A new C20-diterpenoid alkaloid from the lateral roots of Aconitum carmichaeli

Abstract A new C20-diterpenoid alkaloid carmichaedine (1) and six known alkaloids (2–7) were isolated from the lateral roots of Aconitum carmichaeli. Their structures were established on the basis of extensive spectroscopic analyses. Compound 1 exhibited potent antibacterial activity against Bacillus subtilis with minimum inhibitory concentration of 8 μg/mL.


Introduction
Aconitum carmichaeli Debx. (Ranunculaceae) is widely distributed and cultivated in China and other Eastern Asian countries, such as Japan and Korea (Shim et al. 2005). The processed lateral roots of A. carmichaeli, which is known as 'Fu Zi' in traditional Chinese medicine, has been clinically used for keeping heart pumping and rescuing the heart deficiency since ancient times . Previous studies have revealed that diterpenoid alkaloids, which exhibited various pharmacological activities (Liu & Katz 1994;Atta-ur-Rahman et al. 2005;Guo et al. 2014;Shyaula et al. 2015), are the main active constituents of this drug. More than 50 diterpenoid alkaloids have been isolated from the raw and prepared forms of 'Fu Zi' (Zhou et al. 2015). As part of our continuous work on the discovery of bioactive alkaloids from Aconitum species (Yin et al. 2014(Yin et al. , 2015, a phytochemical investigation on the lateral roots of A. carmichaeli was carried out, which led to the isolation of a new vakognavine-type C 20 -diterpenoid alkaloid carmichaedine (1) (Figure 1), together with six known alkaloids, aconitine (2) (Yin et al. 2013), 3-deoxyacontine (3), mesaconitine (4), hypaconitine (5) (Gao et al. 2012), songorine (6) (Csupor et al. 2006) and 14-acetylneoline (7) (Yin et al.2013). Their structures were established on the basis of extensive spectroscopic analyses. Herein, the isolation, structural elucidation and antibacterial activity of these compounds are described.
The relative configuration of 1 was deduced from the vicinal coupling constants and a ROESY experiment ( Figure S1). The coupling constant between H-11 with H-9 (J = 9.0 Hz) indicated a 1,2-diaxial relationship between them, implying that H-11 was β-oriented (Liu et al. 2009). The ROESY correlations of H-7 with H-14 and H-15, H-2 with H-5, and H-13 with H-17 revealed the α-orientations of H-7 and H-13, and the β-orientation of H-2 (Zhou et al. 2005). Hence, the structure of 1 was fully established and named as carmichaedine.
The results of antibacterial activity assay indicated that 1 possesses potent antibacterial activity against Bacillus subtilis with minimum inhibitory concentration (MIC) of 8 μg/mL, while the positive control kanamycin showed antibacterial activity against B. subtilis with MIC of 8 μg/mL.

General procedure
Melting points were determined on a XRC-1 Melting Point Apparatus (Sichuan University Science Factory, Chengdu, China). Optical rotations were measured with a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan). A Nicolet Magna-IR 550 spectrometer (Thermo Nicolet, Madison, USA) was used for scanning IR spectroscopy with KBr pellets. NMR spectra were acquired with either a Bruker AM-400 spectrometer or a Bruker DRX-600 spectrometer in CDCl 3 using TMS as the internal reference. ESI-MS analyses were recorded with Agilent G3250AA (Agilent, Santa Clara, USA) and Auto Spec Premier P776 spectrometer (Waters, Milford, USA). Silica gel (200-300 and 300-400 mesh; Qingdao Haiyang Chemical Factory, Qingdao, China) and Sephadex LH-20 (GE Healthcare, Fairfield, USA) were used for column chromatography (CC). Fractions were monitored by TLC and visualised by spraying with modified Dragendorff's reagent.

Plant material
The lateral roots of A. carmichaeli were collected from Kunming City in Yunnan Province of China in December 2015, and identified by assistant professor Shu-Da Yang from School of Pharmaceutical Science & Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University. A voucher specimen (2015-fz-1) is deposited in the School of Chemical Science and Technology in Yunnan University of China.

Antibacterial activity assay
The pathogenic bacteria B. subtilis, collected from Yunnan Institute of Microbiology, was selected for antibacterial assay. The antibacterial activity assay was performed in 96-well sterilised microplates using a microdilution method described previously (Yin et al. 2015). The 18-h-old bacterial cultures from B. subtilis were added to sterile LB broth medium to reach 1 × 10 5 colony-forming units/mL. Compound 1 and positive control were dissolved in DMSO and their final concentrations ranged from 512 to 1 μg/mL using a twofold serial dilution method. The wells containing test strains and diluted compounds were incubated at 37 °C (24 h). The wells containing a culture suspension and DMSO were run as negative controls. Kanamycin was introduced as positive control in the experiments on account of its prominent antibacterial activity against B. subtilis. All experiments were repeated three times. The growth of test strains was observed with a CX21BIM-set5 microscope. The MICs were determined as the lowest concentrations that produce complete growth inhibition of the tested micro-organisms.

Conclusion
A new vakognavine-type C 20 -diterpenoid alkaloid carmichaedine (1) and six known alkaloids (2-7) were isolated from the lateral roots of A. carmichaeli, whose structures were established on the basis of extensive spectroscopic analyses. Carmichaedine (1) exhibited potent antibacterial activity against B. subtilis with MIC of 8 μg/mL.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Natural Science Foundation of China [grant number 81460648].