1. Introduction

The lateral roots of Aconitum carmichaelii Debx. (Ranunculaceae), named "fu zi" in Chinese, is an indispensable herbal medicine for the treatment of various diseases, such as cardianeuria, arrhythmia, neuralgia, rheumatalgia, and inflammations, in oriental countries [1-4]. Chemical and pharmacological investigations have resulted in isolation of more than a hundred of compounds from extracts of the principal and lateral roots (tubers) of A. carmichaelii, of which aconitane-type C₁₉-diterpenoid alkaloids were reported as the main active constituents [2-12]. However, in previous chemical studies organic solvents (benzene, CHCl₃, methanol, and ethanol) were mainly applied for extracting the plant materials including raw and prepared "fu zi" [2-11]. These extraction procedures differ from a practical utilization by decocting with water. Therefore, as part of a program to systematically study the chemical diversity of traditional Chinese medicines and their biological effects [13-33], we investigated an aqueous decoction of the raw lateral roots of A. carmichaelii. As results of our investigation, four new hetisan-type, three new napeline-type, and a new arcutine-type C₂₀-diterpenoid alkaloids, twenty-one new aconitane-type C₁₉-diterpenoid alkaloids, two new 2-(quinonylcarboxamino)benzoates, and seven new aromatic acid derivatives, along with solvent-/base-/acid-dependent transformation and equilibration between alcohol iminium and aza acetal forms of the napeline-type C₂₀-diterpenoid alkaloids, have been reported [34-38]. Described herein are isolation and structural characterization of four unprecedented aconitane-type C₁₉-diterpenoid alkaloid glycosides with isomeric L-arabinosyls (1-4) (Fig. 1), from the same decoction.

2. Results and discussion

Compound 1 was isolated as a colorless gum with [α]_D²⁰ -11.6 (c 0.69, MeOH). Its IR spectrum exhibited absorption bands assignable to hydroxyl (3356 cm^-1) groups. The molecular formula of 1 was elucidated as C₂₉H₄₇NO₁₀ by (+)-HR-ESIMS at m/z 570.3283 [M+H]⁺ (calcd. for C₂₉H₄₈NO₁₀, 570.3273), combined with NMR spectroscopic data (Table 1). The 1H NMR spectrum of 1 in MeOH-d₄ showed resonances attributable to an N-CH₂CH₃ unit at δ_H 3.30 and 3.16 (dq, 1H each, J = 12.6 and 7.2 Hz, H₂-20) and 1.37 (t, 3H, J = 7.2 Hz, H₃-21); three methoxy groups at δ_H 3.40 (s, OCH₃-6), 3.33 (s, OCH₃-16), and 3.31 (s, OCH₃-18); four oxymethines at δ_H 4.34 (brd, J = 6.6 Hz, H-6), 4.23 (dd, J = 4.2 and 4.8 Hz, H-14), 4.00 (brs, H-1), and 3.28 (m, H-16); one nitrogen-bearing methine at δ_H 3.25 (brs, H-17); an oxymethylene at δ_H 3.52 (2H, s, H₂-18); and a nitrogen-bearing methylene at δ_H 3.40 and 3.01 (d, 1H each, spectroscopic data analysis (Table 1). In the ¹H-¹H COSY spectrum of 1, homonuclear vicinal coupling correlations of H-1/H₂-2/H₂-3, H-5/H-6/H-7, H-14/H-9/H-10/H₂-12/H-13/H-14, H₂-15/H-16, and H₂-20/H₃-21 revealed five vicinal spin systems of the aglycone moiety (Fig. 2, thick lines). In the HMBC spectrum of 1, two-and three-bond correlations (Fig. 2, red arrows) from H₂-3 to C-4 and C-19; from H-5 to C-4, C-18, and C-19; from H₂-18 to C-3, C-4, C-5, and C-19; and from H₂-19 to C-3, C-4, C-5, and C-18 indicated a J = 12.0 Hz, H₂-19); as well as partially overlapped resonances due to four aliphatic methylenes (H₂-2, H₂-3, H₂-12, and H₂-15) and five aliphatic methines (H-5, H-7, H-9, H-10, and H-13) between δ_H 2.37 and 1.54. In addition, the spectrum showed typical signals for a pentose moiety, consisting of four oxygen-bearing methines at δ_H 4.38 (d, J = 7.2 Hz, H-1'), 3.61 (dd, J = 7.2 and 9.0 Hz, H-2'), 3.52 (dd, J = 3.0 and 9.0 Hz, H-3'), and 3.78 (m, H-4') and an oxygen-bearing methylene at δ_H 3.85 (dd, J = 3.0 and 12.6 Hz, H-5'a) and 3.56 (dd, J = 1.8 and 12.6 Hz, H-5'b). The ¹³C NMR and DEPT spectra showed 29 carbon signals (Table 1) corresponding to the above units and three quaternary carbons including an oxygen-bearing carbon at δ_C 74.8 (C-8). As compared with those of the alkaloids previously isolated from "fu zi", the spectroscopic data suggest that 1 is an unprecedented glycosidic C₁₉-diterpenoid alkaloid [12, 34-38], of which the structure was elucidated by 2D NMR data analysis.

The hydrogen resonances and corresponding hydrogen-bearing carbon resonances in the NMR spectra were assigned by HSQC spectroscopic data analysis (Table 1). In the ¹H-¹H COSY spectrum of 1, homonuclear vicinal coupling correlations of H-1/H₂-2/H₂-3, H-5/H-6/H-7, H-14/H-9/H-10/H₂-12/H-13/H-14, H₂-15/H-16, and H₂-20/H₃-21 revealed five vicinal spin systems of the aglycone moiety (Fig. 2, thick lines). In the HMBC spectrum of 1, two-and three-bond correlations (Fig. 2, red arrows) from H₂-3 to C-4 and C-19; from H-5 to C-4, C-18, and C-19; from H₂-18 to C-3, C-4, C-5, and C-19; and from H₂-19 to C-3, C-4, C-5, and C-18 indicated a linkage of the quaternary C-4 with C-3, C-5, C-18, and C-19. The HMBC correlation from H-7 to C-8, C-9, and C-15; from H-9 to C-7, C-8, and C-15; from H₂-15 to C-7, C-8, and C-9; together with their chemical shifts, demonstrated that the oxygen-bearing quaternary C-8 was linked to C-7, C-9 and C-15. The HMBC correlation from H-1 to C-10, C-11, and C-17; from H-5 to C-10, C-11, and C-17; from H-10 to C-1, C-5, C-11, and C-17; and from H-17 to C-5, C-10, C-11 indicated that the quaternary C-11 connected to C-1, C-5, C-10 and C-17. The connection between C-13 and C-16 was established by the HMBC correlations from H₂-12 to C-14 and C-16, from H-13 to C-15, from H-14 to C-16, and from H-16 to C-12 and C-14, though the vicinal coupling correlation between H-13 and H-16 was not observed in the ¹H-¹H COSY spectrum possibly due to a perpendicular dihedral angle between the two hydrogens. In addition, the HMBC correlations from H-6 to C-17; from H-17 to C-6, C-8, C-19, and C-20; from H₂-19 to C-17 and C-20; from H₂-20 to C-17 and C-19; together with their chemical shifts, demonstrated that C-17 connected with C-7 and via the nitrogen atom to both C-19 and C-20. The HMBC correlations from OCH₃-6 to C-6, from OCH₃-16 to C-16, and from OCH₃-18 to C-18 located the three methoxy groups at the corresponding carbons. Furthermore, the ¹H-¹H COSY spectrum of 1 exhibited the correlations of H-1'/H-2'/ H-3'/H-4'/H₂-5'. This, combined with the HMBC correlation from H-1' to C-5' and C-14, from H₂-5' to C-1', and from H-14 to C-1' demonstrated that there was a pentopyranosyloxy unit at C-14. The hydroxyl group at C-8 was deduced from the chemical shift of this carbon and the molecular formula requirement. Accordingly, the planar structure of 1 was determined as shown, wherein the planar structure of the aglycone is identical to the co-occurring neoline [39, 40].

In the NOESY spectrum of 1, the NOE correlations between H-1 with H-10 and H-12a, between H-3a and H₂-18, between H-5 with H-10 and H₂-18, between H-6 and H-9, between H-10 with H-12a and H-14, and between H-14 with H-9 and H-13 indicated that these hydrogens were oriented on the same side of the ring system (Fig. 3). The NOE correlations between H-3b with H-19b, between H-16 with H-12b and H₃-21, and between H-17 with H-12b and H₃-21 revealed that these hydrogens were oriented on the other side of the ring system. These NOE correlations indicated that the relative configuration of the aglycone is identical to neoline [39, 40]. Additionally, the NOE correlations between H-1' with H-3' and H-5'b, together with the coupling constant values of J_{1', 2'} (7.2Hz) and J_{3', 4'} (3.0Hz), demonstrated that the pentopyranosyloxy in 1 is an α-arabinopyranosyloxy [41], while the NOE correlations between H-1' and H-14 confirmed the location of the sugar unit. Altogether, 1 was deduced as neoline 14-O-α-arabinopyranoside, of which the absolute configuration was further assigned by acid hydrolysis. The aglycone and sugar, which were isolated from the hydrolysate of 1 by CC over Sephadex LH-20 (MeOH), displayed the 1H NMR spectroscopic features (Figs. S13-S17 in Supporting information) and the specific rotation values {aglycone:[α]_D²⁰ +21.8 (c 0.05, MeOH); sugar: [α]_D²⁰ +104.8 (c 0.01, H₂O)} well consistent with those of the authentic samples of neoline {[α]_D²⁰ +21.6 (c 0.16, MeOH)} and L-arabinose {[α]_D²⁰ +113.3 (c 0.29, H₂O)}. Especially the absolute configuration of neoline was previously determined by chemical transformation and X-ray crystallography [42-44]. Thus, the structure of compound 1 was determined as neoline 14-O-α-L-arabinopyranoside and named aconicarmichoside A.

Compound 2, a colorless gum with [α]_D²⁰ +46.3 (c 0.54, MeOH), exhibited spectroscopic features similar to those of 1. However, as compared with the NMR spectroscopic data of 1, the H-14 and C-13 resonances in 2 were shielded by Δδ_H -0.15 and Δδ_C -1.8, respectively, whereas the anomeric proton resonance (H-1') was significantly deshielded by Δδ_H +0.53 (Table 1). Additionally, the coupling constants of H-1' (J1', 2') was changed from 7.2Hz in 1 into 3.0Hz in 2. This suggests that the anomeric carbon (C-1') of the arabinopyranosyl in 2 has a b-configuration instead of the a-configuration in 1. The suggestion was supported by shielded shifts of the C-1', C-3', and C-5' resonances in the ¹³C NMR of 2 by Δδ_C -2.8, -2.8, and -2.0, respectively, as compared with those of 1. This was further confirmed by 2D NMR data analysis and acid hydrolysis of 2 (Figs. 2 and 3). Especially, the homonuclear vicinal coupling correlations of H-1'/H-2'/H-3'/H-4'/H₂-5' in the ¹H-¹H COSY spectrum and the long-range heteronuclear correlation from H-1' to C-5', from H-5' to C-1', from H-1' to C-14 and from H-14 to C-10 in the HMBC spectrum, together with the coupling constants of J_{2', 3'} (9.0Hz) and J_{3', 4'} (3.5Hz), proved the presence and location of the β-arabinopyranosyloxy in 2. Using the same method as described for 1, neoline {[[α]_D²⁰ +20.9 (c 0.08, MeOH)} and L-arabinose {[α]_D²⁰ +106.2 (c 0.03, H₂O)} was isolated and identified from the hydrolysate of 2. Therefore, the structure of compound 2 was determined as neoline 14-O-β-L-arabinopyranoside and named aconicarmichoside B.

Compound 3, a colorless gum with [α]_D²⁰ -20.6 (c 0.31, MeOH), is another isomer of 1 and 2 as indicated by spectroscopic data. Comparison of the NMR spectroscopic data of 1-3 (Table 1) demonstrated that the anomeric hydrogen and carbon of the pentosyl in 3 resonated with deshielded chemical shifts at δ_H 5.01 (H-1') and δ_C 109.5 (C-1'), respectively, while the H-1' resonance appeared as a singlet. This suggests that the pentosyl in 3 is an α-arabinofuranosyl, which was confirmed by 2D NMR data analysis of 3 (Figs. 2 and 3). Especially the ¹H-¹H COSY crosspeaks of H-1'/H-2'/H-3'/H-4'/H₂-5' and the HMBC correlations from H-1' to C-4' and from H-4' to C-1', as well as the coupling constants of J_{2', 3'} (2.0Hz) and J_{3', 4'} (3.5Hz), indicated the presence of α-arabinofuranosyl in 3. Meanwhile, the HMBC correlations from H-1' to C-14 and from H-14 to C-1' verified the location of the sugar unit at C-14 in 3. Moreover, neoline {[α]_D²⁰ +22.3 (c 0.08, MeOH)} and L-arabinose {[α]_D²⁰ +107.6 (c 0.04, H₂O)} were isolated and identified from the hydrolysate of 3. Thus, the structure of compound 3 was determined as neoline 14-O-α-L-arabinofuranoside and named aconicarmichoside C.

Compound 4 was isolated as a colorless gum with [α]_D²⁰ +37.1 (c 0.50, MeOH). The spectroscopic data of 4 indicated that this compound is one more isomer of 1-3, in which the anomeric proton (H-1') had a more deshielded chemical shift (δ_H 5.08) and a coupling constant value of 4.8 Hz (J_{1', 2'}) (Table 1). This, combined with 2D NMR data analysis (Figs. 2 and 3), suggested replacement of the α-arabinofuranosyl in 3 by a β-L-arabinofuranosyl in 4. The suggestion was proved by acid hydrolysis of 4. From the hydrolysate of 4, neoline {[α]_D²⁰ +20.7 (c 0.07, MeOH)} and Larabinose {[α]_D²⁰ +110.1 (c 0.03, H₂O)} were isolated and identified as the sole aglycone and sugar. Therefore, the structure of compound 4 was determined as neoline 14-O-β-L-arabinofuranoside and named aconicarmichoside D.

Interestingly, the specific rotations of 1 and 3 with the α-L-arabinosyls are reversed to those of 2 and 4 with the β-L-arabinosyls, though both the aglycone and sugar have the positive specific rotations. This suggests that the specific rotations of the neoline 14-O-β-L-arabinosides are dominated by the C-1' configurations of the L-arabinosyls.

Because trifluoroacetic acid (TFA) was used in the final HPLC purification steps, the basicity of the diterpenoid alkaloids suggests that 1-4 were possibly obtained as trifluoroacetates or complexes [34, 36-38, 45-47]. However, in the ESIMS of 2 and 4, ion peaks at m/z 592 (positive mode) and 604 and/or 568 (negative mode), ascribable to [alkaloid + Na]⁺ and [alkaloid + HCl-H]^- and/ or [alkaloid-H]^-, respectively, supported the occurrence of the free bases and/or an equilibration of interactions between the alkaloids and acids (Figs. S18 and S44 in Supporting information). Although the ¹³C resonances of TFA^- (or TFA) were not observed in the ¹³C NMR spectra of 1-4 (Figs. S7, S23, S37, and S51 in Supporting information), the ¹⁹F NMR spectra displayed a ¹⁹F resonance attributable to TFA^- (or TFA) around δ_F -76.5, indicating the presence of TFA^- (or TFA) in the samples. Subsequent quantitative ¹⁹F NMR analysis of 1-4, using benzene (C₆H₆) and hexafluorobenzene (C₆H₆) as internal standards, determined that approximate ratios of the alkaloids and TFA in the samples were 1:1.2-1:1.7 (Figs. S59-S66 in Supporting information). The absence of the 13C resonances of TFA^- (or TFA) in the ¹³C NMR spectra should be due to comprehensive reasons, including spin couplingsplit of the ¹³C resonances by the ¹⁹F nucleus, conjugation between different carboxylic species of TFA, and interactions of the carboxylic species of TFA with the alkaloid and solvent in the solution state. This is similar to that observed in our previous studies [34, 36-38]. Since TFA came from the experimental procedures and since a variety of acids are exist in the bio-system, under in vivo hydrophilic conditions these diterpenoid alkaloids should interact with the acids to increase their solubility, bioavailability, and transportations, as well as to play biological functions. In addition, in specific and/or local alkali microenvironments of the bio-systems the free base forms, along with potential equilibrations of interactions with the acids, would also play potential roles.

In the in vitro bioassays, compounds 1 and 2 inhibited lipopolysaccharide (LPS)-induced NO production in cultivated murine microglial cell line BV-2 with 28.3% and 32.6% inhibitions at 10mmol/L, respectively, while the positive control curcumin gave 87.3% inhibition at the same concentration [48]. Compound 2 also showed antiviral activity against the influenza virus A/ Hanfang/359/95 (H3N2) with IC₅₀ and SI values of 33.3mmol/L and 3.0, respectively, the positive control, ribavirin, gave IC₅₀ = 1.71 mmol/L and SI = 680.8 [49]. This indicates that isomerization of the arabinosyl plays a role for the activities. Other assays, including influence onsynchronized Ca²⁺ oscillation in cultivated cardiac muscle cell, activity against KCNQ2 potassium channel in CHOK1 cells, protection against L-glutamic acid-induced SK-N-SH neuroblastoma cell damage, inhibition against Fe²⁺-cysteine induced rat liver microsomal lipid peroxidation, antiviral activity against Coxsackie virus B3 (CVB3), and cytotoxicity against several human cancer cell lines were also performed in this study. However, 1-4 were inactive at a concentration of 10mmol/L in each assay.

3. Conclusion

Four aconitine-type C₁₉-diterpenoid alkaloids 1-4 were isolated and characterized from the aqueous extract of the lateral roots of A. carmichaelii ("fu zi"). Although a great number of diterpenoid alkaloids, covering C_20^-, C_19^-, and C_18^-categories, have been isolated from plant species of the Ranunculaceae, Rosaceae, Asteraceae, Garryaceae, Escalloniaceae, and Polygonaceae families during the past century, no diterpenoid alkaloid glycoside was reported as a natural or synthetic product [50-53]. This is abnormal because glycosidation of the free hydroxyl group(s) in many structures of the diterpenoid alkaloids would occur as commonly observed in the other types of natural products. Therefore, the discovery of this study amends and proves the natural occurrence of diterpenoid alkaloid glycosides, while 1-4 are the first examples of the unprecedented glycosidic diterpenoid alkaloids. In addition, 1-4 represent also the first examples that the glycosidic natural products with the four isomeric L-anabinosyls are simultaneously isolated from the same plant species. Although weak inhibitory activities against NO-production and influenza virus A/Hanfang/359/95 (H3N2) were found for 1 and/or 2, the potential activities of the unique C₁₉-diterpenoid alkaloid isomers and contributions to the clinic effects of "fu zi" are still expected from future evaluations with an assistance of semi/total synthesis to obtain enough amount of the samples.

4. Experimental 4.1. General experimental procedures

Optical rotations were measured on a P-2000 polarimeter (JASCO, Tokyo, Japan). UV spectra were recorded on a V-650 spectrometer (JASCO). CD spectra were measured on a JASCO J-815 CD spectrometer (JASCO). IR spectra were recorded on a Nicolet 5700 FT-IR Microscope spectrometer (FT-IR Microscope Transmission) (Thermo Electron Corporation, Madison, WI, USA). 1D and 2D NMR spectra were obtained at 600 or 500 MHz for ¹H, 150 or 125 MHz for ¹³C, respectively, on a SYS 600 MHz, an Inova 500 MHz (Varian Associates Inc., Palo Alto, CA, USA), a Bruker 600 MHz (Bruker Corp., Karlsruhe, Germany), or a WNMR-I 500 MHz (Wuhan Zhongke Niujin Magnetic Resonance Technology Co., Ltd., Wuhan, China) spectrometer, with TMS or solvent peaks as references. ESIMS and HR-ESIMS data were obtained on Agilent 1100 Series LC-MSD-Trap-SL and Agilent 6520 Accurate-Mass QTOFL CMS spectrometers (Agilent Technologies, Ltd., Santa Clara, CA, USA), respectively. Column chromatography (CC) was performed with silica gel (200-300 mesh, Qingdao Marine Chemical Inc., China), reversed phase C-18 silica gel (W. R. Grace & Co., Maryland, USA), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). HPLC separation was performed on a system consisting of a Waters 600 controller, a Waters 600 pump, and a Waters 2487 dual absorbance (Waters Corporation, Milford, MA, USA) or a Smartline RI (Knauer, Berlin, Germany) detector, using an Ultimate XB-Phenyl column (250 mm × 10 mm i.d.) packed with phenyl-silica gel (5mm) (Welch, shanghai, China). TLC was conducted on precoated silica gel GF₂₅₄ plates. Spots were visualized under UV light (254 or 365 nm) or by spraying with 7% H₂SO₄ in 95% EtOH followed by heating or with a Dragendorff's reagent. Unless otherwise noted, all chemicals were obtained from commercially available sources and were used without further purification.

4.2. Plant material

See Refs. [34-38].

4.3. Extraction and isolation

For extraction and preliminary fractionation of the extract, see refs 34-38. Fraction C2-2 (200 g) was separated by CC over Sephadex LH-20 (CHCl₃-MeOH, 1:1) yielded C2-2-1-C2-2-8. Fraction C2-2-4 (9.5 g) was chromatographed over silica gel (150 g) eluting with a gradient of petroleum ether-Me₂CO-diethylamine (5:2:1-2:2:1) to give C2-2-4-1-C2-2-4-7. Fraction C2-2-4-6 (2.65 g) was separated by CC over silica gel, eluting with a gradient of CHCl₃ (saturated with ammonia water)-MeOH (20:1-5:1), to give C2-2-4-6-1-C2-2-4-6-11, of which C2-2-4-6-6 (1.5 g) was further fractionated by CC over reversed phase C-18 silica gel (30-50% MeOH in H₂O) to give C2-2-4-6-6-1-C2-2-4-6-6-3. Fraction C2-2-4-6-6-1 (1.2 g) was subjected to preparative TLC [CHCl₃ (saturated with ammonia water)-MeOH, 5:1] to yield C2-2-4-6-6-1-1 and C2-2-4-6-6-1-2. Further separation of C2-2-4-6-6-1-1 (0.9 g) by reversed phase HPLC (Ultimate XB-Phenyl column, 15% MeCN in H₂O containing 0.1% TFA, 2 mL/min) yielded C2-2-4-6-6-1-1-1-C2-2-4-6-6-1-1-5, of which C2-2-4-6-6-1-1-5 was purified by reversed phase HPLC using the same column and the mobile phase of 42% MeOH in H₂O containing 0.1% TFA and the flow rate 2 mL/min to afford 4 (6.5 mg, t_R = 25 min). Fraction C2-2-4-6-6-2 (0.3 g) was chromatographed over silica gel eluting with CHCl₃ (saturated with ammonia water)-MeOH (5:1), to give C2-2-4-6-6-2-1-C2-2-4-6-6-2-5, of which C2-2-4-6-6-2-4 (39 mg) was separated by HPLC (Ultimate XB-Phenyl column, 45% MeOH in H₂O containing 0.1% TFA, 2 mL/min) to yield C2-2-4-6-6-2-4-1 and C2-2-4-6-6-2-4-2, which were separately purified by HPLC using the same column. Using the mobile phase of 35% MeOH in H₂O containing 0.1% TFA (2 mL/min), 3 (4 mg, t_R = 49 min) was obtained from the former, and using the mobile phase of 20% MeOH in H₂O containing 0.1% TFA (2 mL/min), 2 (7 mg, t_R = 44 min) was obtained from the latter. Fraction C2-2-4-6-7 (110 mg) was separated by preparative TLC [CHCl₃ (saturated with ammonia water)-MeOH (5:1)] to afford C2-2-4-6-7-1-C2-2-4-6-7-3, of which C2-2-4-6-7-1 (20 mg) was purified by reversed phase HPLC (Ultimate XBPhenyl column, 19% acetonitrile in H₂O containing 0.1% TFA, 2 mL/ min) to yield 1 (9 mg, t_R = 24 min).

Aconicarmichoside A (1): Colorless gum; [α]_D²⁰ -11.6 (c 0.69, MeOH); IR ν_max 3356, 2935, 1678, 1448, 1291, 1201, 1128, 1010, 942, 836, 801, 721 cm^-1; 1H NMR (MeOH-d₄, 600 MHz) data, see Table 1; ¹³C NMR (MeOH-d₄, 150 MHz) data, see Table 1; (+)-ESIMS m/z 570 [M+H]⁺; (+)-HR-ESIMS m/z 570.3283 [M+H]⁺ (calcd. for C₂₉H₄₈NO₁₀, 570.3273).

Aconicarmichoside B (2): Colorless gum; [α]_D²⁰ +46.3 (c 0.54, MeOH); IR ν_max 3369, 2935, 1678, 1451, 1356, 1202, 1133, 1082, 1011, 938, 886, 837, 800, 721 cm^-1; 1H NMR (MeOH-d₄, 500 MHz) data, see Table 1; ¹³C NMR (MeOH-d₄, 125 MHz) data, see Table 1; (+)-m/ z ESIMS 592 [M+Na]⁺, 570 [M+H]⁺; (-)-ESIMS m/z 604 [M+HCl-H]^-; (+)-HR-ESIMS m/z 570.3273 [M+H]⁺ (calcd. for C₂₉H₄₈NO₁₀, 570.3273).

Aconicarmichoside C (3): Colorless gum; [α]_D²⁰ -20.6 (c 0.31, MeOH); IR ν_max 3393, 2924, 2851, 1679, 1438, 1352, 1205, 1134, 1076, 1048, 984, 962, 841, 802, 724 cm^-1; 1H NMR (MeOH-d₄, 500 MHz) data, see Table 1; ¹³C NMR (MeOH-d₄, 125 MHz) data, see Table 1; (+)-ESIMS m/z 570 [M+H]⁺; (+)-HR-ESIMS m/z 570.3268 [M +H]⁺ (calcd. for C₂₉H₄₈NO₁₀, 570.3273).

Aconicarmichoside D (4): Colorless gum; [α]_D²⁰ +37.1 (c 0.50, MeOH); IR ν_max 3360, 2937, 1678, 1445, 1359, 1310, 1201, 1126, 1041, 939, 899, 835, 801, 721 cm^-1; 1H NMR (MeOH-d₄, 600 MHz) data, see Table 1; ¹³C NMR (MeOH-d₄, 150 MHz) data, see Table 1; (+)-ESIMS m/z 592 [M+Na]⁺, 570 [M+H]⁺; (-)-ESIMS m/z 604 [M +HCl-H]^-, 568 [M-H]^-; (+)-HR-ESIMS m/z 570.3277 [M+H]⁺ (calcd. for C₂₉H₄₈NO₁₀, 570.3273).

4.4. Acid hydrolysis of 1-4

Compounds 1-4 (1-2 mg) were separately dissolved in acetonitrile (0.1 mL), then refluxed with 2 mol/L HCl (2.0 mL) at 95 ℃ for 3 h. The reaction mixture was concentrated under reduced pressure and the residue was chromatographed by CC over Sephadex LH-20 (MeOH) to yield aglycone and sugar. The aglycone (0.5-1.2 mg) gave specific rotation{[α]_D²⁰ +20.7 to +22.3 (c 0.05-0.1, MeOH)} and 1H NMR spectroscopic data (MeOH-d₄) consistent with those of neoline obtained from the same extract, while the sugar (0.1-0.4 mg) showed [α]_D²⁰ values of +104.8 to +110.1 (c 0.01-0.04, H₂O) and 1H NMR spectroscopic data (D2O) are in good agreement with those of an authentic L-arabinose, which was also isolated from the plant extract.

Acknowledgments

Financial support from the National Natural Science Foundation of China (NNSFC, Nos. 81630094 and 30825044) is acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at 10.1016/j.cclet.2017.04.026.