,† Lian-Hai Shan,† Feng Gao,*,† and Xian-Li Zhou*,†,‡ †

School of Life Science and Engineering and ‡Key Laboratory of Advanced Technology of Materials, Ministry of Education, School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031, People’s Republic of China § School of Chemistry and Chemical Engineering, China West Normal University, Nanchong 637002, People’s Republic of China S Supporting Information *

ABSTRACT: Twenty-five diterpenoid alkaloids were isolated from the roots of two Aconitum species. The structures of seven new C19-diterpenoid alkaloids, apetaldines A−G (1−7), and 10 known alkaloids (8−17) from Aconitum apetalum and eight known alkaloids (18−25) from Aconitum franchetii var. villosulum were elucidated via HRESIMS, IR, and NMR data. Alkaloids 1−10, 15, 16, and 18−25 were screened for their antifeedant activity. Among the compounds tested, chasmanthinine (19) showed highly potent antifeedant activity with an effective concentration for 50% feeding reduction (EC50) at 0.07 mg/cm2. The antifeedant structure−activity relationship of the diterpenoid alkaloids is also discussed.

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potential use as botanical pesticides (insecticides, antifeedants, and growth inhibitors), the extracts from A. apetalum were studied. Seven new C19-diterpenoid alkaloids, apetaldines A−G (1−7) (Figure 1), and 10 known alkaloids, talassicumine A (8),15 aconorine (9),15 acobretine E (10),16 cammaconine (11),15 8-O-ethylcammaconine (12),17 3-deoxyaconitine (13),18 aconitine (14),18 taurenine (15),19 songorine (16),14 and songomine (17),20 were isolated from the roots of A. apetalum. Based on the results of a previous investigation,21 five compounds were determined to be chasmaconitine (18), chasmanthinine (19), talatisamine (20), indaconitine (24), and leueandine (25) by matching the spectroscopic data among 11 known diterpenoid alkaloids isolated from A. f ranchetii var. villosulum, a traditional Chinese herb distributed in Sichuan, China. Further research on this herb resulted in the isolation of ezochasmanine (21),22 pseudaconine (22),23 and leucanthumsine A (23).24 Herein, the isolation, structure elucidation, and antifeedant activity against Spodoptera exigua (Hübner) of these diterpenoid alkaloids are reported.

est insects can cause severe problems by killing agricultural crops, damaging harvested food, and deteriorating the natural environment. Many strategies, including chemical insecticides, push−pull strategies, natural enemies, and pathogenic microbes, have been exploited to control these pests.1,2 There is a growing demand for novel and effective alternatives that are nontoxic, safe, and biodegradable. Recently, the use of botanical pesticides has been emphasized as alternatives to the conventional control of insects by synthesized pesticides, which have many disadvantages, such as high toxicity, long-term persistence, and propensity of bioaccumulation.3 As well-known toxic natural products, diterpenoid alkaloids were found to exhibit strong disinfestant activity and nicotinic acetylcholine receptor (nAChR) inhibition against Spodoptera eridania and Musca domestiea.4 Since that discovery, numerous studies have been conducted to explore the potency of diterpenoid alkaloids against insect pests.5−14 In 2004, González-Coloma and co-workers investigated the antifeedant activity of 43 C19 and 21 C20 diterpenoid alkaloids from Aconitum, Delphinium, and Consolida plants against Spodoptera littoralis and Leptinotarsa decemlineata and established a preliminary structure−activity relationship (SAR) of the pesticidal diterpenoid alkaloids.7,8 Although more than 1300 diterpenoid alkaloids have been reported, none have been tested for their antifeedant activity against Spodoptera exigua (Hübner), a polyphagous pest that damages numerous kinds of cultivated crops. Aconitum apetalum (Ranunculaceae) is a perennial herb mainly distributed in the grasslands of the Xinjiang Uygur region and shows remarkable antifeedant and pesticidal activity. As part of an effort to search plant-derived chemicals with © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The molecular formula of compound 1 was established as C36H50N2O8 based on its HRESIMS and 13C NMR data. The IR data indicated the presence of an NH group (3318 cm−1) and an aromatic functionality (1589 and 1527 cm−1). The NMR data revealed the presence of an N-ethyl group [δH 1.07 (3H, t, J = 7.2 Hz); δC 49.3 t, 13.6 q], an acetoxy group [δH 2.01 (3H, s); δC 171.5 s, 21.5 q], two methoxy groups (δH 3.27, Received: May 2, 2017 Published: November 20, 2017 3136

DOI: 10.1021/acs.jnatprod.7b00380 J. Nat. Prod. 2017, 80, 3136−3142

Journal of Natural Products

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Figure 1. Structures of compounds 1−25.

Figure 2. Key HMBC and 1H−1H COSY correlations of compounds 1−7.

3.32, each 3H, s; δC 56.4 q, 56.4 q), and an ethoxy group [δH 1.06 (3H, t, J = 7.2 Hz); δC 55.7 t, 16.4 q]. In addition, the signals from an NH group [δH 11.02 (1H, br s)], an orthodisubstituted benzene unit [δH 8.69 (1H, d, J = 7.8 Hz), 7.54 (1H, t, J = 7.8 Hz), 7.09 (1H, t, J = 7.8 Hz), 7.95 (1H, d, J = 7.8 Hz); δC 114.9, 141.8, 120.5, 134.8, 122.5, 130.6, 168.3], and an acetyl group (δH 2.22 s; δC 169.1 s, 25.6 q) indicated that compound 1 possessed an o-acetamidobenzoate moiety

(−OCOC6H4-o-NHAc).15 Analyses of the NMR and HRESIMS data revealed that 1 was an aconitine-type diterpenoid alkaloid.15 The substituted benzene moiety was located at C-18 (δC 70.9) based on the HMBC correlations of H-18α (δH 3.99) and H-6′ (7.95) with C-7′ (δC 168.3) and the amido proton with C-1′ (δC 114.9), C-3′ (120.5), and C-8′ (169.1). In the HMBC spectrum (Figure 2), one set of 1H−13C long-range correlations of H-14 (δH 4.72) with C-13 (δC 45.2), C-9 (43.2), 3137



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the HMBC (Figure 2) correlations of H-7 and H-15 with C-8 and of H-13 and H-16 with C-14. Thus, the structure of apetaldine C (3) was established as shown in Figure 1. Compound 4 was found to be a demethylated analogue of talassicumine A (8)15 through spectroscopic analysis. Comparison of the NMR data of 4 and 8 showed that the chemical shift of C-1 was shielded (ca. ΔδC 13.3 ppm) and that of C-2 was deshielded (ca. ΔδC 3.7 ppm), respectively, which suggested that a hydroxy group in 4 replaced the C-1 methoxy group in 8. This replacement was supported by 2D NMR and HRESIMS data. Therefore, all of the available evidence suggests the structure of apetaldine D (4) as depicted in Figure 1. Compound 5 had a molecular formula of C31H40N2O6, as established from HRESIMS data. The NMR data strongly suggested that 5 was a C19-diterpenoid alkaloid bearing an Nethyl group, a methoxy group, and a disubstituted double bond [δH 5.65 (1H, d, J = 8.4 Hz); 5.91 (1H, dd, J = 8.4, 7.2 Hz); δC 130.0, 132.0].26 The Δ15(16) double bond was assigned based on the HMBC correlations of H-15 (δH 5.65) with C-7 (δC 42.7) and C-9 (46.6) and of H-16 (δH 5.91) with C-13 (δC 39.2) and C-14 (74.7). A key correlation of 1-OCH3 with C-1 suggested the presence of a methoxy group at C-1. Further, the chemical shift of H-14β at δH 4.08 (t, 1H, J = 4.2 Hz) indicated the presence of a hydroxy group at C-14. Therefore, the structure of apetaldine E (5) was confirmed as shown in Figure 1. Compound 6 was obtained as a white, amorphous powder with a molecular formula of C32H44N2O7 based on its HRESIMS data. Its NMR data were similar to those of 1 except for the absence of the N-ethyl and acetoxy groups. In addition, the 1H NMR signal at δH 4.06 and the 13C NMR signal at δC 75.5 suggested a hydroxy group at C-14 in 6. The tertiary carbon signal at δC 78.8 revealed that C-8 carried an ethoxy group, which was also supported by the HMBC correlations. Considering the above evidence, the structure of apetaldine F (6) was elucidated as shown in Figure 1. The HRESIMS data of compound 7 indicated a molecular formula of C32H42N2O7. The characteristic NMR data of 7 strongly suggested it to be a C19-diterpenoid alkaloid possessing two methoxy groups and an ethoxy group. Distinctive allylic signals at δH 7.36 (1H, d, J = 1.2 Hz) and δC (162.8 d) confirmed the presence of an imino group. Comparing the 13C NMR data of 7 and 6 showed that the chemical shifts of C-4, C-17, and C-19 in 7 were deshielded approximately ΔδC 11.2, 5.1, and 114.6 ppm, respectively. The deshielded H-19 signal in the NMR spectrum suggested that compound 7 was a rare imino-containing alkaloid with an NC (19)-H moiety similar to bulleyanitine A.29 The structure of apetaldine G (7) was confirmed by extensive analyses of its 1D and 2D NMR spectra. The antifeedant activity of compounds 1−10, 15, 16, and 18−25 was tested against S. exigua (Hübner) Linne (Table 3) using azadirachtin A as a positive control. Chasmanthinine (19) showed the strongest antifeedant activity, with a low EC50 (effective concentration for 50% feeding reduction; 0.07 mg/ cm2). Compounds 1, 5, 18, 23, and 24 also displayed higher potencies (EC50 values of 0.45, 0.28, 0.20, 0.18, and 0.41 mg/ cm2) than those of compounds 2, 4, 6, 8, and 15 (EC50 values of 0.94, 0.64, 0.68, 0.76, and 0.66 mg/cm2). Interestingly, alkaloids 7, 9, 16, and 20 (EC50 values of 9.23, 5.65, 60, and 50 mg/cm2) were inactive against S. exigua (Hübner). Based on comprehensive analysis of the structural features and antifeedant activities, the following SAR of these antifeedant diterpenoid alkaloids was determined. (1) An amine (−NH−) moiety makes a greater contribution to the antifeedant activity

and the carbonyl carbon (δC 171.5) suggested that the acetoxy group was connected at C-14 (δC 75.8). In addition, the locations of the methoxy groups at C-1 and C-16 and the ethoxy group at C-8 were confirmed by analysis of the HMBC correlations. The relative configuration of compound 1 shown in Figure 3 was deduced from the NOESY and 1H−1H COSY connections.

Figure 3. Key NOESY correlations of compound 1.

The NOESY cross-peak between H-16/H-17 indicated the αorientation of H-16.25 Additionally, the cross-peak between H5/H-10 showed that H-10 is β-oriented. Furthermore, the characteristic protons signals (δH 3.12, 1H, dd, J = 10.2, 7.2 Hz; 4.72, 1H, t, J = 4.8 Hz) could be assigned to H-1β and H-14β.26 Since the absolute configuration of the aconitine-type skeleton was repeatedly confirmed by the X-ray crystallographic analysis of analogues isolated from species of the same genus,27,28 it is proposed that the absolute configuration of this skeleton was retained in 1. Therefore, the structure and absolute configuration of apetaldine A (1) was defined as shown in Figure 1. The molecular formula of apetaldine B (2) was determined as C34H46N2O8 based on the HRESIMS ion at m/z 611.3322 [M + H]+ (calcd for C34H47N2O8, 611.3332). Its 13C NMR data showed 34 carbon signals (Table 1), and its 1H NMR data revealed an N-ethyl group [δH 1.08 (3H, t, J = 7.2 Hz)], an acetoxy group [δH 2.04 (3H, s)], two methoxy groups (δH 3.23, 3.28, each 3H, s), and an o-acetamidobenzoate group. The NMR data for 2 were similar to those for 1 except that the ethoxy group in 1 was replaced by a hydroxy group in 2 at C-8, which was supported by the HMBC correlations of H-6, H-9, and H-14 with C-8. In addition, the methoxy groups were assigned at C-1 (δC 85.5) and C-16 (δC 81.7) based on the HMBC correlations of the methoxy proton signals with C-1 and C-16. The 1H−1H COSY correlations of H-1/H-2α and H15α/H-16 also supported the location of the methoxy groups. Accordingly, the observation of an HMBC cross-peak between H-18α and C-7′ suggested the location of the o-acetamidobenzoate group at C-18. Thus, the structure of apetaldine B (2) was determined as shown in Figure 1, and the full assignment of its spectroscopic data was achieved based on 1D and 2D NMR analyses. HRESIMS data implied the molecular formula of 3 to be C34H44N2O8 (m/z 609.3174 [M + H]+). Two methoxy groups (δH 3.28, 3.32, each 3H, s), an N-ethyl group [δH 1.12 (3H, t, J = 7.2 Hz)], an acetoxy group [δH 1.97 (3H, s)], and an oacetamidobenzoate group were observed in its 1H NMR data. The spectroscopic data also suggested that this compound is structurally similar to 1 apart from the replacement of the 8ethoxy and 14-acetoxy groups in 1 with acetoxy and carbonyl groups in 3, respectively. This replacement was supported by 3138



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Table 1. 1H NMR Spectroscopic Data for Compounds 1−7 (600 MHz, CDCl3, δH in ppm, J in Hz) position 1 2a 2b 3a 3b 5 6a 6b 7 9 10 12a 12b 13 14 15a 15b 16 17 18a 18b 19a 19b 21a 21b 22 1-OMe 8-OEt 8-OEt 8-OEt 8-OAc 14-OAc 16-OMe NH 3′ 4′ 5′ 6′ 9′ a

1 3.12 2.05 2.32 1.47 1.85 1.66 1.39 2.01 2.40 2.38 2.40 1.91 2.43 1.96 4.72 1.99 2.13 3.24 2.84 3.99 4.08 2.10 2.60 2.36 2.52 1.07 3.32 3.33 3.36 1.06

dd (10.2, 7.2) m m br t (13.2) m d (7.2) dd (15.0, 7.2) m m t (5.4) m m m m t (4.8) m m m br s ABq (11.4) ABq (11.4) ABq (11.4) ABq (11.4) m m t (7.2) s m m t (7.2)

2 3.15 2.06 2.31 1.48 1.86 1.70 1.54 1.96 2.13 2.34 2.63 1.85 2.15 1.87 4.82 1.83 2.43 3.19 3.05 3.96 4.10 2.13 2.63 2.41 2.56 1.08 3.28

dd (10.8, 6.6) m m br t (12.6) m d (7.2) dd (15.0, 7.2) dd (15.0, 7.2) d (7.2) t (6.0) m m m m t (4.8) m m m br s ABq (10.8) ABq (10.8) ABq (13.8) ABq (13.8) m m t (7.2) s

3 3.24 dd (10.8, 6.6) 2.07 m 2.34 m 1.44 m 1.88 m 1.75 d (7.8) 1.50 m 1.99 m 1.96 m 2.42 t (6.0) 3.35 m 2.05 m 2.20a 2.53 m 2.46 2.61 3.80 3.57 3.99 4.04 2.20 2.64 2.43 2.62 1.12 3.32

m m td (5.4, 1.8) br s ABq (10.8) ABq (10.8) ABq (11.4) ABq (11.4) m m t (7.2) s

4 3.78 1.59 1.68 1.53 1.92 1.93 1.60 1.98 2.52 2.13 2.39 1.91 2.50 2.45 4.13 2.15 2.22 3.37 2.67 4.00 4.14 2.21 2.25 2.46 2.58 1.12

5

br s m m m m d (7.8) m m m t (6.0) m m m m t (4.2) m m m br s ABq (10.8) ABq (10.8) m m m m t (7.2)

3.14 1.95 2.39 1.48 1.87 1.68 1.57 2.01 2.13 2.29 1.97 1.88 2.48 2.45 4.08 5.65

dd (10.2, 6.6) m m br t (12.6) m d (7.2) dd (14.4, 7.2) dd (14.4, 7.2) d (7.2) t (5.4) m m m m t (4.2) d (8.4)

5.91 2.99 3.97 4.13 2.15 2.61 2.41 2.48 1.05 3.26

dd (8.4, 7.2) br s ABq (11.4) ABq (11.4) ABq (11.4) ABq (11.4) m m t (7.2) s

3.44 m 3.46 m 1.16 t (7.2)

6

7

3.31 m 1.72 m 1.90 m 1.68 m 1.87 m 1.85 d (7.2) 1.66 m 1.98 dd (15.0, 7.2) 2.37 d (7.2) 2.15 t (6.0) 1.92 m 1.68 dd (14.4, 7.8) 1.87 m 2.34 m 4.06a 2.05 dd (14.5, 6.0) 2.38 dd (14.5, 6.0) 3.28a 3.22 br s 3.96 ABq (10.8) 4.06 ABq (10.8) 2.53 ABq (13.2) 2.88 ABq (13.2)

3.27 1.54 1.86 1.53 1.78 1.79 1.43 2.00 2.45 2.13 1.86 1.67 1.88 2.39 4.04 2.10 2.36 3.40 3.96 4.34 4.38 7.36

dd (10.2, 6.6) m m m m d (7.2) dd (14.4, 7.2) dd (14.4, 7.2) d (7.2) t (6.0) m dd (12.6, 4.8) m m dd (9.6, 4.8) m m m br s ABq (11.4) ABq (11.4) d (1.2)

3.37 3.41 3.43 1.12

3.36 3.28 3.38 1.08

s m m t (7.2)

s m m t (7.2)

1.97 s 2.01 s 3.27 s 11.02 s 8.69 d (7.8) 7.54 t (7.8) 7.09 t (7.8) 7.95 d (7.8) 2.22 s

2.04 s 3.23 s 11.04 s 8.70 d (7.8) 7.48 t (7.8) 7.01 t (7.8) 7.96 d (7.8) 2.23 s

3.28 s 11.00 s 8.71 d (7.8) 7.56 t (7.8) 7.10 t (7.8) 7.95 d (7.8) 2.24 s

3.40 s 11.01 s 8.71 d (7.8) 7.57 t (7.8) 7.10 t (7.8) 7.96 d (7.8) 2.24 s

11.05 s 8.71 d (7.8) 7.55 t (7.8) 7.10 t (7.8) 7.97 d (7.8) 2.24 s

3.29 s 11.02 s 8.71 d (7.8) 7.55 t (7.8) 7.08 t (7.8) 7.95 d (7.8) 2.24 s

3.23 s 11.00 s 8.70 d (7.8) 7.55 t (7.8) 7.08 t (7.8) 7.96 d (7.8) 2.22 s

Indicates overlapped.

alkaloids will contribute to the development of potential botanical pesticides.

than an imine (−NC) moiety when the same parent alkaloids are de-ethylated. (2) Esterification and/or etherification of HO-8 and/or HO-14 in a C19-diterpenoid alkaloid enhance the antifeedant activity. (3) Elimination of the MeO16 group in a C19-diterpenoid alkaloid to form a Δ15(16) double bond improves the antifeedant activity. (4) Substituting with an anthranilic acid scaffold at C-18 of a C19-diterpenoid alkaloid also improves the activity. (5) Alkaloids substituted with a cinnamoyl group exhibited more potent activity than those substituted with a benzoyl group. (6) Oxygenation at C-13 strengthens the antifeedant potency. In summary, 25 structurally diverse diterpenoid alkaloids were isolated from the roots of two Aconitum species, and their antifeedant activities against S. exigua (Hübner) were evaluated for the first time. Chasmanthinine (19) exhibited significantly higher antifeedant activity than the other alkaloids, suggesting it as a new lead compound for further investigation. The seven new structural diterpenoid alkaloids identified from this study and SAR of antifeedant activity among these diterpenoid

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in CHCl3 using a PerkinElmer polarimeter with a sodium lamp operating at 598 nm and 20 °C. IR spectra were obtained using a Thermo Fisher Nicolet 6700 spectrometer. HRESIMS data were measured using a Q-TOF micro mass spectrometer (Waters). NMR spectra were recorded on a Bruker AV 600 spectrometer. Plant Material. A. apetalum was collected from Yili, Xinjiang, China, in August 2014 and was authenticated by Professor Qing-Er Yang of the Institute of Botany, Chinese Academy of Sciences. A voucher specimen (C. Ren & L. Wang 748) was deposited at the School of Life Science and Engineering, Southwest Jiaotong University, Sichuan, China. For A. f ranchetii var. villosulum, see ref 21. Extraction and Isolation. The powdered roots of A. apetalum (4.3 kg) were extracted with 95% EtOH (3 × 20 L) at room temperature for 4 days. The crude extract (360 g) was suspended in H2O, the pH was adjusted to 3.0 using 10% HCl solution, and the suspension was extracted with light petroleum (4 × 2 L) and EtOAc 3139



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Table 2. 13C NMR Spectroscopic Data for Compounds 1−7 (150 MHz, CDCl3, δC in ppm) position

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21 22 1-OCH3 8-COCH3 8-COCH3 8-OCH2CH3 8-OCH2CH3 14-COCH3 14-COCH3 16-OCH3 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′

85.3 26.4 32.8 38.1 45.9 24.4 41.1 76.9 43.2 38.3 49.2 29.1 45.2 75.8 36.3 83.5 61.5 70.9 52.8 49.3 13.6 56.4

2 d t t s d t d s d d s t d d t d d t t t q q

85.5 26.2 32.9 38.2 46.1 25.3 46.4 73.7 45.5 35.5 49.0 28.6 45.0 77.0 41.0 81.7 62.1 70.8 52.8 49.4 13.7 56.5

55.7 t 16.4 q 171.5 s 21.5 q 56.4 q 114.9 s 141.8 s 120.5 d 134.8 d 122.5 d 130.6 d 168.3 s 169.1 s 25.6 q

3 d t t s d t d s d d s t d d t d d t t t q q

4

85.7 d 25.8 t 32.8 t 38.4 s 46.0 d 25.5 t 43.0 d 92.1 s 52.6 d 41.7 d 49.0 s 24.9 t 46.6 d 215.1 s 31.9 t 86.1 d 63.2 d 70.4 t 52.7 t 49.4 t 13.7 q 56.4 q 170.5 s 22.7 q

72.1 29.7 30.1 36.9 41.7 24.3 43.9 79.0 45.4 40.0 49.1 26.7 40.2 75.7 38.1 83.2 63.4 70.5 56.4 48.5 13.1

1 2 3 4 5 6 7 8 9 10 15

EC50 (mg/cm2) (95% confidence limits) 0.45 0.94 1.18 0.64 0.28 0.68 9.23 0.76 5.65 1.75 0.66

(0.29, (0.46, (0.68, (0.36, (0.12, (0.39, (5.12, (0.54, (3.26, (1.14, (0.38,

0.71) 1.91) 2.04) 1.14) 0.63) 1.19) 16.62) 1.07) 9.80) 2.70) 1.14)

85.6 d 26.2 t 33.0 t 38.3 s 46.0 d 24.0 t 42.7 d 74.4 s 46.6 d 46.0 d 48.7 s 33.3 t 39.2 d 74.7 d 132.0 d 130.0 d 63.0 d 71.1 t 53.0 t 49.5 t 13.6 q 56.5 q

56.4 t 16.3 q 170.9 s 21.5 q 56.3 q 114.9 s 141.8 s 120.6 d 134.9 d 122.5 d 130.6 d 168.3 s 169.2 s 25.7 q

56.4 q 114.8 s 141.9 s 120.7 d 135.0 d 122.6 d 130.6 d 168.3 s 169.2 s 25.7 q

56.7 q 114.8 s 141.9 s 120.7 d 135.1 d 122.7 d 130.5 d 168.5 s 169.2 s 25.7 q

compd 16 18 19 20 21 22 23 24 25 azadirachtin A

EC50 (mg/cm2) (95% confidence limits) ≈60 0.20 0.07 ≈50 2.09 1.79 0.18 0.41 3.32 0.02

(0.08, 0.47) (0.03, 0.18) (1.29, (1.25, (0.10, (0.09, (2.37, (0.01,

115.0 s 141.9 s 120.6 d 134.9 d 122.6 d 130.6 d 168.3 s 169.3 s 25.7 q

6 83.2 25.0 28.7 37.8 43.1 24.5 47.3 78.8 45.2 45.2 49.3 28.7 40.1 75.5 36.3 82.7 57.5 70.8 48.2

7 d t t s d t d s d d s t d d t d d t t

83.3 d 24.7 t 27.4 t 49.0 s 42.0 d 24.3 t 48.1 d 77.4 s 44.8 d 45.5 d 48.9 s 28.5 t 38.5 d 75.3 d 33.8 t 82.1 d 62.6 d 67.7 t 162.8 d

56.6 q

56.6 q

56.4 t 16.3 q

56.2 t 16.2 q

56.0 q 114.8 s 141.9 s 120.6 d 135.0 d 122.6 d 130.5 d 168.4 s 169.2 s 25.7 q

56.4 q 114.5 s 141.9 s 120.6 d 135.1 d 122.6 d 130.5 d 168.1 s 169.1 s 25.6 q

fractions (Frs.) A1 (900 mg), A2 (5.5 g), A3 (3.2 g), A4 (4.6 g), A5 (4.5 g), A6 (4.3 g), A7 (1.5 g), and A8 (4.2 g) based on TLC analyses. Fr. A1 was loaded onto a silica gel column and eluted with light petroleum− acetone−Et2NH (15:1:0.1 to 0:1:0.1) to obtain compounds 1 (28 mg), and 3 (36 mg). Fr. A2 was separated on a silica gel column (light petroleum−acetone−Et2NH, 9:1:0.1 to 0:1:0.1) to afford Frs. B1 (100 mg), B2 (110 mg), B3 (165 mg), B4 (130 mg), B5 (370 mg), B6 (312 mg), B7 (146 mg), and B8 (194 mg). Compounds 2 (35 mg), 4 (23 mg), and 8 (27 mg) were obtained by purifying Fr. B3 by silica gel column chromatography (light petroleum−acetone−Et2NH, 8:1:0.1 to 0:1:0.1). Frs. B5 and B7 were separated on silica gel (light petroleum− acetone−Et2NH, 12:1:0.1 to 0:1:0.1) to afford compounds 5 (24 mg), 6 (31 mg), 14 (9.2 mg), and 15 (22.5 mg). Fr. A4 was isolated on a silica gel column and eluted with light petroleum−acetone−Et2NH (6:1:0.1 to 0:1:0.1) to give compounds 10 (10 mg), 16 (28 mg), and 11 (6 mg). Repeated chromatography of Frs. A5 and A6 on a silica gel column (CHCl3−MeOH, 40:1 to 0:1) yielded compounds 7 (28 mg), 9 (24 mg), 12 (5.6 mg), 13 (7 mg), and 17 (8.4 mg). A. f ranchetii var. villosulum. The total alkaloid extracts (100 g) were chromatographed on a silica gel column, eluting with a CH2Cl2− MeOH gradient system (100:1 to 1:100), to afford Frs. A−F (in ref 21). Fr. C (8 g) was loaded onto a silica gel column and eluted with light petroleum−acetone−Et2NH (20:1:0.1 to 0:1:0.1) to furnish Frs. C1−C3. Frs. C2 and C3 were separated on a silica gel column (light

Table 3. Antifeedant Activities of the Compounds against Spodoptera exigua (Hübner) (n = 3) compd

5 d t t s d t d s d d s t d d t d d t t t q

3.39) 2.57) 0.33) 2.10) 4.67) 0.07)

(4 × 2 L), successively. The pH of the aqueous layer was adjusted to 9.4 using an aqueous ammonia solution and was extracted with CH2Cl2 (4 × 2 L) to obtain the alkaloid extract (40 g). The alkaloid extract (40 g) was chromatographed on a silica gel column, eluting with a CH2Cl2−MeOH gradient system (80:1 to 0:1), to afford 3140



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petroleum−acetone−Et2NH, 25:1:0.1 to 0:1:0.1) to yield compounds 21 (21 mg), 22 (38 mg), and 23 (26 mg). Apetaldine A (1): white, amorphous powder; [α]20 D −15 (c 0.5, CHCl3); IR (KBr) νmax 3318, 2968, 2924, 1686, 1589, 1527, 1448, 1296, 1090, 758 cm −1; 1H NMR (600 MHz, CDCl3) data, see Table 1; 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS at m/z 639.3649 [M + H]+ (calcd for C36H51N2O8, 639.3645). Apetaldine B (2): white, amorphous powder; [α]20 D +25 (c 0.5, CHCl3); IR (KBr) νmax 3317, 2963, 2925, 1704, 1686, 1526, 1261, 1090, 757 cm−1; 1H NMR (600 MHz, CDCl3) data, see Table 1; 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS at m/z 611.3322 [M + H]+ (calcd for C34H47N2O8, 611.3332). Apetaldine C (3): white, amorphous powder; [α]20 D +14 (c 0.2, CHCl3); IR (KBr) νmax 3319, 2960, 2923, 1757, 1731, 1687, 1589, 1448, 1261, 1240, 1090, 758 cm−1; 1H NMR (600 MHz, CDCl3) data, see Table 1; 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS at m/z 609.3174 [M + H]+ (calcd for C34H45N2O8, 609.3176). Apetaldine D (4): white, amorphous powder; [α]20 D +5 (c 0.4, CHCl3); IR (KBr) νmax 3319, 2924, 2853, 1686, 1589, 1526, 1463, 1296, 1088, 758 cm−1; 1H NMR (600 MHz, CDCl3) data, see Table 1; 13 C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS at m/z 583.3382 [M + H]+ (calcd for C33H47N2O7, 583.3383). Apetaldine E (5): white, amorphous powder; [α]20 D −9 (c 0.2, CHCl3); IR (KBr) νmax 3318, 2925, 2815, 1686, 1589, 1526, 1448, 1296, 1091, 757 cm−1; 1H NMR (600 MHz, CDCl3) data, see Table 1; 13 C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS at m/z 537.2965 [M + H]+ (calcd for C31H41N2O6, 537.2965). Apetaldine F (6): white, amorphous powder; [α]20 D +12 (c 0.5, CHCl3); IR (KBr) νmax 3319, 2925, 2856, 1686, 1589, 1526, 1448, 1296, 1088, 758 cm−1; 1H NMR (600 MHz, CDCl3) data, see Table 1; 13 C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS at m/z 569.3232 [M + H]+ (calcd for C32H45N2O7, 569.3227). Apetaldine G (7): white, amorphous powder; [α]20 D +45 (c 0.7, CHCl3); IR (KBr) νmax 3320, 2926, 2821, 1688, 1589, 1526, 1448, 1296, 1239, 758 cm −1; 1H NMR (600 MHz, CDCl3) data, see Table 1; 13C NMR (150 MHz, CDCl3) data, see Table 2; HRESIMS at m/z 567.3071 [M + H]+ (calcd for C32H43N2O7, 567.3070). Antifeedant Bioassays. The S. exigua (Hübner) colony (Henan Jiyuan Baiyun Industry Co., Ltd.) was reared on cabbage foliage and maintained at 24 ± 1 °C and >70% relative humidity with a photoperiod of 16:8 h (L:D) in a growth chamber. The antifeedant properties of the test compounds were evaluated using the choice leaf-disc method.30,31 The test compounds and positive control were dissolved in acetone, and the concentrations were adjusted to 1000 mg/mL and then diluted to 500, 250, 125, and 62.5 mg/mL. The choice experiments were conducted with newly emerged third-instar larvae of S. exigua (Hübner). Fresh cabbage leaves were cut into leaf discs (2 cm diameter) and treated on the upper surface with 15 μL of the test substance emulsions or deionized water containing acetone and Tween-20 as a control. All the leaves were airdried. Two leaves from the treated group and another two leaves form the control group were alternately placed in a Petri dish (15 cm in diameter) that contained a 2% agar bed with a diameter of 2−3 mm. Four healthy and starved 6 h instars were placed in each dish. In addition, the two sets of leaves were fed separately in the above laboratory environment. Each treatment had three replicates. When 50−70% of the leaves had been consumed, feeding was stopped, as shown in Figure 4. The area consumed was determined using an LI3000 portable area meter (American Lincoln Co. Ltd.), and the percentage of food reduction (%FR) in each dish was determined using the equation %FR = (CK − T)/CK × 100 (CK is the control leaf disc area eaten and T is the treated leaf disc area eaten). Compounds with %FR > 50% were tested in a dose−response experiment to calculate their relative potency (EC50) by linear regression analysis (%FR of the logarithmic concentration).

Figure 4. Antifeedant activities against Spodoptera exigua (Hübner).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00380. 1D and 2D NMR spectra for compounds 1−7 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-28-66367232. Fax: +86-28-66367260. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Xian-Li Zhou: 0000-0002-1690-0578 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grants from NSFC (81773605), the Research Foundation for the Educational Commission of Sichuan Province (15TD0048), the Science and Technology Innovation Seedling Project of Sichuan Province (2015014), and the Interdisciplinary Frontier Basic Research Project of SWJTU (2682017QY04).

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REFERENCES

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