1. Introduction

Aconitum nagarum var. heterotrichum, called 'xiao bai cheng' in Chinese, is a variant of A. nagarum. This variety is distinguished from the typical one with the indumentum of dense spreading yellowish glandular hairs on both the inflorescence axis and the pedicel, mainly distributing in Dengchuan, Gongshan and Qiaojia etc. in Yunnan Province . Its roots are highly toxic, and also can be used for treating lumbar muscle strain, rheumatism and other diseases by natives [2]. Previous phytochemical investigations of this plant revealed that C₁₉-diterpenoid alkaloids were the primary constituents [3-5]. As a part of an ongoing phytochemical investigation of A. nagarum var. heterotrichum, four new diterpenoid alkaloids, named nagaconitines A-D (1-4, Fig. 1), were isolated from the roots of this medicinal plant. Moreover, their activities against cancer cell line SK-OV-3 were measured in vitro, and compounds 3-4 showed antitumor effect. This paper described their isolation, structural elucidation and antitumor results.

2. Results and discussion

Nagaconitine A (1) was isolated as a colorless powder and showed positive reaction with the Dragendorff's reagent. The molecular formula of 1 was determined to be C₃₆H₅₁NO₁₂ based on ESI-MS ([M + H]⁺; m/z 690) and HR-ESIMS (m/z 690.3484 [M + H]⁺, calcd. for 690.3478). IR spectrum showed the absorption for hydroxyl (3482, 3476 cm^-1), conjugated ester carbonyl (1721 cm^-1) and aromatic ring (1631, 1452 cm^-1). The ¹H and ¹³C NMR data displayed the presence of one N-ethyl group (δ_H 2.35, 2.86, m, each 1H; 1.07, t, 3H, J = 7.1 Hz; δ_C 46.9 t, 13.3 q), one benzoyl group (δ_H 8.02, d, 2H, J = 7.2 Hz; 7.57, dd, 1H, J = 7.2, 7.2 Hz; 7.45, dd, 2H, J = 7.2, 7.2 Hz; δ_C 165.9 s, 133.3 d, 129.7 d, 129.7 s, 128.7 d) and four OMe groups (δ_H 3.75, 3.28, 3.24, 3.15, each 3H, s; δ_C 61.3, 59.0, 58.2, 55.9 q×4). Compound 1 revealed 36 resonances in the ¹³C NMR (DEPT) spectrum (Table 1) attributing to 5 methyls, 8 methylenes, 16 methylidynes and 7 quaternary C-atoms, suggesting that compound 1 might be a C₁₉- diterpenoid alkaloid [7, 8], bearing four OMe groups, one NCH₂CH₃, one OBz, and one OCO (CH₂) ₃OH (4-hydroxylbutyryl) unit. The 4- hydroxylbutyryl unit was confirmed by the cross-peaks between the signal at δ_H 1.56, 1.92 (each 1H, m, H-2') and C-1' (δ_C 175.0 s), C- 3' (δ_C 27.0 t), and C-40 (δ_C 61.5 t) in the HMBC spectrum (Fig. 2). The 1H and ¹³C NMR data (Tables 1 and 2) of compound 1 were almost the same as those of aconitine [9] except that the 4-hydroxylbutyryl unit signal appeared in compound 1 instead of an OAc group, suggesting that there was only one 4-hydroxylbutyryl group at C-8. The 2D NMR spectra including HMQC, HMBC, COSY and ROESY resulted in the assignments of all the protons and carbon atoms of compound 1 (Tables 1 and 2).

Compound 1 had the same relative configuration as aconitine, based on their almost same ¹H NMR and ¹³C NMR data, and ROESY spectrum (Fig. 3). Thus, the structure of compound 1 was determined as 8- (4'-hydroxylbutyryl) aconitine, named Nagaconitine A (1).

Nagaconitine B (2) had a molecular formula of C₃₂H₄₃NO₉ in agreement with ESI-MS ([M + H]⁺ m/z 586) and HR-ESIMS (586.3011 [M + H]⁺, calcd. for 586.3007) analyses. IR bands exhibited hydroxyl (3441 cm^-1), romatic ring (1632, 1452 cm^-1) and carbonyl (1714 cm^-1) functions. In the ¹H NMR spectra, four OMesinglets (δ_H 3.76, 3.30, 3.25, 3.25, s, each 3H), an N-ethyl group (δ_H 2.40, m; 2.88, m; 1.05, t, J = 7.1 Hz, N-CH₂CH₃) and a benzoyl group (δ_H 7.97, d, J = 7.2 Hz; 7.61, dd, J = 7.2, 7.2 Hz; 7.45, dd, J = 7.2, 7.2 Hz) were observed, which exhibited characteristic features of an aconitine type C₁₉-diterpenoid alkaloid. Comparing the ¹H and ¹³C NMR spectral data (Tables 1 and 2) of compound 2 with those of 13-deoxyludaconitine [10] showed high similarity except that a C = O signal at δ_C (211.7 s) at C-15 appeared in 2, and the chemical shift due to C-15 was downfield shifted from δ_C 41.4 (t) to δ_C 211.7 (s), indicating that there was an C = O group located at C-15. This was futher confirmed by the long-rang correlations between H-16 (δ_H 3.84, and C-13 (δ_C 38.7 d), and C-15 (δ_C 211.7 s) in the HMBC spectrum. Accordingly, compound 2 was established to be 15-oxo-13-deoxyludaconitine.

Nagaconitine C (3) was elucidated as C₂₈H₄₃NO₉ by ESI-MS ([M + H]⁺ m/z 538) and HR-ESIMS (538.3011 [M + H]⁺, calcd. for 538.3007) experiments. The ¹H NMR and ¹³C NMR data displayed the signals of three OMe groups at δ_H 3.31, 3.31, 3.22 (each s, 3H), one N-ethyl signalat δ_H 1.13 (t, J = 7.2 Hz) and twoOAcgroupsat δ_H 2.13, 1.92 (each 3H, s). Careful analyses of the ¹H and ¹³C NMR spectral data suggested that the structrue of compound 3 was similar to that of delphisine [11]. The main diference between the two compounds was that compound 3 contained one OH group at C-15. The long-range HMBC spectrum (Fig. 2) from H-15 (δ_H 5.14, 1H, d, J = 8.5 Hz) to C-8 (δ_C 74.2 s) and C-16 (δ_C 81.7 d) confirmed the location of the OH group. The correlations between H-16, assumed to be a-oriented with reference to the aconitine type C₁₉- diterpenoid alkaloid [12], and H-15 were not observed in the ROESY spectrum, indicating the β-orientation of H-15. Hence, Nagaconitine C (3) was elucidated as 15-hydroxyldelphisine (3).

Nagaconitine D (4) was obtained as a colorless powder with a molecular formula C₂₄H₃₁NO₆ deduced from its ESI-MS ([M + H]⁺ m/z 430) and HR-ESIMS (430.2224 [M + H]⁺, calcd. for 430.2221) analyses. The ¹H NMR spectral of 4 exhibited three Me signals for a quaternary Me group (δ_H 1.41, s, H-18) and two OAc groups (δ_H 2.06, 2.03, s× 2). Its ¹³C NMR spectral data (Table 1) exhibited 24 C-atom signals, indicating that compound 4 might be a C₂₀-diterpenoid alkaloid [13], bearing two OAc groups. The ¹H and ¹³C NMR data (Tables 1 and 2) of compound 4 were almost identical with those of spiradine C [14] except that an OH group and an OAc signal appeared in compound 4. The OAc group was assigned to C-2 according to the HMBC spectrum in which the cross-peaks between H-2 (δ_H 5.22, br. s) and C-1 (δ_C 27.8 t), C-3 (δ_C 38.7 t), and C = O (δ_C 169.7 s) emerged. Moreover, the OH group locating at C-14 was also confirmed by the HMBC correlation between H-20 (δ_H 3.23 br. s) and H-13 (δ_H 2.00, 2.41, dd, J = 9.3, 5.4 Hz), and C-14 (δ_C 78.5 s) (Fig. 2). Consequently, Nagaconitine C (4) was defined as 14-hydroxyl-2-acetoxyspiradine C (4).

Compounds 1-4 were tested against human ovarian cancer cell line SK-OV-3 (Table 3). As a result, compounds 3-4 showed significant cytotoxicity against the tested cell line with inhibition of 31% (IC₅₀ 43.78 mmol/L) and 24% (IC₅₀ 32.14 μmol/L), respectively, as compared to the reference drug cisplatin (89%) with the IC₅₀ values of 11.58 μmol/L.

3. Conclusion

Four new diterpenoid alkaloids, nagaconitines A-D (1-4), were isolated from the roots of Aconitum nagarum var. heterotrichum. Their structures were elucidated by extensive spectroscopic examinations. Compounds 3-4 showed inhibition of cancer cell line SK-OV-3.

4. Experimental 4.1. General experimental procedures

Optical rotations were determined on a Jasco model 1020 polarimeter (Horiba, Tokyo, Japan). UV spectra were measured on a Shimadzu UV2401PC spectrophotometer (Shimadzu, Kyoto, Japan). IR (KBr discs, cm^-1) spectra were recorded on a Bio-Rad FTS-135 spectrometer (Bio-Rad, Hercules, California, USA). 1D and 2D NMR were recorded on Bruker AM-400, Bruker DRX-500 or AVANCE III-600 spectrometers (Bruker, Bremerhaven, Germany). Mass spectra were run on a VG Spec-3000 spectrometer (VG, Manchester, UK) and Waters AutoSpec Premier P776 (Waters, USA). Silica gel (200-300 mesh) for column chromatography and TLC plates (GF₂₅₄) were obtained from Qingdao Makall Chemical Company (Makall, Qingdao, China). Al₂O₃ for column chromatography was purchased from Shanghai Wusi Chemical Reagents Company, Ltd. (Wusi, Shanghai, China). Fractions were visualized by silica gel plates sprayed with dragendorff0s reagent.

4.2. Plant material

The roots of Aconitum nagarum var. heterotrichum were collected in Weixi county of Yunnan Province, China, in September 2014, and authenticated by Prof. Qing-Er Yang from South China Botanical Garden, Chinese Academy of Sciences. A voucher specimen (No. YAU20140906) had been deposited in the College of Agriculture and Biotechnology, Yunnan Agricultural University.

4.3. Extraction and isolation

The air-dried roots of A. nagarum var. heterotrichum (11 kg) were powdered and extracted three times with MeOH for 2 h under reflux. The crude extract was dissolved with 8 L 2% HCl solution, then basified to pH 9.0 with ammonia (25%) and extracted with CHCl₃ to obtain crude alkaloidal extract (256 g). The alkaloidal extract was performed on silica column chromatography (SiO₂ CC, 1500 g, 12×100 cm), eluting with petroleum ether (30-60℃) -acetone-diethylamine (30:1:1, 20:1:1, 20:3:1, 20:6:1, 20:10:1, 10:20:1, v/v) to afford six fractions (A-F). Fr. C (43 g) was further separated to obtain five subfractions (C1-C5) by SiO₂ CC with petroleum ether-acetone-diethylamine (20:1:1, each 500 mL). Fr. C2 (8.7 g) was performed on Al₂O₃ CC (2.0×35 cm, 30 g) and eluted with petroleum ether-EtOAc (6:1, each 100 mL) to yield compound 2 (25 mg). Fr. C3 (3.6 g) was applied to Al₂O₃ CC (4×40 cm, 140 g) with an eluent of petroleum ether-EtOAc (5:1, each 50 mL), and then purified through Sephadex LH - 20 (CHCl₃/ MeOH, 1:1) to produce compounds 3 (63 mg) and 4 (37 mg). Fr. D (39 g) was further separated to obtain four subfractions (D1-D4) by Si CC with petroleum ether-acetone-diethylamine (15:3:1, v/v, each 500 mL) as the eluent. Fr. D2 (8.2 g) was applied to Al₂O₃ CC (5.0×45 cm, 320 g) with an eluent of petroleum ether-EtOAc (2:1, each 150 mL), and further purified through Sephadex LH-20 (CHCl₃/MeOH, 1:1) to provide compound 1 (43 mg). All obtained compounds had a degree of purity > 90%, based on the TLC method in three different solvent systems exhibiting one spot with dragendorff0s reagent, and NMR spectra with the smooth baseline and no impurity peak.

Nagaconitine A (1) : Colorless powder; [α]_D²⁵ : + 2.00 (c 0.10, MeOH); UV (MeOH) : 230 (4.08) nm; IR (KBr, cm^-1) v_max: 3482, 3476, 2931, 1721, 1631, 1452, 1098, 712. ¹H NMR and ¹³C NMR data see Tables 1 and 2. HR-ESIMS at m/z 690.3484 [M + H]⁺ (calcd. for C³⁶H⁵²NO¹², 690.3478).

Nagaconitine B (2) : Colorless powder; [α]_D²⁵ :-72.4 (c 0.10, MeOH); UV (MeOH) : 230 (4.10) nm; IR (KBr, cm^-1) v_max: 3441, 2930, 1714, 1632, 1452, 1100, 714. ¹H NMR and ¹³C NMR data see Tables 1 and 2. HR-ESIMS at m/z 586.3011 [M + H]⁺ (calcd. for C₃₂H₄₄NO₉, 586.3007).

Nagaconitine C (3) : Colorless powder; [α]_D²⁵ : + 48.47 (c 0.10, MeOH); UV (MeOH) : 202 (3.28) nm; IR (KBr, cm^-1) v_max: 3454, 2938, 1740, 1105. ¹H NMR and ¹³C NMR data see Tables 1 and 2. HR-ESIMS at m/z 538.3011 [M + H]⁺ (calcd. for C₂₈H₄₄NO₉, 538.3007).

Nagaconitine D (4) : Colorless powder; [α]_D²⁵ :-13.73 (c 0.10, MeOH); UV (MeOH) : 202 (3.73) nm; IR (KBr, cm^-1) v_max: 3468, 3434, 2944, 1737, 1029. ¹H NMR and ¹³C NMR data see Tables 1 and 2. HR-ESI-MS at m/z 430.2224 [M + H]⁺ (calcd. for C₂₄H₃₂NO₆, 430.2221).

4.4. Antitumor assay in vitro

Compounds 1-4 were tested for their acitvities against human ovarian cancer cell line SK-OV-3 by the MTT method [6]. Briefly, 100 mLofcellsuspension (5×10⁴ cells/mL) wasseededinto96-well microtiter plates and cultured for 24 h before the compound was added. Then, differentconcentrations of compounds 1-4 were added to the plates, the cells were cultivated for 48 h, and 20 mL of MTT (5 mg/mL) was added to each well. After 4 h, the culture medium was removed and the formazan crystals were completely dissolved with 150 μL DMSO in each well by vigorously shaking the plate. Finally, the absorbance was measured at 570 nm on a Molecular Devices ELX-800 Microplate spectrophotometer (Omega Bio-Tek, Inc., Norcross, GA, USA). Cisplatin was used as a positive control.

Acknowledgment

This work was supported by the National Natural Science Foundation of China (Nos. 21362045, 81560622) and the project of Sciences and Technology Department of Yunnan Province (No. 2012CG007). The authors are grateful to the staff of analytical group at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences for the spectral data.