Alkaloids from the stem of Ephedra equisetina

Abstract Two new cyclotrypyamine alkaloids equisetinines A and B, as well as three known alkaloids (3–5) were isolated from the stems of Ephedra equisetina Bunge. Their structures were characterized by spectroscopic methods, and the absolute configurations of the new compounds were determined by interpretation of their electronic circular dichroism. Anti-asthmatic activities of compounds were evaluated by releasing β-Hex in C48/80-induced RBL-2H3 cells, and compound 5 exhibited significant anti-asthmatic activities. Graphical Abstract

As a research interest [1], alkaloids from Ephdra had many pharmacological activities such as antipyretic, diaphoretic [2], excitatory central nerve [8], and anti-inflammatory effects [9]. In this article, five alkaloids, containing two new cyclotrypyamine alkaloids were identified from the stem of E. equisetina. And anti-asthmatic activities of all compounds were evaluated by releasing b-Hex in C48/80-induced RBL-2H3 cells ( Figure 1).

Results and discussion
Compound 1 was obtained as white needle crystals with ½a 20 D -100 (c 0.6, CH 3 OH). And its molecular formula was determined to be C 15 H 14    3 0 167.9 a1 H NMR spectra measured at 500 MHz in CD 3 OD; 13 C NMR spectra measured at 125 MHz in CD 3 OD. b1 H NMR spectra measured at 500 MHz in DMSO; 13 C NMR spectra measured at 125 MHz in DMSO. 53.5), two methylenes (d C 46.1, 45.1), and two methines (d C 85.0, 61.4). The NMR data aforementioned were close to product 6 synthesized by Xiao [10]. The main difference among them was that an extra carbonyl at C-3 0 instead of a methyl group attached to C-3 0 and a hydroxy connected to C-6, which was determinted by key HMBC correlations from H-8 [d H 7.43 (1H, d, J ¼ 7.6 Hz)] to C-6 (d C 88.1) and from H-4 [d H 5.00 (1H, d, J ¼ 9.2 Hz)] to C-3 0 (d C 167.9) ( Figure 2). And the calculated electronic circular dichroism (ECD) curve of the (2S,4S,6S) isomer matched well with the experimental ECD curve of compound 1, suggesting that the absolute configuration of compound 1 was (2S,4S,6S) ( Figure 3(A)). Therefore, the structure of compound 1 was determined and named as equisetinine A.
Compound 2 was isolated as white needle crystals with ½a 20 D -89 (c 0.4, CH 3 OH). Its molecular formula was deduced as C 14 H 12 N 2 O 5 based on HRESIMS at m/z 311.0641 [M þ Na] þ . Comparison of the NMR data of 1 with those of 2 (Table 1) revealed that they shared the same skeleton. A detailed analysis of the 1 H and 13 C NMR spectral data of 2, strongly suggested that there was a hydroxyl instead of a methoxyl group attached to ester group in 2. The use of different deuterated reagents resulted in slightly different chemical shift values. Then, the calculated ECD curve of the (2S,4S,6S) isomer matched well with the experimental ECD curve of compound 2, suggesting that the absolute configuration of compound 2 was (2S,4S,6S) (Figure 3(B)). Therefore, the structure of compound 2 was assigned as equisetinine B.
According to the structures of compounds 1 and 2, it is presumed that both are products obtained from tryptophan by a series of reactions, the specific reaction  process of which may be shown in Figure 4. In addition, three known alkaloids were identified as seco-((S)-Pro-(R)-Val) (3) [11], ephedrine (4), pseudoephedrine (5) [12], by comparing their spectroscopic and physical data with those of related references. Then, the isolated compounds were subjected to evaluate their anti-asthmatic activities by releasing b-Hex in C48/80-induced RBL-2H3 cells. As shown in Table 2, compared with the model group, compound 5 exhibited significant anti-asthmatic activities with the b-Hex release rate was 0.88 ± 0.130, however, compounds 1-4 had no anti-asthmatic effects.

ECD spectra calculations
The conformations of 1 and 2 were determined by GMMX software using the MMFF94 force field. The geometry optimizations and predictions of the ECD spectra of the conformers were carried out using density functional theory (DFT) at the B3LYP/6-311G (d, p) level in the Gaussian 16W [13]. SpecDis software, version 1.71 was used to simulate the ECD curves according to Boltzmann distribution theory [14].

In vitro anti-asthmatic evaluation
RBL-2H3 cells used in in vitro experiments were purchased from ATCC (MD, USA). They were cultured in DMEM and 10% heat-inactivated fetal calf serum at 37 C in a water-saturated 5.0% CO 2 incubator (Thermo Scientific, MA, USA). Cells were divided into control (NC), model (M, C48/80, 20 lg ml À1 ), each administration group (10 lM þ C48/80, 20 lg ml À1 ) and total enzyme group (1% Trinton x-100). After 30 min, 50 ll cell supernatant was added with 50 ll b-hexaminoglycosidase substrate solution of 1 mmol L À1 , and the reaction was incubated at 37 C for 1 h. Then, 150 ll Na 2 CO 3 /NaHCO 3 (pH ¼ 10.7) was added to terminate the reaction. The absorbance was measured at 405 nm with a microplate analyzer, and the b-hexaminoglycosidase release rate was calculated.