Fitoterapia 94 (2014) 120–126

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Fitoterapia

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New diterpenoid from coreanum and their anti-arrhythmic effects on cardiac sodium current

Bei-Ni Xing a, Si-Si Jin b, Hao Wang a, Qing-Fa Tang c, Jing-Han Liu a, Rui-Yang Li a, Jing-Yu Liang a,⁎, Yi-Qun Tang b,⁎⁎, Chun-Hua Yang d,⁎⁎⁎ a School of Chinese Medicine, Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China b Research Division of Pharmacology, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China c School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, People's Republic of China d Department of Biology, Georgia State University, Atlanta, GA 30303, USA article info abstract

Article history: Two new diterpenoid alkaloids, Guan-Fu base J (GFJ, 1) and Guan-Fu base N (GFN, 2) along Received 18 November 2013 with nineteen known alkaloids (3–21) were isolated from the roots of Aconitum coreanum Accepted in revised form 13 January 2014 (Lèvl.) Rapaics, which is the raw material of a new approval anti-arrhythmia drug “Acehytisine Accepted 15 January 2014 Hydrochloride”. The structures of isolated compounds were established by means of 1D, 2D Available online 4 February 2014 NMR spectroscopic and chemical methods. All isolates obtained in the present study were evaluated for their inhibitory effects on blocking the ventricular specific sodium current Keywords: using a whole-cell patch voltage-clamp technique. Among these 21 compounds, Guan-Fu base Aconitum coreanum S (GFS, 3) showed the strongest inhibitory effect with an IC50 value of 3.48 μM, and only Diterpenoid hetisine-type C diterpenoid alkaloids showed promising IC values for further development. Guan-Fu base J 20 50 © 2014 Elsevier B.V. All rights reserved. Guan-Fu base N Anti-arrhythmic effect

1. Introduction to treat paroxysmal supraventricular tachycardia in 2005 approved by China Food and Drug Administration [4]. Aconitum coreanum (Lèvl.) Rapaics is widely distributed in Cardiovascular diseases cause a worldwide mortality northeastern China. The roots of this , “Guan Bai Fu” in larger than both infectious diseases and cancer combined Chinese, were extensively used for the treatment of various [5]. About 25% of cardiovascular-related deaths are due to kinds of disorders including cardiovascular diseases in ancient arrhythmias, arising primarily from disturbances in the China [1]. Pharmacological studies had demonstrated that transmembrane ion fluxes carried by Na+,Ca2+,K+ [6,7]. extracts from the roots of A. coreanum showed anti-arrhythmic, Among the cardiac different ionic currents known to effects, as well as anti-inflammatory activities [2]. generate the cardiac action potential (AP), the sodium Previous phytochemical investigations of this herb led to the current (INa) plays a crucial role which is responsible for the isolation of several diterpenoid alkaloids (DAs), sitosterols, early depolarization and duration of the AP and involved for and organic acids [3]. Guan-fu base A (GFA, 16), one of the the propagation of the electrical impulse from one cell to the dominate DAs in this herb, had been developed into a new other [8,9]. Consequently, the INa could be proved a useful anti-arrhythmia drug (Acehytisine Hydrochloride Injection), candidate target for new potential therapeutic agents in arrhythmia. In our previous study, several known DAs had been ⁎ Corresponding author. Tel.: +86 13605154996. isolated from the roots of A. coreanum [10,11]. As part of a ⁎⁎ Corresponding author. Tel.: +86 25 83271070. programmed continuously seeking potential bioactive con- ⁎⁎⁎ Corresponding author. Tel.: +1 404 4937694. E-mail addresses: [email protected] (J.-Y. Liang), [email protected] stituent from A. coreanum, two new DAs (1 and 2) and (Y.-Q. Tang), [email protected] (C.-H. Yang). nineteen known DAs (3–21) were isolated and elucidated

0367-326X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2014.01.022 B.-N. Xing et al. / Fitoterapia 94 (2014) 120–126 121

mainly by spectral methods. All the purified DAs were (2.5 × 50 cm, 400 mesh) using CHCl3\MeOH in a gradient evaluated for their efficacy on sodium current blocking (50:1 to 20:1) elution mode to yield 4 fractions (Frs. C1–C4). using a whole-cell patch voltage-clamp technique. In this Fr. C1 was loaded on silica gel column (1.0 × 20 cm, 400 paper, we report the isolation and structure elucidation of mesh) using CHCl3\MeOH (20:1) as eluents to afford these new DAs, as well as their anti-arrhythmic activities. compounds 1 (21 mg) and 18 (14 mg). Fr. C2 was submitted to silica gel column (1.0 × 20 cm, 400 mesh) eluting with a

2. Experimental gradient of CHCl3\MeOH (20:1 to 15:1) to give compounds 3 (6 mg), 4 (7 mg) and 8 (14 mg). Fr. C3 was loaded on silica gel 2.1. Gerneral methods column (1.0 × 20 cm, 400 mesh) eluted with petroleum ether–acetone (6:1) and further purified by Sephadex LH-20 Melting points (uncorrected) were obtained on an X-4 to afford 9 (17 mg) and 13 (21 mg). Fr. C4 was separated micro-melting point detector (Beijing Taike Instrument on Sephadex LH-20 column eluted with CHCl3\MeOH (1:1) Corp., Beijing, China). UV spectra were recorded on a JASCO to give 5 (8 mg) and 6 (8 mg). Fraction D (2.8 g) was fur-

V-550 UV/VIS spectrophotometer (JASCO, Inc., Japan). IR ther subjected to C18 low-pressure column (3.0 × 45 cm, spectra were measured on a Nicolet-Impact-410 FT-IR 40–63 μm) eluted with MeOH\H2O in gradient mode (1:9 spectrometer, KBr pellets, in cm−1 (Nicolet Instrument to 8:2) to yield 4 fractions (Frs. D1–D4). Fr. D1 was loaded on

Corp., USA). Optical rotations were tested on Rudolph C18 low-pressure column (2.5 × 20 cm, 40–63 μm) eluted Research Analytical AUTOPOL® IV automatic polarimeter with MeOH\H2O (1:9 to 3:7) and further purified by (Rudolph Research Analytical, Hackettstown, NJ, USA). Sephadex LH-20 to give 2 (12 mg), 17 (18 mg), 19 (9 mg)

HR-ESI-MS data were carried out on Agilent 6210 ESI/TOF and 20 (70 mg). Fr. D2 was separated on C18 low-pressure mass spectrometer (Agilent, Inc., USA). 1D and 2D NMR column (2.5 × 20 cm, 40–63 μm) eluting with a gradient of 1 spectra were recorded on Bruker AV-500 spectrometer ( H: MeOH\H2O (3:7 to 1:1) to afford 7 (11 mg) and 16 (56 mg). 500 MHz, 13C: 125 MHz), δ values in parts per million Fr. D4 was recrystallized using MeOH to give 10 (28 mg). Fr. F relative to internal TMS; J values in Hz (Bruker Corp., (4.3 g) was further separated by C18 low-pressure column Germany). Silica gel (200–300 mesh; Qingdao Marine (2.5 × 20 cm, 40–63 μm) eluted with MeOH\H2Oingradient Chemical Group Corp., Qingdao, China), Sephadex LH-20 mode (1:9 to 3:7) to yield 3 fractions (Frs. F1–F3). Compounds

(Pharmacia Biotech AB, Uppsala, Sweden) and RP-C18 15 (77 mg) was obtained by recrystallizing Fr. F3 in methanol. (40-63 μm, Merck, Darmstadt, Germany) were used for Fr. F1 was further separated by C18 low-pressure column column chromatography. TLC was performed using precoat- (2.5 × 20 cm, 40–63 μm) using MeOH–H2O (1:9) to afford 11 ed silica gel plates (Qingdao Marine Chemical Group Corp., (10 mg) and 21 (47 mg).

Qingdao, China). Spots on chromatograms were detected Guan-Fu base J (1): white powder (CHCl3); mp 246– with Dragendorff's reagent. 247 °C; [α]25 D: +16.67 (c 1.0, CHCl3); IR (KBr) νmax: 2928, 1740, 1380, 1239, 1122, 1015, 981 cm−1. 1H (500 MHz, 13 2.2. Plant material CDCl3) and C NMR (125 MHz, CDCl3) data, see Table 1; + HR-ESI-MS m/z 388.2128 [M + H] (calcd for C22H30NO5: The air-dried roots of A. coreanum were provided by the 388.2124).

Good Agriculture Practice (GAP) construction base, Jilin Guan-Fu base N (2): white powder (CHCl3); mp 194– Province, China, in October 2011, and were authenticated 196 °C; [α]25 D: +32.91 (c 1.0, CHCl3); IR (KBr) νmax: 3357, by professor Min-Jian Qin (Department of Pharmacognosy, 3038, 1727,1665, 1388, 1205, 937 cm−1; 1H (500 MHz, 13 China Pharmaceutical University). The voucher specimen CDCl3) and C NMR (125 MHz, CDCl3) data, see Table 1; + (No. 20111025) was deposited at the Department of Natural HR-ESI-MS m/z 416.2441 [M + H] (calcd for C24H34NO5: Medicinal Chemistry, China Pharmaceutical University, Nanjing, 416.2437). China. 2.4. Ventricle myocyte preparation 2.3. Extraction and isolation Male guinea pigs, 300 ± 20 g, were purchased from The air-dried and powdered roots of A. Coreanum (10 Kg) Qinglongshan Experiment Animal Center (permission for were extracted with 95% EtOH at room temperature (10 L × 3, production No. SCXK 2012–2008, Nanjing, Jiangsu province, each 3 days). The extracting solutions were combined and China). All experiments were performed in accordance with evaporated under reduced pressure to yield a brown syrup the procedures of the Laboratory Animal Management (590 g). The brown syrup was suspended in water (2 L) and Regulations of Jiangsu Province. Ventricular myocytes from then successively extracted by alkalinized CHCl3 (adjusted guinea pig hearts were enzymatically dissociated by the to pH 9 by ammonia water, 4 L × 3), the CHCl3 extracting procedure described previously [12] and in Supplemental solution was then evaporated to obtain a crude alkaline extract Methods. The isolated myocytes were maintained at room (43 g). Based on silica gel TLC pilot test, crude alkaline extract temperature in the KB medium for at least 1h before was subjected to silica gel column chromatography (4.0 × electrophysiological recording.

80 cm, 200–300 mesh), using CHCl3\MeOH (100:1, 50:1, 25:1, 10:1, 5:1) as eluents to yield 7 fractions (Frs. A–G). 2.5. Data acquisition and analysis Fr.A(0.6g)andFr.B(0.7g)wererecrystallizedinMeOHto give compounds 12 (28 mg) and 14 (17 mg), respectively. The whole-cell patch-clamp technique was used for Fr. C (3.7 g) was further separated by silica gel column electrophysiological recording. Sodium current was recorded 122 B.-N. Xing et al. / Fitoterapia 94 (2014) 120–126

Table 1 significance of differences between two group means, and 1 13 δ a Hand C NMR spectra data of compounds 1 and 2 (CDCl3, in ppm, J in Hz) . one-way ANOVA was used for multiple groups. P b 0.05 was Position 1 2 considered to indicate statistically significant differences.

δ δ δ δ H C H C 3. Results and discussion 1 1.56 dd (15.0, 4.2) 28.2 t 1.70 dd (15.0, 4.7) 29.7 t 2.39 d (15.0) 2.33 d (15.0) The EtOH extract of the roots of A. coreanum was 2 5.20 t (2.2) 69.1 d 5.21 t (4.6) 69.1 d 3 1.59 dd (16.8, 4.7) 37.1 t 1.59 m 37.2 t suspended in water and partitioned with CHCl3. The CHCl3 1.90 d (16.8) 1.82 m soluble fraction was repeatedly subjected to column chro- – – 4 37.1 s 37.1 s matography on silica gel, reversed-phase C18, and Sephadex 5 1.57 m 57.1 d 1.56 s 59.3 d LH-20 to afford two new diterpenoid alkaloids, along with 6 3.20 m 63.2 d 3.18 br.s 63.2 d nineteen known compounds (Fig. 1). 7 1.29 m 32.6 t 1.73 d (13.7) 29.5 t 1.99 dd (13.8, 2.5) 2.23 dd (13.8, 3.4) Compound 1 was a white powder, and it showed a positive 8 – 39.7 s – 46.4 d reaction with Dragendorff's reagent. Its molecular formula was 9 1 .66 d (3.2) 51.9 d 1.28 s 59.9 d deduced to be C22H29NO5 from the quasi-molecular ion at m/z 10 – 43.8 s – 43.4 s + 388.2128 [M + H] (calcd for C22H30NO5: 388.2124) in its 11 4.94 d (3.1) 93.2 d 4.33 dd (8.8, 4.0) 67.2 d 12 1.74 d (12.9) 35.5 t 2.67 br.s 51.1 d HR-ESI(+)-MS. The IR spectrum exhibited absorptions of ester −1 −1 1 1.94 dt (12.9, 3.7) carbonyl (1727 cm ) and ether (1239 cm ) groups. The H 13 3.83 dd (3.5, 1.8) 73.9 d 3.72 br.s 75.9 d NMR spectrum of 1 (Table 1) exhibited characteristic signals 14 – 74.0 s – 79.6 s for three groups of methyl protons including two singles at δH 15 1.44 d (12.9) 37.9 t 5.53 s 125.8 d 1.01, 1.16 (both 3H, s) and one acetyl methyl at δ 2.05 (3H, s), 1.54 d (12.9) H δ 16 – 69.7 s – 136.5 s three oxygenated methine protons at H 3.83 (1H, brs), 4.94 13 17 1.16 s 28.1 q 1.87 s 21.9 q (1H, d, J = 3.1 Hz), and 5.20 (1H, brs). Analysis of its CNMR 18 1.01 s 29.4 q 1.03 s 29.3 q (Table 1) and HSQC spectroscopic data revealed that 1 19 2.61 d (12.1) 63.8 t 2.57 d (12.1) 63.1 t contained 22 carbon resonances, including three methyl, six 2.88 d (12.1) 2.89 d (12.1) 20 3.15 s 72.3 d 3.27 s 69.5 d methylene, seven methine, five aliphatic quaternary carbons 13 21 – 169.8 s – 176.2 s and one carbonyl carbon. The C NMR and MS features of 1 22 2.05 s 21.6 d 2.50 dt (14.0, 7.0) 34.3 d showing twenty-two carbons composed by twenty carbons on 23 1.16 d (7.0) 18.9 q the skeleton and two from the acetyl group indicated that 1 24 1.16 d (7.0) 18.9 q was a C20-diterpenoid alkaloid. The absence of IR signals for a Assignments were based on the COSY, NOESY, DEPT, HSQC and HMBC double bond suggested that 1 was not a hetisine-type C20 experiments. Overlapped proton NMR signals are reported without desig- diterpenoid alkaloid [13]. Most chemical shifts of the carbon nated multiplicity. and proton signals of 1 were found to be similar with those

reported data of kusnezoline (C20H27NO3), which was an kusnezoline-type diterpenoid alkaloid isolated from Aconitum at room temperature (~23 °C). Glass patch electrodes were kusnezoffi [14], except for the chemical shift of C-2 and C-14 pulled using a two-step puller (HEKA PIP5; HEKA Elektronic, shifted to downfield from δC 67.0 52.9 in kusnezoline to δC Lambrecht/Pfalz, Germany) and had a tip resistance of 69.1 74.0 in 1, respectively. The HMBC correlations (Fig. 2)

1–3MΩ when filled with pipette solution. The bath solution from the oxymethine proton at δH 5.20 (H-2) to the carbonyl contained (in mM): CaCl2 1.8, NaCl 30, MgCl2 1, CsCl 105, carbon resonance at δC 169.8 (C-21) suggested the presence of glucose 5, HEPES 5 (pH 7.4). In addition, 5 μM nimodipine a acetyl group at C-2 position. On the other hand, the HMBC was used to block the L-type Ca2+ channels. The intracellular cross-peaks of H-13 with C-14, of H-20 with C-14, and of H-9 pipette solution contained (in mM): CaCl2 1, CsCl 120, MgCl2 with C-14 with the oxygen-bearing carbon resonance at δC 73.0 5, Na2ATP 5, TEACl 10, EGTA 11, and HEPES 5 (pH 7.3). When confirmed the existence of one hydroxyl group at C-14 position. a gigaseal (N10 GΩ) was obtained, the cell membrane was So 1 was identified as 2-acetyl-14-hydroxy-kusnezoline. ruptured by a gentle suction to establish the whole-cell The NOESY correlations (see Fig. 2) were observed between configuration. The sodium channel membrane currents were H-22 and H-20, indicated that 2-acetyl was orientated the recorded using a patch clamp EPC-10 amplifier and stored on C-2 α position. Thus, 1 was further elucidated as 2α-acetyl-14- the hard disk of a PC compatible computer. Pipette potentials hydroxy-kusnezoline, which was a new compound and named were zeroed before the pipette contacted the cell. The cell Guan-Fu base J. membrane capacitance was directly performed using Pulse Compound 2 was a white powder, and it showed a software (v8.61, HEKA, Lambrecht, Germany). The series positive reaction with Dragendorff's reagent. The molecular resistance was electrically compensated to minimize the formula of C24H33NO5 as deduced from the HRESIMS capacitive surge on the current recording. When measuring quasi-molecular ion at m/z 416.2441 [M + H]+ (calcd for action potential, recording pipettes were filled with a C24H34NO5: 416.2437). The IR spectrum showed the absor- solution containing (in mM): potassium aspartate 80, KCl bance of hydroxyl (3357 cm−1), ethylene (1665 cm−1) and −1 40, MgSO4 2, Na2ATP 5, EGTA 15 and HEPES 5 (pH 7.2). ester carbonyl (1727 cm ) groups. The NMR data (Table 1) The experimental data are presented as means ± SEM. of 2 showed the presence of two methyl groups (δH 1.03, 3H, Nonlinear curve fitting was performed using Origin7.5 s, δC 29.3 q; δH 1.87, 3H, s, δC 21.9 q), one isobutyl group (δH (OriginLab Corp., Northampton, MA) paired and/or Student's 1.15, 3H, s, δC 18.9 q; δH 1.17, 3H, s, δC 18.9 q; δH 2.50, 1H, m, t-test was used as appropriate to evaluate the statistical δC 34.3 d; δC 176.2 s), one olefinic resonance (δH 5.53, 1H, s, δC B.-N. Xing et al. / Fitoterapia 94 (2014) 120–126 123

Fig. 1. Chemical structures of compounds 1–21.

13 125.8 d; δC 136.5 s). The C NMR and MS features of 2 of MeN, MeO, and aromatic ring moieties NMR signals showed twenty-four carbons, including twenty carbons on indicated that no oxazolidine ring existed in the skeleton the skeleton, as well as four from the isobutyrate group, further inferred that 2 was a hetisine-type C20-diterpenoid indicating that 2 was a C20-diterpenoid alkaloid. The absence alkaloid [15]. The molecular formula of 2 was C24H33NO5, 124 B.-N. Xing et al. / Fitoterapia 94 (2014) 120–126

Fig. 2. Key HMBC and ROESY ( ) correlations of 1 and 2.

which was same as Guan-Fu base Z [16], indicating that 2 was 15-ene-hetisine. The ROESY (Fig. 2) cross-peaks of H3-23/H-20 an isomer of GFZ. On the basis of the DEPT data, 2 had one and H3-24/H-20, indicated that the α-isobutyl was orientated more methyl and one less methylene compared with GFZ, and at C-2 position; cross-peaks of H2-1α/H-11, H-11/H-17 and the different ethylenic linkage position was resulted from the H-13/H-17 confirmed that the hydroxyl groups at C-11 and absence of an exocyclic double bond and the existence of cyclic C-13 were β-orientation, respectively. Thus, 2 was further double bond. The HMBC spectrum showed correlations of elucidated to be 2α-isobutyl-11β,13β,14-trihydroxy-15-ene-

Me-17 with an olefinic carbon at δC 136.5 (C-16), and of the hetisine, which was a new compound and named Guan-Fu olefinic proton H-15 with the other one at δC 125.8 (C-15), base N. which confirmed their attribution to C-15 and C-16, respec- The other isolated known compounds were identified as tively. So 2 was identified as 2-isobutyl-11,13,14-trihydroxy- Guan-Fu base S (GFS, 3) [17], Guan-Fu base Q (GFQ, 4) [18],

Fig. 3. Blocking property of sodium current by 3. (a) Original current traces of INa at corresponding time points. The membrane capacitance was 67.76 pF. INa was elicited by a 30-ms pulse to −40 mV from the holding potential of −90 mV. (b) Concentration-response relationship fitted to Hill equation to obtain the IC50 value of 3.48 ± 1.08 μmol/l (n = 5 for each concentration). (c) I–V relationships of the mean values of sodium current in the absence and presence of 3 (3 μmol/l, n = 5). (d) The activaton curve was fit to the Boltzmann function: I/Imax = 1 / (1 + exp[(V1 / 2 − V) / slope]). The V1/2 measured before (control) and after the application 3 were −51.47 ± 0.31 mV and −55.21 ± 0.3 mV, respectively, with a slope of 2.24 ± 0.28 mV and 1.39 ± 0.19 mV. (e) Inactivation curve: steady-state inactivation curves of INa were obtained using a double-pulse protocol; a 50-ms condition prepulse of various potentials (from −120 mV to −40 mV, holding potential at −90 mV) was followed by a 30 ms test pulse to −40 mV. The inactivation curve was also fitted using Boltzmann's equation:

I/Imax = 1 / (1 + exp[(V − V1 / 2) / slope]). The V1/2 of INa measured before (control) and after application of 3 was −83.26 ± 0.25 mV and −88.86 ± 0.31 mV, respectively, with the slope of 5.79 ± 0.22 mV and 6.14 ± 0.28 mV. B.-N. Xing et al. / Fitoterapia 94 (2014) 120–126 125

Fig. 4. Effect of 3 on action potential. Action potential was recorded in “current clamp” mode. For a given drug concentration the effect on action potential duration (APD) reached a steady-state within 2 ± 3 min of application to the cell under study. (a) Action potential with normal external Tyrode's solution

(control) and following exposure to 3 μmol/L 3. At 3 μmol/L there was an increase in the APD90 (from 218.7 ± 14.2 ms to 261.2 ± 24.8 ms, n = 3; P b 0.05 compared to control). (b) In a different cell, 10 μmol/L 3 increased the APD90 (from 428.5 ± 35.8 ms to 699.4 ± 43.3 ms, n = 3; P b 0.01 compared to control). The drug produced a dose-dependent prolongation of the action potential.

Guan-Fu base V (GFV, 5) [8], 2-isobutryl-14-hydroxy- hetisine sodium current in Guinea pig ventricular myocytes by binding N-oxide (6) [19], Guan-Fu base X (GFX, 7) [8], Hypaconitine to the open channels and showed a dose-dependent manner (8) [20],Mesaconitine(9)[21], Guan-Fu base K (GFK, 10) [13], (Fig. 3), which suggests that GFS (3) was a promising anti- Guan-Fu base H (GFH, 11) [22], Dihydroatisine (12) [23], arrhythmia agent. Condelphine (13) [22], Talatisamine (14) [24], Hetisine (15) [25], Guan-Fu base A (GFA, 16) [26], acrodine (17) [27], Conflict of interest Guan-Fu base G (GFG, 18) [28], Guan-Fu base Z (GFZ, 19) [16], Guan-Fu base I (GFI, 20) [29] and Tangutisine (21) [30] Authors declared that we have no conflict of interest on throughcomparisonofthespectroscopicdatawiththose this manuscript. reported in the literatures. According to their basic skeletons and specific functional substituents, all isolates were divided Acknowledgments into four groups: hetisine-type C20-DAs(2,3,5–7, 15–17, 19–21), kusnezoline-type C -DAs (1, 10), atisine-type C -DAs 20 20 This study was supported by Project Program of State Key (11, 12) and -type C -DAs(8,9,13,14). 19 Laboratory of Natural Medicines, China Pharmaceutical Uni- The inhibitory effects on sodium current of compounds versity (JKGQ 201118), National Natural Science Foundation of 1–21 were tested using a whole-cell patch voltage-clamp China (Grant No. 81202431), and the Natural Science Founda- technique. Acehytisine Hydrochloride (crude drug of Guan-Fu tion of Guangdong Province (Grant No. S2012010009576). base A injection, batch: 20120513) was used as positive control (IC =78.26μM). GFS (3, IC =3.48μM) showed 50 50 Appendix A. Supplementary data strong inhibitory effect on sodium current, GFQ (4, IC50 = 82.65 μM), Hetisine (15, IC =75.72μM), GFA (16, IC = 50 50 1D and 2D NMR, and HR-ESI-MS of 1 and 2 can be found in 41.17 μM) and GFG (18, IC50 =23.81μM) showed moderate the online version. Supplementary data to this article can be anti-arrhythmic activities, while the tested other compounds found online at http://dx.doi.org/10.1016/j.fitote.2014.01. exhibited only weak activities (IC50 N 100 μM). Interestingly, 022 significant blocking activities on sodium current were exerted only by five hetisine-type C20-DAs, which indicated that References hetisine-type C20-DAs were the major anti-arrhythmic com- ponents of the herb A. coreanum. Effects of Guan-Fu base S (3) [1] Li H, Sun M, Xu J, Li H, Zang M, Cui Y. Immunological response in H22 on sodium current and action potential were showed at Figs. 3 transplanted mice undergoing Aconitum coreanum polysaccharide and 4, respectively. treatment. Int J Biol Macromol 2013;55:295–300. Comparison of the data from the Hetisine (15), GFA (16) [2] Tang Q-F, Yang C-H, Ye W-C, Liu J-H, Zhao S-X. Preparative isolation and purification of chemical components from Aconitum coreanum by high- and GFG (18) showed that increasing the number of acetyl speed counter-current chromatography coupled with evaporative light groups at C-2, C-11 and C-13 positions (Fig. 1) could increase scattering detection. Phytochem Anal 2008;19:155–9. the inhibitory effect. The IC50 of GFN (2) was above 100 μM [3] Meng X-Y, Yang C-H, Liu J-H, Wang H. Aconitum coreanum: chemical indicated that the C =C cyclic double bond made no compounds and their pharmacological actions. Prog Pharm Sci 15 16 2013;37:390–5. contribution to the inhibition as compare to the data from [4] Huang X, Yang Y, Zhu J, Dai Y, Pu J. The effects of a novel anti-arrhythmic those exocyclic double bond DAs, while in the light of the drug, acehytisine hydrochloride, on the human ether-a-go-go related data from GFS (3) enlightened us that the C =C cyclic gene K channel and its trafficking. Basic Clin Pharmacol Toxicol 2009; 2 3 104:145–54. double bond was crucial in the improvement of activity of [5] Health Statistics and Health Information Systems. inhibitory effect. [6] Ravens U, Poulet C, Wettwer E, Knaut M. Atrial selectivity of Collectively, a series of diterpenoid alkaloids including 4 antiarrhythmic drugs. J Physiol 2013;591:4087–97. [7] Tang Y-Q, Yu P, Zhao N, Yang Q, Yin Y-M, Le X-Y, et al. The multiple ion type skeletons with different substitute groups at various channel blocker CPUY11018 prevents aconitine-induced arrhythmias. positions were isolated from A. coreanum.GFS(3) suppressed Drug Dev Res 2012;73:214–21. 126 B.-N. Xing et al. / Fitoterapia 94 (2014) 120–126

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