A new diterpenoid alkaloid from Aconitum hemsleyanum

Abstract A new C19-diterpenoid alkaloid named hemsleyaline (1), along with fourteen known alkaloids (2-15), were isolated from the roots of Aconitum hemsleyanum Pritz. (Ranunculaceae), a herbal medicine in southwest China. Their structures were established on the basis of extensive spectroscopic analyses. Compound 1 showed mild cholinesterase inhibitory effect with IC50 value of 471 ± 9 μM. Graphical Abstract


Results and discussion
Compound 1 was obtained as a white powder, whose molecular formula was deduced to be C 34 H 49 NO 9 by HR-ESI-MS at m/z 616.3472 [M þ H] þ . The 1 H NMR spectrum of 1 displayed signals of an N-ethyl group (d H 1.09, 3H, t, J ¼ 7.2 Hz), five methoxyl groups, and an anisyl group (d H 3.85, 3H, s; 6.91, 2H, d, J ¼ 8.6 Hz; 8.02, 2H, d, J ¼ 8.6 Hz). The 13 C NMR spectrum revealed that 1 contains nineteen carbons except for the above groups, including five methylenes, eleven methines and three characteristic quaternary carbons at d C 43.0 s, 50.9 s, 78.6 s. The data summarized above, in combination with biogenetic consideration, suggested that 1 might be an aconitine-type C 19 -DA (Liang et al. 2017;Wang and Chen 2010).
The 13 C NMR spectrum of 1 revealed seven oxygenated carbons (d C 72.0 d, 75.8 d, 77.3 t, 78.6 s, 82.7 d, 83.1 d, 83.4 d), corresponding to the molecular formula, which indicated the presence of a hydroxyl group except for the anisyl and methoxyl groups. A signal at d H 3.83 (1H, dd, J ¼ 4.8 Hz, 2.8 Hz) was attributed to H-3b, suggestting the presence of OH-3a, which was supported by 1 H-1 H COSY correlation of H-2/H-3 ( Figure  1S) Zhao et al. 2017). The characteristic methylene (d H 3.47, 3.62, each 1H, ABq, J ¼ 8.8 Hz) was undoubtedly attributed to H-18, indicating a methoxy group substituted at C-18, which was further confirmed by HMBC correlation of OCH 3 -18/C-18. The HMBC correlations of OCH 3 -1/C-1, OCH 3 -6/C-6 and OCH 3 -16/C-16 suggested that three methoxy groups was placed at C-1, C-6 and C-16, respectively. The a-orientation of OCH 3 -1 and OCH 3 -6, and the b-orientation of OCH 3 -16 were established by the ROESY correlations of H-1/H-5, H-6/H-9 and H-13/OCH 3 -16, respectively ( Figure 1S) (Yin et al. 2018). The last methoxy group substituted at oxygenated quaternary carbon C-8 was confirmed by HMBC correlation of OCH 3 -8/C-8, and was further supported by the fact that chemical shift of OCH 3 -8 (d C 48.4 q) was upfield approximately 8 $ 10 when compared with methoxy groups substituted at methines (Wang 1982). Finally, the anisyl group was placed at C-14 on the basis of HMBC correlation of H-14/OCO-14. Therefore, the structure of compound 1 was determined as hemsleyaline.
AChE inhibitors are potential candidates for the treatment of Alzheimer's disease. Several kinds of DAs have been reported to exbihit considerable AChE inhibitory effects, such as denudatine-type, hetisine-type C 20 -DAs, and lycaconitine-type C 19 -DAs (Ahmad et al. 2018;Ahmad et al. 2017;Nisar et al. 2009), which encourage the further extensive exploration on the inhibition effects of DAs on AChE. However, hemsleyaline (1) only showed a moderate AChE inhibitory effect with a IC 50 value of 471 ± 9 lM. Attaur et al. (2000) also repoted two C 19 -aconitine-tpye DAs, faleoconitine and pseudaconitine showed AChE inhibitory effect with IC 50 values of 293 ± 3.8 lM and 278 ± 3.6 lM, respectively, while the other one 3 0 -methoxyacoforestinine was found to be inactive on inhibition of this enzyme (Attaur et al. 2000). The data from this study and those from earlier studies imply that C 19 -aconitine-tpye DAs might be ineffective in inhibition of AChE when compared with C 20 -DAs, but more research is needed to confirm this.

General experimental procedures
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 Bruker AM-400 or DRX-500 spectrometer (Bruker, Karlsruhe, Germany) 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 or 300 $ 400 mesh; Qingdao Haiyang Chemical Factory, Qingdao, China) were used for column chromatography (CC). Fractions were monitored by TLC and visualised by spraying with modified Dragendorff's reagent.

Plant material
A. hemsleyanum roots were collected from Hezhang County in Guizhou Province of China in August 2017, and identified by associate professor Shuda Yang from School of Pharmaceutical Science in Kunming Medical University. A voucher specimen (No. 2017-AH-1) is deposited in Zunyi Medical University.

AChE inhibition assay
AChE inhibition effect was determined spectrophotometrically using acetylthiocholine as substrate by modifying a prvious reported method (Attaur et al. 2000). The reaction was carried out in 100 mM sodium phosphate buffer (PB) at pH 8.0 at 25 C. Briefly, 140 lL of PB, 20 lL AChE, and 20 lL test compound solution were mixed and incubated for 30 min. 10 lL of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) was added, and then the reaction was initiated by adding 10 lL of acetylthiocholine. The hydrolysis of acetylthiocholine was determined by monitoring the formation of the yellow 5-thio-2nitrobenzoate anion as a result of the reaction of DTNB with thiocholine released by the enzymatic hydrolysis of acetylthiocholine at a wavelength of 412 nm. The concentration of the test compound that inhibited the enzyme activity by 50% (IC 50 ) was determined by interpolation using increasing levels of these substances in the assays. Huperzine A was used as a positive control.

Conclusion
A new aconitine-type C 19 -diterpenoid alkaloid hemsleyaline (1) and fourteen known alkaloids (2-14) were isolated from the roots of A. hemsleyanum, whose structures were established on the basis of extensive spectroscopic analyses. Hemsleyaline (1) exhibited mild cholinesterase inhibitory effect with IC 50 value of 471 ± 9 lM.

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