Diterpenoid Alkaloids from the Aerial Parts of Aconitum flavum Hand.-Mazz

Sixteen diterpenoid alkaloids (DAs), including six aconitine-type alkaloids (5 and 9 − 13), seven 7,17-seco-aconitine-type alkaloids (1 − 4, 6 − 8), two napelline-type alkaloids (14 and 15) as well as one veatchine-type alkaloid (16), were isolated from the aerial parts of Aconitum flavum Hand.-Mazz. In which, flavumolines A − D (1 − 4) were four new ones, and flavumoline E (5) was reported as natural compound for the first time. Their chemical structures were elucidated by the analysis of extensive spectroscopic data. The inhibitory activities of these isolates on Cav3.1 low voltage-gated Ca2+ channel, NO production in LPS-activated RAW264.7cells, five human tumor cell lines, as well as acetylcholinesterase (AChE) were tested. Supplementary Information The online version contains supplementary material available at 10.1007/s13659-021-00302-3.


Introduction
Aconitum species represent a large genus in the Ranunclaceae family [1]. It is estimated that there are more than 350 species of Aconitum all over the world, which are widely distributed in the northern temperate zone, of which 173 species are endemic to mainland China [1]. Previously chemical and pharmacological studies shown that the diterpenoid alkaloids (DAs) were the main pharmacological constituents of this genus [2]. So far, around 1300 natural DAs, categorized into C 20 -, C 19 -, and C 18 -families depending on the number of contiguous carbon atoms, have been reported [3], [4], [5]. Meanwhile, they have been the targets of medicinal chemists for a broad range of confirmed pharmacological properties including analgesic, antiarrhythmic, anti-inflammatory, hypotensive, neuroprotective and so on [6].
Aconitum flavum Hand.-Mazz, known as a perennial herb, is mainly distributed in Qinghai, Gansu, and other northwest places in China [7]. As one of the most vital Chinese traditional ethnic minority folk medicine, the roots were commonly used for the treatment of traumatic injuries, rheumatic arthritis, and some other inflammations [7]. Previously phytochemical investigations of this plant have resulted in the isolation of more than 20 DAs from the roots, such as aconitine, mesaconitine, and deoxyaconitine, which were not only considered as the predominant toxic components of this folk medicine, but simultaneously, the significant active constituents [8]. In this study, sixteen DAs including four new 7,17-seco-aconitine-type DAs (flavumolines A − D, 1 − 4), one new natural compound (5), together with 11 known analogues (6 − 16) were identified. These compounds could be divided into four different types: six aconitines (5 and 9 − 13), seven 7,17-seco-aconitines (1 − 4 and 6 − 8), two napellines (14 and 15) as well as one veatchine-type alkaloid (16). All of these isolated compounds were evaluated for their inhibitory effects on Ca v 3.1 low voltage-gated Ca 2+ channel, NO production in LPS-activated RAW264.7 cells, five human tumor cell lines, and acetylcholinesterase (AChE). Reported herein, the isolation, structural determination, and biological activity of these compounds were thoroughly described.
The molecular formula of compounds 2 (C 30 H 37 NO 8 ) and 3 (C 31 H 41 NO 8 ) were established on the analysis of HRESIMS and NMR data. The NMR data of 2 showed the presence of six methylenes, eight methines, three quaternary carbons, and a characteristic trisubstituted intra-annular double bond, in addition to a benzoyl, an aldehyde group and three methoxy groups. All of these spectroscopic data suggested that 2 was an analogue of 13-hydroxylfranchetine (6) [9]. The main difference was the presence of a N-CHO moiety in compound 2 instead of N-ethyl group in 6, which was verified by 1D NMR data and HMBC correlations. The HMBC correlations (Fig. 2) from the singlet for aldehyde group (δ H 7.95, s) to C-19 and C-17 suggested that the aldehyde group was placed at N-atom. On the analysis of ROESY data, the correlations of H-14/H-9, H-16/H-12α/H-17, H-1/H-10 confirmed the α-orientation of benzoyl group at C-14, β-orientation of methoxy at C-16, and α-orientation of methoxy at C-1, respectively. Compared with 2, compound 3 possessed a typical N-ethyl moiety, which were elucidated by the HMBC correlations from H-21 to C-17, C-19. A hydroxyl located at C-3 (δ C 71.0, d) confirmed by the 1 H-1 H COSY correlation of H-2/H-3 and HMBC correlations of H-3 with C-2/5/19. Additionally, the ROESY correlations of H-3/H-1/H-5 confirmed α-orientation of 3-OH. Therefore, the structures of flavumoline B (2) and flavumoline C (3) were confirmed as shown above (Fig. 1).
Biosynthetically, the configurations of H-5, 9, 10, 8-OAc, 13-OH could be ascribed to β-orientation and H-17 to α-orientation, respectively, due to the caged core of aconitine-type C 19 -DA [23]. Then, the configurations of two methoxy groups and one benzoyl at C-6, 16, and 14 were confirmed as α, β, and α, respectively, according to the correlations between H-6/H-5, H-16/H-12α/H-17 and H-14/H-9 (Fig. 3). However, the configuration of 15-OH has not been confirmed yet because of the simultaneous correlations of H-15 with H-7/H-16/16-OCH 3 and 15-OH with H-7/H-16/16-OCH 3. Eventually, the structure of 5 was identified as shown in Fig. 1, which was reported as a natural compound for the first time according to the literature on synthesis [24].
Since diterpenoid alkaloids in Aconitum were reported to commonly treat traumatic injury, arrhythmia, and rheumatism, in which ion channels or inflammation were involved in the pathophysiological process and inhibitors of ion channels or NO release were considered as potential agents for the treatment of these diseases [25], these isolated compounds were evaluated for their inhibitory effects on T-type ion channels using the whole-cell recording patch clamp method, NO production in LPS-activated RAW264.7 cells using Griess assay [26], on five human tumor cell lines [27], as well as acetylcholinesterase (AChE) [28]. As a result, compound 8 (30 μM) exhibited 64.5% inhibitory rate on Ca v 3.1 low voltage-gated Ca 2+ channel. Compounds 3, 4, 5, 6, 7, and 11 showed potential inhibitory effects on NO production ranging from 20% to 32% at 50 μM. Additionally, compound 5 showed potential inhibitory effects on four human tumor cell lines HL-60, A-549, SMMC-7721, MCF-7 with IC 50 value as 16.88, 33.11, 23.97, 24.21 μM in vitro, and no compound showed inhibitory effect on AChE.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1020 polarimeter. UV spectra were detected on a Shmadzu UV-2401PC spectrometer. IR spectra were determined on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. All 1D and 2D NMR spectra were recorded on Bruker DRX-600 spectrometers using TMS as an internal standard. Unless otherwise specified, chemical shifts (δ) were expressed in ppm with reference to the solvent signals. ESIMS and HRESIMS analysis were carried out on Waters Xevo TQS and Aglient G6230 TOF mass spectrometers, respectively. Semi-preparative HPLC was performed on a Waters 2695 HPLC with a 5C 18 -MS-II column [4.6 × 150 mm]. Silica gel (100-200, 200-300 mesh, Qingdao Marine Chemical Co., Ltd., People's Republic of China), and MCI gel (75-150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan) were used for column chromatography. Fractions were monitored by TLC (GF 254, Qingdao Marine Chemical Co., Ltd.), and spots were visualized under a UV lamp at 254 nm or by spraying the Dragendorff' reagent and heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH.

Extraction and Isolation
Air-dried and powdered plant material (aerial parts) of A. flavum (45 kg) was extracted three times (3 × 150 L) with MeOH at room temperature and then concentrated to 3.88 kg under reduce pressure. The crude extract was suspended in 1% HCl followed by basification with 10% aqueous NH 4 OH (pH 9 − 10) and subsequently, extracted with ethyl acetate to afford crude alkaloids (808 g). The total alkaloids fraction was separated on a silica gel column (CHCl 3 /CH 3 Table 1) and 1 H NMR data (see Table 2 Table 1) and 1 H NMR data (see Table 2

T-Type Ion Channel Inhibitory Activity Assay
All experiments were performed at room temperature (~ 22 °C). Pipettes were fabricated from borosilicate glass