Alkaloids Isolated from the Lateral Root of Aconitum carmichaelii

Two new alkaloids, aconicarmine (1) and aconicaramide (5), were isolated from the EtOH extract of the lateral roots of Aconitum carmichaelii, together with five known compounds: fuziline (2), neoline (3), N-ethylhokbusine B (4), 5-hydroxymethylpyrrole-2-carbaldehyde (6), and oleracein E (7). Their structures were elucidated by physical and NMR analysis. Pyrrole alkaloids were isolated from A. carmichaelii for the first time. In the in vitro assays, compounds 2 and 3 showed activity against pentobarbital sodium-induced cardiomyocytes damage by obviously recovering beating rhythm and increasing the cell viability, while compounds 5 and 7 showed moderate antibacterial activity.


Results and Discussion
The EtOH extract of the lateral roots of A. carmichaelii was suspended in water and successively partitioned with petroleum ether, EtOAc, and n-BuOH. Separation of the n-BuOH fraction by column chromatography provided compounds 1−7 ( Figure 1).  revealed 22 carbon resonances corresponding to the above protonated units and four quaternary carbons (one oxygen-bearing,  C 80.3). The above-mentioned spectroscopic data suggested that 1 was a C 20 -diterpenoid alkaloid with an atisine-denudatine skeleton and an N-ethyl group [13]. Detailed comparison of the NMR data and the molecular composition of 1 with those of 11-epi-16α,17dihydroxylepenine [14] indicated that compound 1 was an isomer of the latter. The 13 C-NMR spectrum showed high similarity between them, except that the signal of C-11 (δ C 72.3) in 1 was deshielded by 7.5 ppm compared to that of 11-epi-16α,17-dihydroxylepenine possessing an α hydroxy group at C-11. This revealed 1 was an 11β-hydroxyepimer [14], which was proved by 2D NMR experiments, including the ROESY analysis. The five OH groups could be located at C-1, C-11, C-15, C-16, and C-17, respectively, according to their HMBC correlations ( Figure 2). In the ROESY spectrum, the correlations of H-11 and H-15 with H-13 and H-14, the same as those of lepenine [15], verified the OH-11 and OH-15 groups were β-oriented. Moreover, the correlations of H-1/H-5, H-1/H-9, and H-9/H-17 demonstrated the α-configuration of OH-1 and OH-16 ( Figure 3). Accordingly, Compound 1 was established to be 16α,17-dihydroxylepenine and named aconicarmine.   Comparison of the NMR data between 5 and 6 indicated that they differed in the presence of resonances attributable to an additional prolyl moiety (δ C 23.6, 29.9, 42.7, 57.5, 169.2) in 5 [17]. In addition, almost no downfield shift of H-6 was observed in 5 as compared with that of 6, suggesting that the prolyl unit was attached to N instead of OH-6. This conjecture was refined by a 1 H-1 H COSY correlation observed between the exchangeable proton (δ H 4.39) and H-6 (δ H 4.65), as well as no HMBC correlation of H-6 with the carbonyl (C-6′).
The protective activities of the compounds against cardiomyocyte damage induced by pentobarbital sodium in primary cultured neonatal rat cardiomyocytes were investigated by the MTT method. The results showed that pentobarbital sodium induced a significant inhibition of MTT reduction. At concentrations of 10 μM, 1 μM, and 0.1 μM, compounds 2 and 3 increased the cell viability obviously (

Plant Material
The lateral root of A. carmichaelii was collected in July of 2010 from the culture field in Jiangyou, Sichuan postal code, China. Plant identity was verified by Prof. Min Li (Chengdu University of TCM, Sichuan, China). A voucher specimen (SFZ-0710) was deposited at the School of Pharmacy, Chengdu University of TCM, Chengdu, China.

Cardiomyocyte Protection Assay
Neonatal rat cardiomyocytes were cultured in 96-well plates with DMEM media supplemented with 15% FBS. Cultures were maintained in a 37 °C humidified incubator with 5% CO 2 . On the fifth day when the cardiomyocytes were in the growth with rhythmical beating, they were exposed to the medium containing pentobarbital sodium at a concentration of 8 mg/mL. After 8 min, the medium was replaced with serum free medium including compounds at concentrations of 10 μM, 1 μM, and 0.1 μM, respectively, and incubated for 24 h. Then, 10 μL of MTT solution (5 mg/mL) was added and incubated for 4 h. Absorbance was measured at both 570 nm and 655 nm, and cell viability was evaluated with the deviations between them.

Antibacterial Activity Experiments
All bacteria were obtained from clinical samples and stored in the Department of Pharmacology of Chengdu University of TCM. The in vitro antibacterial activity was determined by the standard agar dilution method, according to NCCLS (National Committee for Clinical Laboratory Standard). 2 μL of cultures of test strains at the concentration of 1 × 10 6 CFU/mL were inoculated on Mueller Hinton agar containing different concentrations of the test compounds. The MIC values were determined after incubation at 35-37 °C for 18-24 h.

Conclusions
Two new alkaloids aconicarmine (1) and aconicaramide (5) were isolated from the lateral roots of A. carmichaelii, together with five known alkaloids. Compounds 5 and 6 were the first report of pyrrole alkaloids from A. carmichaelii. Compounds 2 and 3 showed activity against pentobarbital sodium-induced cardiomyocytes damage by recovering beating rhythm and increasing the cell viability obviously. Compounds 5 and 7 showed moderate antibacterial activity.