Potentially Cardiotoxic Diterpenoid Alkaloids from the Roots of

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Potentially Cardiotoxic Diterpenoid Alkaloids from the Roots of Aconitum carmichaelii Xingxing Zong,†,§ Xiaojing Yan,†,§ Jian-Lin Wu,† Zhongqiu Liu,‡ Hua Zhou,† Na Li,*,† and Liang Liu*,†

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†

State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macao 999078, Special Administrative Region of the People’s Republic of China ‡ International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, People’s Republic of China S Supporting Information *

ABSTRACT: Aconitum carmichaelii is a traditional Chinese herbal medicine used for the treatment of pain and inflammation in the joints. However, the strong cardiotoxicity hinders its use. Although diester- and monoester-type diterpenoids, e.g., aconitine, mesaconitine, and hypacaonitine, are commonly considered as the toxic components, the toxicity of A. carmichaelii cannot be completely explained by the compounds reported. To investigate further the cardiotoxic compounds and their potential mechanism, the chemical constituents were first isolated by column chromatography and identified using mass spectrometry and NMR spectroscopy. Two new hetisine-type (1 and 2) and four new aconitine-type alkaloids (3−6) were assigned. The cardiac cytotoxicity assessed on H9c2 cells indicated that the new compound 4 as well as six known alkaloids (7 and 9−13) exhibited significant toxicities. A preliminary structure−toxicity relationship study suggested that substitution at C-8 and C-10 both have a significant influence on cardiotoxicity, and such toxicity decreased in the order OBz-8, OBu-8, and OMe-8. The presence of an OH-10 group abolished the toxicity. Finally, it was found that ion channel disorder and induction of mitochondrial-mediated cell apoptosis are the possible mechanisms of cardiotoxicity among the compounds studied.

their possible cardiotoxicity mechanisms, especially to test the contribution of direct mitochondrial function impairment to cytotoxicity.11−14 In addition, the cells have the versatility of allowing the study of action potentials and cardiac electrophysiology, which are similar to embryonic or neonatal cells.15 Thus, the H9c2 cell line was chosen in the present study to evaluate the cardiac cytotoxicity of the A. carmichealii diterpenoid alkaloids and to investigate further their potential cardiotoxic mechanism. Furthermore, preliminary structure− toxicity relationships were also investigated. The potential mechanisms were assessed using assays of intracellular Ca2+ concentration, membrane potential, and cell apoptosis and mitochondrial membrane potential (ΔΨm).

Aconitum carmichaelii Debeaux (Ranunculaceae) is an important traditional Chinese medicine and possesses antiinflammatory, analgesic, and antitumor activities.1−3 Previous studies have showed that C19-diester diterpenoid alkaloids, e.g., aconitine, mesaconitine, and hypaconitine, as well as C19monoester diterpenoids, e.g., benzoylaconine, benzoylmesaconine, and benzoylhypaconine, are the major bioactive components.4 However, C19-diester diterpenoid alkaloids also show strong cardiotoxicity, which causes severe acute arrhythmias that may lead to death. Although about a hundred aconitine alkaloids have been reported, the physiological activity and toxicity of A. carmichaelii cannot be completely explained by the compounds reported. In order to determine the possible toxic components, the alkaloids present were first isolated and identified in this study. Then, their cytotoxic effects were assessed using an in vitro cell model. The cardiac H9c2 (rat embryonic myocardium) cell line derived from the embryonic rat heart myocardium has been applied as an in vitro model to study cardiac biochemical5 and pathophysiological processes, including myocardial ischemia injury, reactive oxidative stress, tissue differentiation, and metabolic capacity of the heart.6−8 Importantly, this model is utilized frequently to evaluate the cardiotoxicity of compounds9,10 and to investigate © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION In order to find new potentially bioactive or toxic components, the roots of A. carmichaelii were investigated using various chromatography techniques. Eleven alkaloids, including six new compounds (1−6), and five known compounds (7, 12− 15) were isolated and characterized structurally by combinaReceived: December 7, 2018

A

DOI: 10.1021/acs.jnatprod.8b01039 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

characteristic exocyclic double bond unit [δH 5.15 (1H, br s), 5.02 (1H, br s); δC 149.9, 112.6], which implied that 1 might be a hetisine-type alkaloid. The methine signal [δH 3.04 (1H, br s), δC 67.3, position-20] showed HMBC correlations with C-6, C-8, H-1, H-5, H-9, and H-14 as well as two oxymethines at δH 4.24 [(1H, m), δC 90.7, position-19] and δH 4.21 [(1H, m), δC 75.9, position-13] (Figure 1). Furthermore, the oxymethine at δH 4.24 showed HMBC correlations with the methyl at δH 0.98 [(3H, s), H3-18], C-3, and H-5, while the oxymethine at δH 4.21 gave 1H−1H COSY cross-peaks with H12 and H-14 as well as an HMBC cross-peak with C-16. These observations confirmed that compound 1 has the characteristic structure of a hetisine-type alkaloid, i.e., the connection of C14−C-20−N(C-19)−C-6. Moreover, a hydroxy group and an acetoxy group were located at C-19 and C-13, respectively, as deduced from the chemical shifts and HMBC correlation between H-13 and the acetyl group (Table 1 and Figure 1). Another oxymethine at [δH 4.05 (1H, br s), δC 70.6] that correlated with the protons and carbons of the exocyclic double bond in the HMBC spectrum was assigned at C-15. The NOE correlation between H-5 and H-9 (Figure 1) implied that compound 1 has the same configuration as found in other hetisine-type alkaloids; that is, the connection of C19−N−C-20−C-14 is under the plane of rings A and B, and CH3-4, H-6, H-14, and H-20 are β-, β-, β-, and α-oriented, respectively. The NOE correlation between H-19 and H-6 indicated an α-oriented OH-19. H-13 and H-15 were α-

tions of spectroscopic methods including HRESIMS and NMR.

Compound 1 was obtained as a white, amorphous powder. Its molecular formula was deduced as C22H29NO4 on the basis of a protonated molecular ion at m/z 372.2176 [M + H]+ in the HRESIMS (calcd 372.2169). The 1H and 13C NMR data indicated the presence of an acetyl group [δH 1.97 (3H, s), δC 170.4, 21.1] (Table 1). Except for the resonances of an acetyl group, there were 20 carbon signals, i.e., one methyl, five methylenes, nine methines, three quaternary carbons, and a

Table 1. 1D and 2D NMR Spectroscopic Data for Compounds 1 and 2a 1 position

δC, type

1a 1b 2a 2b 3a 3b 4 5 6 7a 7b 8 9 10 11a 11b 12 13 14 15 16 17a 17b 18 19 20 OCOCH3 OCOCH3

26.3, CH2 19.1, CH2 33.7, CH2 42.7, 60.8, 61.1, 31.5,

C CH CH CH2

46.3, 42.7, 49.4, 23.1,

C CH C CH2

38.1, CH 75.9, CH 52.7, CH 70.6, CH 149.9, C 112.6, CH2 22.2, CH 90.7, CH 67.3, CH 170.4, C 21.1, CH3

δH (J in Hz)

HMBC

1.83, 1.19, 1.72, 1.65, 1.60, 1.23,

m m m m m m

20

1.37, 3.63, 2.08, 1.62,

br s br s dd (13.8, 2.3) m

19, 20

2 b

1

1

H− H COSY 2a 2b 1a, 3a 1b 2a

NOESY 1b 1a, 5 20 3b 3a, 5

8, 14

6

1b, 3b, 6, 7a, 9 5, 7a, 18, 19 5, 6 6

1.82, m

5, 8, 14, 20

11b

5

1.85, 1.63, 2.37, 4.21, 1.67, 4.05,

m m br s m m br s

12

12 9, 12 11a, 11b, 13 12, 14 13 17a, 17b

12 12 11a, 11b, 13, 17b 12, 14, 20 13, 15, 20 14, 17a

5.15, 5.02, 0.98, 4.24, 3.04,

br s br s s m br s

12, 15, 16

15 15

15 12 6, 19 6, 18 2a, 13, 14

1.97, s

7b

9, 13, 14, 15, 16, 17 16, 20, OCOCH3 9, 13, 20 7, 8, 9, 12, 16, 17

3, 4, 5, 19 3, 5, 20 6, 8, 13, 19

δC, type 25.9, CH2 18.9, CH2 33.3, CH2 42.8, 60.3, 61.9, 31.4,

C CH CH CH2

46.5, 42.8, 49.6, 22.7,

C CH C CH2

41.7, CH 72.9, CH 54.7, CH 70.2, CH 149.5, C 112.2, CH2 21.3, CH 91.3, CH 67.7, CH

δH (J in Hz) 1.88, 1.23, 1.67, 1.62, 1.65, 1.31,

m m m m m m

1.51, 3.65, 2.14, 1.61,

br s br s dd (13.8, 2.4) m

1.80, m 1.84, 1.60, 2.27, 3.37, 1.62, 3.97,

m m br s m m br s

5.18, 5.05, 1.01, 4.28, 2.94,

br br s br br

s s s s

OCOCH3

a

The spectra of compounds 1 and 2 were acquired in CDCl3 and CD3OD, respectively. bHMBC correlations were from the proton(s) stated to the indicated carbon. B



Article

Figure 1. Key 1H−1H COSY (), HMBC (→), and NOESY (↔) for compound 1.

chemical shift was downfield shifted compared with common C19-monoester diterpenoid alkaloids,16 which suggested that it is substituted by a benzoyl group. Therefore, compound 3 was determined as 8β,14α-dibenzoyloxy-3α,10β,13β,15α-tetrahydroxy-1α,6α,16β,18-tetramethoxy-N-methylaconitane. Compound 4 gave a molecular formula of C38H47NO10 from the protonated molecule at m/z 678.3270 (calcd 678.3273) in the HRESIMS, which was two oxygens less than that of compound 3. The DEPT data showed compound 4 to contain one methylene more and one oxygenated quaternary carbon less than 3 (Table 2). The methylene [δH 1.68 (1H, m), 1.62 (1H, m); δC 34.8] correlated with H-2 in the 1H−1H COSY spectrum as well as with H-1, H-18, and H-19 in the HMBC spectrum; thus the methylene was proposed to occur at C-3. The methine at δH 2.22 (1H, m) gave 1H−1H COSY crosspeaks with H-9 and H-12 as well as HMBC correlations with C-8, C-11, C-12, and C-17, which indicated that no hydroxy group was substituted at C-10 (Table S3, Supporting Information). Consequently, compound 4 was assigned as 8β,14α-dibenzoyloxy-13β,15α-dihydroxy-1α,6α,16β,18-tetramethoxy-N-methylaconitane. A protonated molecular ion of compound 5 at m/z 556.3278 (calcd 556.3269, [M + H]+) in the HRESIMS provided the molecular formula of C32H45NO7, which was C6H2O3 less than that of compound 4. The 1D NMR spectra showed only one set of benzoyl group signals, and one oxymethine carbon and one oxygenated quaternary carbon were replaced by a methylene and a methine (Table 2). Four methoxy group signals gave HMBC cross-peaks with C-1, C-8, C-16, and C-18 (Table S4, Supporting Information), respectively, which meant that no methoxy group was substituted at C-6 and that a methoxy occurred at C-8 in 5. The correlations of a methylene with H-5 and H-7 in the 1H−1H COSY spectrum and with C7, C-8, C-11, and C-17 in the HMBC spectrum confirmed the absence of any OCH3-6 substituent. The methine resonance at δH 2.54 (1H, m) correlated with H-14 in the 1H−1H COSY spectrum, as well as H-9 and H-10 in the HMBC spectrum, respectively. Therefore, the methine was assigned at C-13, and there was an absence of any hydroxy group substitution at this position. Accordingly, compound 5 was determined as 14αbenzoyloxy-N-ethyl-15α-hydroxy-1α,8β,16β,18-tetramethoxyaconitane. The molecular formula of compound 6, C32H45NO8, indicated the presence of one oxygen more than in compound

oriented, which were deduced from their NOE correlation with H-20 and H-14, respectively. Consequently, compound 1 was identified as 13β-acetoxy-15β,19α-dihydroxyhetisane. A protonated molecular ion of compound 2 at m/z 330.2069 (calcd 330.2064) in the HRESIMS provided the molecular formula as C20H27NO3, which was C2H2O less than that of 1. The 1D NMR data indicated the presence of 20 carbon signals, comprising one methyl, five methylenes, nine methines, three quaternary carbons, and an exocyclic double bond unit [δH 5.18 (1H, br s), 5.05 (1H, br s); δC 149.5, 112.2]. Therefore, 2 was also assigned as a hetisine-type alkaloid (Table 1). No signals for an acetyl group were detected. Moreover, H-13 was upfield shifted from 4.21 to 3.37 ppm, which confirmed that a hydroxy group rather than an acetoxy group is substituted at C-13. Similar to 1, an additional two hydroxy groups were found to be substituted at C-15 and C-19, respectively. NOESY cross-peaks indicated that 2 has the same configurations as 1 (Table S1, Supporting Information). Consequently, compound 2 was assigned as 13β,15β,19αtrihydroxyhetisane. The elemental formula of compound 3 was determined as C38H47NO12 from an observed protonated molecular ion at m/ z 710.3169 (calcd 710.3171, [M + H]+). The 1H and 13C NMR data showed the presence of signals for two sets of benzoyl units, four methoxy groups, and an N-methyl group (Table 2). Except for the above signals, 19 carbon signals remained; thus compound 3 was postulated to be a C19diterpenoid alkaloid. Also occurring were one oxymethylene, six oxymethines, and three oxygenated quaternary carbons. The HMBC correlations of the four methoxy groups at C-1, C6, C-16, and C-18 supported the presence of methoxy group substituents at these carbons (Table S2, Supporting Information). Similarly, a benzoyloxy group was found to connect with C-14 from the HMBC cross-peak between H-14 and the benzoyl group. The remaining two oxymethines were assigned at positions C-3 and C-15 from the 1H−1H COSY correlations with H-2 and the HMBC cross-peak with C-18 as well as 1H−1H COSY correlations with H-16 and the HMBC cross-peaks with C-8 and C-16. The HMBC cross-peaks of two oxygenated quaternary carbons with H-1 and H-12 as well as H-9, H-12, and H-14, respectively, indicated the hydroxy group substitutions at C-10 and C-13. The remaining quaternary carbon at δ 89.9 was deduced to be at C-8 from the HMBC correlations with H-6, H-7, and H-14. The C


Journal of Natural Products Table 2. 1H and

13

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C NMR Spectroscopic Data (600 MHz, CDCl3) for Compounds 3−6 3

position

δC, type

4 δH (J in Hz)

1 2

76.9, CH 33.5, CH2

3

71.2, CH

3.76, 2.38, 2.07, 3.80,

4 5 6

43.2, C 42.5, CH 83.2, CH

2.39, m 4.14, d (6.0)

7 8 9 10 11 12

43.5, 89.9, 54.1, 78.7, 55.7, 46.8,

CH C CH CH C CH2

13 14 15 16 17 18

74.7, 78.4, 79.7, 89.5, 62.8, 76.5,

C CH CH CH CH CH2

19

49.6, CH2

20

42.4, CH3

5.43, 4.66, 3.38, 3.01, 3.65, 3.49, 2.80, 2.46, 2.40,

56.0, CH3 58.2, CH3 61.1, CH3 59.0, CH3 166.3, C 130.6, C 129.5, CH 128.2, CH 133.0, CH 167.1, C 129.1, C 129.2, CH 128.0, CH 132.9, CH

21 OCH3-1 OCH3-6 OCH3-8 OCH3-16 OCH3-18 OCOC6H5 1′ 2′, 6′ 3′, 5′ 4′ OC′OC6H5 1″ 2″, 6″ 3″, 5″ 4″

m m m dd (9.0, 4.8)

3.11, br s 3.07, dd (4.8, 1.2)

3.34, m 2.12, dd (16.2, 1.8) d (5.4) m m s d (9.0) d (9.0) d (11.4) d (11.4) s

δC, type 85.1, CH 26.3, CH2 34.8, CH2 39.3, C 48.2, CH 83.2, CH 44.1, 92.3, 44.7, 41.2, 50.0, 36.2,

CH C CH CH C CH2

74.2, 79.1, 78.8, 90.2, 62.3, 80.1,

C CH CH CH CH CH2

5 δH (J in Hz)

3.11, 2.27, 2.02, 1.68, 1.62,

m m m m m

2.99, m 2.22, m

56.6, CH3 58.0, CH3

3.33, s 2.88, s

3.77, s 3.28, s

61.0, CH3 59.0, CH3 166.3, C 129.8, C 129.6, CH 128.1, CH 132.4, CH 167.4, C 129.2, C 129.1, CH 128.1, CH 132.7, CH

3.77, s 3.26, s

7.77, d (7.8) 7.14, t (7.8) 7.30, m

38.4, C 45.8, CH 23.6, CH2

3.19, m 2.22, m

3.34, s 2.91, s

7.88, d (7.8) 7.21, t (7.8) 7.39, t (7.8)

32.7, CH2

3.09, m

42.6, CH3

56.0, CH2

85.8, CH 26.5, CH2

2.16, d (6.6) 4.11, d (6.6)

4.95, 4.63, 3.41, 3.19, 3.62, 3.12, 2.59, 2.42, 2.43,

δC, type

d (4.8) dd (5.4, 3.0) d (5.4) m d (8.4) m d (8.4) m br s

33.3, 81.4, 37.1, 45.2, 49.0, 29.6,

CH C CH CH C CH2

37.1, 76.2, 76.3, 93.0, 61.9, 79.9,

CH CH CH CH CH CH2

53.0, CH2 49.4, CH2 13.5, CH3 56.2, CH3 49.5, CH3 56.9, CH3 59.4, CH3 166.2, C 129.9, C 129.7, CH 128.4, CH 133.8, CH

7.86, m 7.18, m 7.38, m

6 δH (J in Hz)

3.16, 2.33, 2.04, 1.83, 1.47,

m m m m m

1.63, 1.89, 1.44, 2.92,

d (7.2) m m d (7.8)

2.54, m 2.01, m 2.62, 2.04, 2.54, 4.98, 4.41, 3.14, 3.00, 3.14, 3.06, 2.57, 2.02, 2.66, 2.41, 1.09, 3.33,

m m m dd (4.8, 4.2) d (6.6) m s m d (7.2) d (11.4) m m m t (7.2) s

3.19, s 3.43, s 3.32, s

8.05, d (7.8) 7.46, t (7.8) 7.58, m

δC, type 85.7, CH 26.5, CH2 32.7, CH2 38.5, C 45.7, CH 23.7, CH2 33.6, 81.9, 43.9, 41.9, 49.7, 36.7,

CH C CH CH C CH2

75.3, 79.9, 79.0, 94.1, 62.4, 79.8,

C CH CH CH CH CH2

53.0, CH2 49.5, CH2 13.7, CH3 56.5, CH3 49.1, CH3 62.3, CH3 59.6, CH3 166.6, C 130.6, C 129.9, CH 128.4, CH 132.8, CH

δH (J in Hz) 3.12, 2.30, 1.99, 1.79, 1.43,

m m m m m

1.60, 1.78, 1.42, 2.80,

d (7.8) m m m

2.63, m 2.10, m 2.82, m 2.08, m 4.82, 4.49, 3.25, 3.02, 3.06, 3.03, 2.55, 2.01, 2.64, 2.40, 1.08, 3.30,

d (5.4) d (6.0) d (6.0) m d (7.2) m d (11.4) m m m t (7.2) s

2.99, s 3.72, s 3.28, s

8.03, d (7.8) 7.43, t (7.8) 7.52, m

7.70, m 7.13, m 7.34, m

1α,6α,8β,16β,18-pentamethoxyaconitane (12), 14α-benzoyloxy-N-ethyl-13β,15α-dihydroxy-1α,6α,8β,16β,18-pentamethoxyaconitane (13), 14α-benzoyloxy-3α,10β,13β,15α-tetrahydroxy-1α,6α,8β,16β,18-pentamethoxy-N-methylaconitane (14), and 14α-benzoyloxy-13β,15α-dihydroxy-1α,6α, 8β,16β,18-pentamethoxy-N-methylaconitane (15). Cytotoxicity of Isolated Compounds on H9c2 Cells. It has been reported that C19-diterpenoid alkaloids exhibit cardiotoxicity, so the cytotoxic effects of compounds 3, 4, and 7−15 on H9c2 cells were determined using an MTT assay. The IC50 values are presented in Table S7 (Supporting Information). Compounds 7, 9, 10, and 11 showed IC50 values of 12.9, 19.2, 21.2, and 27.0 μM, respectively. Additionally, compounds 4, 12, and 13 showed less potent toxicities with IC50 values of 44.3, 68.4, and 95.6 μM. The IC50 values of the

5. Similar to 5, the NMR data showed the presence of four methoxy group signals at C-1, C-8, C-16, and C-18, respectively. In addition, one more oxygenated quaternary carbon was observed in 6 instead of methine (Table 2). The oxygenated quaternary carbon was assigned at C-13 from the HMBC correlations with H-10, H-14, and H-16 (Table S5, Supporting Information). Moreover, the downfield shift of OCH3-16 (0.29 ppm) compared with 5 supported the presence of an OH-13 group.13 Thus, compound 6 was determined as 14α-benzoyloxy-N-ethyl-13β,15α-dihydroxy1α,8β,16β,18-tetramethoxyaconitane. In addition, five known compounds were also isolated and were determined as 8β,14α-dibenzoyloxy-3α,13β,15α-trihydroxy-1α,6α,16β,18-tetramethoxy-N-methylaconitane (7), 14α-benzoyloxy-N-ethyl-3α,13β,15α-trihydroxyD



Article

Figure 2. Intracellular Ca2+ signaling induced by compounds 9 (A) and 10 (B), mesaconitine (C), and hypaconitine (D) as measured by FLIPR. The image (E) and quantification (F) of the intracellular Ca2+ signaling at a concentration of 80 μM were calculated by FLIPR software. Values are expressed as means ± SD from three independent experiments; *p < 0.05, **p < 0.01 vs control group.

other alkaloids were more than 100 μM, which is considered as a threshold value of no significant cardiac cytotoxicity.17 As shown in Table S7 (Supporting Information), all screened compounds belong to the C19-aconitine-type alkaloid class, with varying substitutions at the N atom, C-3, C-8, and C-10. Based on the substitution at C-8, these compounds could be divided into three groups, i.e., OBu-8, OBz-8, and OMe-8. Three alkaloids with an OBu-8 group (9−11) showed strong cardiac toxicity; thus the presence of a butoxy at C-8 and the absence of any substitution at C-10 seem to be the key factors, while OH-3 (9 vs 10) and N-CH3 or N-CH2CH3 (10 vs 11) had no significant influence. The IC50 values of four compounds (3, 4, 7, and 8) with an OBz-8 group ranged from 12.9 to 118.3 μM. It was evident that the presence of OH-10 (3 vs 7) resulted in decreased toxicity, and the influence of an N-CH3 or N-CH2CH3 in the OBz-8 group (3 vs 8) did not play a major role. However, an OH-3 group contributed to the cardiac toxicity (4 vs 7). On the other hand, the comparison between 7 and 11 showed that OBz-8 was more toxic than OBu-8. Kinetic Measurement of Intracellular Ca2+ Signal. Ca2+ is well known to play crucial roles in the pathogenesis of heart dysfunctions, and the disruption of intracellular Ca2+ homeostasis may cause arrhythmia and finally induces cell apoptosis. To determine the cytotoxic mechanisms of the isolated alkaloids, intracellular Ca2+ signals were measured using a high-throughput fluorometric imaging plate reader

(FLIPR). Compared to a control group, compounds 9 and 10 were found to increase intracellular Ca2+ signaling at a concentration of 80 μM (p < 0.01). Moreover, the effects were similar to those of the known cardiotoxic compounds mesaconitine and hypaconitine (Figure 2). Taken together, these data indicated that compounds 9 and 10 increased the intracellular Ca2+ concentration, triggered arrhythmia, and induced cell apoptosis on myocardial H9c2 cells. FLIPR Membrane Potential Assay. To investigate the effects of the isolated alkaloids on ion channels in cell membranes, a FLIPR membrane potential assay was performed. As shown in Figure 3, compounds 9 and 10 increased membrane potential in a concentration-dependent manner. At a concentration of 80 μM, the increases of membrane potential induced by 9 and 10 were greater than those of the known cardiotoxic compounds mesaconitine and hypaconitine. These results suggested that compounds 9 and 10 may lead to arrhythmia through promoting ion channel dysfunction and inducing sustained depolarization. Effects of Isolated Compounds on Cell Apoptosis. Subsequently, to determine the effects of isolated alkaloids on H9c2 cell apoptosis, cells were incubated with DAPI and annexin V-FITC/PI double staining and analyzed by highcontent screening (HCS) (Figure 4A). Data calculated by an In Cell Analyzer 1000 workstation software showed that the nuclear size became smaller (p < 0.01), and both the annexin V-FITC and PI fluorescence intensity significantly increased (p E



Article

Figure 3. Cell membrane potential induced by compounds 9 (A) and 10 (B), mesaconitine (C), and hypaconitine (D) as measured by FLIPR. The image (E) and quantification (F) of the membrane potential at a concentration of 80 μM were calculated by FLIPR software. Values are expressed as means ± SD from three independent experiments; *p < 0.05, **p < 0.01 vs control group; #p < 0.05, ##p < 0.01 vs mesaconitine; &p < 0.05, &&p < 0.01 vs hypaconitine. Infrared (IR) spectra were recorded on an Agilent Cary 600 series FT-IR spectrometer (KBr). 1D and 2D NMR spectra were acquired on a 600 MHz Bruker Ascend NMR spectrometer, and the solvent peak of CDCl3 or CD3OD was used as reference. HRESIMS data were measured using an Agilent Technologies 6550 iFunnel-Q-TOFMS. Column chromatography (CC) was performed using silica gel (75−150 mesh, Grace, USA) and ODS (Grace, USA) as absorbents. High-performance liquid chromatography (HPLC) separation was performed using an Agilent 1100 system consisting of an autosampler, a binary pump, and a UV detector, with a Grace preparative C18 column (250 × 10 mm, 10 μm). MS grade acetonitrile (CH3CN) and water were purchased from J.T. Baker (Danville, PA, USA). MS grade formic acid (FA) and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich Laboratories, Inc. (St. Louis, MO, USA). HPLC grade dichloromethane, n-hexane, ethyl acetate, nbutanol, CH3CN, and methanol were provided by Anaqua Chemicals Supply (Wilmington, DE, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Pen Strp glutamine, and 0.25% trypsin-EDTA were purchased from Gibco Co., Ltd. (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO) was purchased from Guangzhou Chemical Reagent Factory (Guangzhou, Guangdong, People’s Republic of China). Calcium 6 assay kit and membrane potential assay kit were obtained from Molecular Device Co. (Sunnyvale, CA, USA). Compounds 8−11 were isolated and identified in a previous research project.16 Plant Material. The roots of A. carmichaelii were obtained from Hehuachi Medicinal Materials Market in Chengdu, Sichuan Province,

< 0.01) in compounds 9 and 11 (Figure 4B) at a concentration of 40 μM, when compared to a control group. However, mesaconitine and hypaconitine exhibited no obvious difference in nuclear size and annexin V-FITC/PI fluorescence intensity at 40 μM (p > 0.01), which supported that mesaconitine and hypaconitine exert lower cytotoxic effects on H9c2 cells. Collectively, the presently described data indicate that compounds 9 and 11 markedly inhibited H9c2 cell viability through inducing cell apoptosis. Effects of Isolated Compounds on Cell Mitochondrial Membrane Potential (ΔΨm). To further elucidate the mechanism of inducing cell apoptosis, the cellular mitochondrial membrane potential (ΔΨm) was explored by HCS analysis. Compared with the control group, the ΔΨm fluorescent intensity was significantly decreased (p < 0.01) (Figure 5A,B) when cells were exposed to compounds 9 and 11 at concentration of 40 μM for 24 h, respectively. Meanwhile, the mild effect on ΔΨm was detected in mesaconitine and hypaconitine groups. The results suggested that the mitochondrial pathway is involved in compound 9 and 11 induced myocardial cell apoptosis.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a Rudolph Research Autopol I automatic polarimeter. F



Article

Figure 4. Effects of compounds 9 and 11, mesaconitine, and hypaconitine on cell apoptosis. (A) Images of cells stained with DAPI and annexin VFITC/PI dyes were taken using an In Cell Analyzer 6000 system. Quantification of cell nuclear size (B), annexin V-FITC (C), and PI (D) fluorescent intensity were calculated by In Cell analyzer 1000 workstation software. Values are expressed as means ± SD from three independent experiments; *p < 0.05, **p < 0.01 vs control group; #p < 0.05, ##p < 0.01 vs mesaconitine; &p < 0.05, &&p < 0.01 vs hypaconitine. People’s Republic of China, in December 2013, and authenticated by Ying Liu, Chengdu University. A voucher specimen (Chw-02) was deposited at the State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology. Extraction and Isolation. The roots of A. carmichaelii (7.2 kg) were powdered and extracted with methanol at reflux to provide 356 g of residue. This was divided further into n-hexane, ethyl acetate, nbutanol, and water layers by solvent partitioning. The n-butanol extract (54.1 g) was separated into 50 fractions by silica gel CC eluting with CH2Cl2/MeOH (25:1 → 0:100, v/v). Fr.11 (180 mg) was subjected to reversed-phase (RP) preparative HPLC (prepHPLC) to give six subtractions (Fr.11-1 to Fr.11-6) eluting with 0.02% diethylamine (DEA)-containing CH3CN/H2O, with the increase of organic solvent from 65% to 100% in 40 min. Fr.11-2 (45 mg) was further purified by RP prep-HPLC using 0.02% DEA-

containing CH3CN/H2O with the increase of organic solvent from 25% to 50% in 40 min as the mobile phase to provide compound 1 (33.1 mg). Compound 5 (0.9 mg) was purified from Fr.11-4 (8 mg) on a RP prep-HPLC column with 0.02% DEA-containing CH3CN/ H2O (88:12) as the mobile phase. Compound 2 (4.0 mg) was obtained from Fr.12 (180 mg) by RP prep-HPLC using 0.02% DEAcontaining CH3CN/H2O with the increase of organic solvent from 45% to 80% in 40 min. Compound 3 (1.3 mg) was isolated from Fr.18 (85 mg) on a RP prep-HPLC column using 0.02% DEAcontaining 57% CH3CN/H2O. Compound 4 (4.1 mg) was isolated from Fr.9 (85 mg) on a RP prep-HPLC column using 0.02% DEAcontaining 76% CH3CN/H2O. Compound 6 (13.2 mg) was isolated from Fr.13 on an RP prep-HPLC column using 0.02% DEAcontaining 30% CH3CN/H2O. Other compounds also were obtained by ODS CC and prep-HPLC. G



Article

Figure 5. Effects of compounds 9 and 11, mesaconitine, and hypaconitine on mitochondrial membrane potential (ΔΨm). (A) Images of cells stained with DAPI and Rh 123 were taken by an In Cell Analyzer 6000 system. (B) The ΔΨm fluorescent intensity was calculated by In Cell analyzer 1000 workstation software. Values are expressed as means ± SD from three independent experiments; *p < 0.05, **p < 0.01 vs control group; #p < 0.05, ##p < 0.01 vs mesaconitine; &p < 0.05, &&p < 0.01 vs hypaconitine. 13β-Acetoxy-15β,19α-dihydroxyhetisane (1): white powder (CHCl3); [α]22 D +14.3 (c 0.33, CHCl3); IR (KBr) νmax 2929, 2360, 2340, 1731, 1241, 1026, 754 cm−1; 1H (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 1; HRESIMS m/z 372.2176 (calcd for C22H29NO4, 372.2169). 13β,15β,19α-Trihydroxyhetisane (2): white powder (MeOH); [α]22 D −11.2 (c 0.40, MeOH); IR (KBr) νmax 2928, 1718, 1456, 1277, 1101, 1027 cm−1; 1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Table 1; HRESIMS m/z 330.2069 (calcd for C20H27NO3, 330.2064). 8β,14α-Dibenzoyloxy-3α,10β,13β,15α-tetrahydroxy1α,6α,16β,18-tetramethoxy-N-methylaconitane (3): white powder (CHCl3); [α]22 D +18.1 (c 0.26, CHCl3); IR (KBr) νmax 2927, 2360, 1722, 1696, 1288, 1101, 712 cm−1; 1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Table 2; HRESIMS m/z 710.3169 (calcd for C38H47NO12, 710.3171).

8β,14α-Dibenzoyloxy-13β,15α-dihydroxy-1α,6α,16β,18-tetramethoxy-N-methylaconitane (4): white powder (CHCl3); [α]21 D +26.6 (c 0.84, CHCl3); IR (KBr) νmax 3499, 2926, 1720, 1692, 1291, 1119, 1096, 711 cm−1; 1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Table 2; HRESIMS m/z 678.3270 (calcd for C38H47NO10, 678.3273). 14α-Benzoyloxy-N-ethyl-15α-hydroxy-1α,8β,16β,18-tetramethoxyaconitane (5): white powder (CHCl3); [α]20 D −10.7 (c 0.18, CHCl3); IR (KBr) νmax 2931, 1720, 1520, 1276, 1095, 715 cm−1; 1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, CDCl3, 150 MHz), see Table 2; HRESIMS m/z 556.3278 (calcd for C32H45NO7, 556.3269). 14α-Benzoyloxy-N-ethyl-13β,15α-dihydroxy-1α,8β,16β,18-tetramethoxyaconitane (6): white powder (CHCl3); [α]20 D −10.2 (c 0.66, CHCl3); 1H (CDCl3, 600 MHz) and 13CNMR (CDCl3, 150 MHz), see Table 2; HRESIMS m/z 572.3232 (calcd for C32H45NO8, 572.3218). H



Article

Cell Line and Cell Culture. Rat myocardial H9c2 cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were cultured in DMEM and supplemented with 10% FBS and Pen Strep glutamine (100 unit/mL penicillin, 100 μg/mL streptomycin, and 0.292 mg/mL glutamine) at 37 °C with a humidified atmosphere of 5% CO2 in an incubator. The cells were dissociated with 0.25% trypsin-EDTA. Cell Proliferation Analysis. The cytotoxicity assay was conducted by an MTT method. Briefly, H9c2 cells were seeded in 96-well plates (BD Biosciences, North Ryde, Australia) with a 100 μL volume at a density of 1 × 105 cells/well and were incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO2. Each compound was dissolved in DMSO at a concentration of 80 mM and then diluted with complete medium to a series of concentrations (Table S6, Supporting Information). The cells were treated with the compounds for 24 h, and then 20 μL/well of MTT (5 mg/mL) was added followed by incubation for 4 h at 37 °C. The MTT medium was discarded, and the cells were lysed in 150 μL of DMSO. The plates were shaken gently for 10 min to blend the mixture. The optical density (OD) at 490 nm was measured using a microplate reader (SpectraMax, Molecular Device Co., Sunnyvale, CA, USA). All experiments were performed at least three times. The cell inhibition rate (%) was calculated using the equation (1 − absorbance of treated group/absorbance of blank) × 100%. The blank was the well that contained only culture medium but no cells. FLIPR Assay for Intracellular Ca2+ Concentration and Membrane Potential. H9c2 cells were seeded in clear-bottomed black 96-well plates (Corning, NY, USA) at 1 × 105 cells/well. After being grown to confluency, the medium was removed and replaced by 100 μL/well HBSS containing 20 mM HEPES to maintain the signal, and then 100 μL of calcium-sensitive dye indicators or bluemembrane potential dye was added into each well according to the manufacturer’s protocol. The Ca2+ dye-loaded cell plates were incubated for 2 h at 37 °C, while the membrane potential dyeloaded cell plates were incubated for 30 min at 37 °C. Then, the freshly prepared test compounds in HBSS with 20 mM HEPES were added. The intracellular Ca2+ levels and membrane potential were measured by a Molecular Devices fluorometric imaging plate reader, as described previously.18−21 The relative changes in the Ca2+ and membrane potential fluorescence intensity (ΔF/F0) were calculated as the peak change in fluorescence amplitude (ΔF) divided by the initial intensity value (F0). HCS Analysis for Cell Apoptosis and Mitochondrial Membrane Potential (ΔΨm). H9c2 cells were seeded in black clear-bottomed 96-well imaging plates and incubated with the test compounds at a concentration of 40 μM for 24 h. Treated cells were washed with PBS twice and stained with annexin V-FITC (Thermo Fisher Scientific Inc., Waltham, MA, USA) and propidium iodide (PI) (Acros Orgaincs, Geel, Belgium) and rhodamine 123 (Rh123) (Thermo Fisher Scientific Inc.) for 30 min, respectively. After washing with PBS, cells were fixed with 4% paraformaldehyde for 15 min, washed twice with PBS, and labeled with DAPI for 20 min, followed by washing with PBS; PBS was then added to cells. Images were captured by an In Cell Analyzer 6000 system (GE Healthcare). Staining intensity was calculated from captured images using In Cell Analyzer 1000 workstation software (GE Healthcare). Statistical Analysis. All experimental values were presented as mean ± SD. Data were analyzed using SPSS 17.0 software. The significance of difference was determined by one-way ANOVA with LSD tests, and the Student’s t-test was used for the two group’s comparison. Values of p < 0.05 were considered to be statistically significant.

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MS and 1D and 2D NMR spectra for compounds 1−6 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(N. Li) Tel: +853-8897-2405. Fax: +853-2882-5886. E-mail: [email protected]. *(L. Liu) Tel: +853-8897-2799. Fax: +853-2882-7222. E-mail: [email protected]. ORCID

Zhongqiu Liu: 0000-0001-6986-9677 Na Li: 0000-0002-9578-2871 Author Contributions §

X. Zong and X. Yan contributed equally to this research.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by the Macao Science and Technology Development Fund (003/2016/A1).

(1) Jiangsu New Medical College. Dictionary of Traditional Chinese Medicine; Shanghai Science and Technology Publishing House: Shanghai, 1995; pp 228−232 and 1191−1194. (2) Ren, M. Y.; Yu, Q. T.; Shi, C. Y.; Luo, J. B. Molecules 2017, 22, 267−281. (3) Liang, Y.; Wu, J. L.; Li, X.; Guo, M. Q.; Leung, L. H.; Zhou, H.; Liu, L.; Li, N. Tetrahedron Lett. 2016, 57, 5881−5884. (4) Zhou, G.; Tang, L.; Zhou, X.; Wang, T.; Kou, Z.; Wang, Z. J. Ethnopharmacol. 2015, 160, 173−193. (5) Kimes, B.; Brandt, B. Exp. Cell Res. 1976, 98, 367−381. (6) Zordoky, B. N.; El-Kadi, A. O. J. Pharmacol. Toxicol. Methods 2007, 56, 317−322. (7) Force, T.; Kolaja, K. L. Nat. Rev. Drug Discovery 2011, 10, 111− 126. (8) Wang, L. Q.; He, Y.; Wan, H. F.; Zhou, H. F.; Yang, J. H.; Wan, H. T. J. Zhejiang Univ., Sci., B 2017, 18, 586−596. (9) Ellis, C.; Naicker, D.; Basson, K.; Botha, C.; Meintjes, R.; Schultz, R. Toxicon 2010, 55, 12−19. (10) Arbo, M. D.; Silva, R.; Barbosa, D. J.; da Silva, D. D.; Rossato, L. G.; de Lourdes Bastos, M.; Carmo, H. Toxicol. Lett. 2014, 229, 178−189. (11) Yaglom, J. A.; Ekhterae, D.; Gabai, V. L.; Sherman, M. Y. J. Biol. Chem. 2003, 278, 50483−50496. (12) Will, Y.; Dykens, J. A.; Nadanaciva, S.; Hirakawa, B.; Jamieson, J.; Marroquin, L. D.; Hynes, J.; Patyna, S.; Jessen, B. A. J. Toxicol. Sci. 2008, 106, 153−161. (13) Lekes, D.; Szadvari, I.; Krizanova, O.; Lopusna, K.; Rezuchova, I.; Novakova, M.; Novakova, Z.; Parak, T.; Babula, P. Physiol. Res. (Prague, Czech Repub.) 2016, 65, 505−514. (14) Gao, X.; Zhang, X.; Hu, J.; Xu, X.; Zuo, Y.; Wang, Y.; Ding, J.; Xu, H.; Zhu, S. Mol. Med. Rep. 2017, 17, 284−292. (15) Wang, Y. J.; Chen, B. S.; Lin, M. W.; Lin, A. A.; Peng, H.; Sung, R. J.; Wu, S. N. Toxicol. Sci. 2008, 106, 454−463. (16) Zong, X. X.; Yan, G.; Wu, J. L.; Leung, E. L. H.; Zhou, H.; Li, N.; Liu, L. Tetrahedron Lett. 2017, 58, 1622−1626. (17) Lin, Z.; Will, Y. Toxicol. Sci. 2012, 126, 114−127. (18) Schmunk, G.; Nguyen, R. L.; Ferguson, D. L.; Kumar, K.; Parker, I.; Gargus, J. J. Sci. Rep. 2017, 7, 40740−40748. (19) Pratt, D. S.; Mezler, M.; Geneste, H.; Bakker, H. M.; Hajduk, J. P.; Gopalakrishnan, M. S. Comb. Chem. High Throughput Screening 2011, 14, 631−641.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b01039. I



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(20) Vasilyev, D. V.; Shan, Q. J.; Lee, Y. T.; Soloveva, V.; Nawoschik, S. P.; Kaftan, E. J.; Dunlop, J.; Mayer, S. C.; Bowlby, M. R. J. Biomol. Screening 2009, 14, 1119−1128. (21) Finley, M.; Cassaday, J.; Kreamer, T.; Li, X.; Solly, K.; O’Donnell, G.; Clements, M.; Converso, A.; Cook, S.; Daley, C. J. Biomol. Screening 2016, 21, 480−489.

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