Discovery of Three New Monoterpenoid Indole Alkaloids from the Leaves of Gardneria multiflora and Their Vasorelaxant and AChE Inhibitory Activities

Three novel monoterpenoid indole alkaloids gardflorine A (1), gardflorine B (2), and gardflorine C (3) were isolated from the leaves of Gardneria multiflora. Their structures, including absolute configurations, were established on the basis of spectroscopic methods (MS, UV, IR, 1D and 2D NMR) and circular dichroism experiments. All the compounds were evaluated for their vasorelaxant and acetylcholinesterase (AChE) inhibitory activities. Compound 1 exhibited potent vasorelaxant activity, with an EC50 value of 8.7 μM, and compounds 2 and 3 showed moderate acetylcholinesterase (AChE) inhibitory activities, with IC50 values of 26.8 and 29.2 μM, respectively.


Introduction
Monoterpenoid indole alkaloids (MIAs) are important secondary metabolites widely distributed in the members of plant families Apocynaceae, Loganiaceae, and Rubiaceae [1]. These compounds have attracted considerable interest in drug research for their complex structures and diverse biological activities, such as ganglion blocking [2], anticancer [3][4][5], anti-inflammatory [6], antibacterial [7], vasorelaxant [8], and neuroprotective activities [9]. The plant Gardneria multiflora (Loganiaceae family) is widely distributed in the south of the Qinling Mountains-Huaihe River Line and north of the Nanling Mountains in China [10]. The roots and leaves of G. multiflora are widely used as medicine for the treatment of arthrophlogosis and sciatica owing to their effects of expelling wind and activating blood flow [10]. Previous phytochemical investigations of plants from this genus led to the isolation of more than 50 compounds [11,12], most of them were identified as MIAs [13][14][15][16][17].
In search of novel and bioactive alkaloids, we carried out a phytochemical investigation on the constituents of the leaves of G. multiflora. In our present study, three new alkaloids (Figure 1), named gardflorine A (1), gardflorine B (2), and gardflorine C (3), were isolated from the leaves of G. multiflora. Their structures were elucidated by using spectroscopic methods and electronic circular dichroism (ECD) calculation. Additionally, compound 1 exhibited potent vasorelaxant activity, with an EC 50 value of 8.7 µM, while, compound 1 exhibited potent vasorelaxant activity, with an EC50 value of 8.7 μM, while, compounds 2 and 3 showed moderate AChE inhibitory activities, with IC50 values of 26.8 and 29.2 μM, respectively. Herein, we describe the isolation, structural elucidation, and biological activities of 1-3.

Results and Discussion
Compound 1 was obtained as white oil, and its molecular formula was established as C20H24N2O3 by HR-ESI-MS at m/z 341.1859 [M + H] + (calcd for C20H25N2O3, 341.1860). The IR spectrum of 1 indicated the presence of amino group (3355 cm −1 ), carbonyl group (1736 cm −1 ), and aromatic ring (1609 and 1464 cm −1 ). The 1 H NMR spectrum of 1 (Table 1) showed signals for an ortho-disubstituted benzene ring [δH 7.08 (1H, dd, J = 7.5, 1. 2), and one methyl (δC 11.8). With the aid of 2D NMR spectra, the 1 H and 13 C NMR signals of 1 were assigned as shown in Table 1.

General Experimental Procedures
UV and IR spectra were obtained on a JASCO V-550 spectrophotometer and a JASCO FI/IR-480 Plus Fourier transform infrared spectrometer, respectively. CD spectra were recorded on a Chirascan spectropolarimeter. Optical rotations were determined with a JASCO P-1020 Automatic Polarimeter. HR-ESI-MS data were obtained using an Agilent 6210 ESI/TOF mass spectrometer. NMR experiments were performed on Bruker AV-600 and AV-400 spectrometers. HPLC was carried out on an Agilent 1260 chromatograph and a semi-preparative chromatograph with a DAD detector.

Plant Materials
The leaves of Gardneria multiflora were collected in Yangchang Town, Longli County, Guizhou province, in June 2018 and identified by Dr. Ying Zhang of Jinan University. A  In order to explore the scientific connotation of the traditional use of G. multiflora, the vasorelaxant and AChE inhibitory activities in vitro of compounds 1-3 were evaluated. Among them, compound 1 exhibited potent vasorelaxant activity, with an EC50 value of 8.7 μM (EC50 = 0.1 μM for positive control phentolamine mesylate). Moreover, compounds 2 and 3 exhibited moderate AChE inhibitory activities, with IC50 values of 26.8 and 29.2 μM, respectively (Supplementary Materials).

General Experimental Procedures
UV and IR spectra were obtained on a JASCO V-550 spectrophotometer and a JASCO FI/IR-480 Plus Fourier transform infrared spectrometer, respectively. CD spectra were recorded on a Chirascan spectropolarimeter. Optical rotations were determined with a JASCO P-1020 Automatic Polarimeter. HR-ESI-MS data were obtained using an Agilent 6210 ESI/TOF mass spectrometer. NMR experiments were performed on Bruker AV-600 and AV-400 spectrometers. HPLC was carried out on an Agilent 1260 chromatograph and a semi-preparative chromatograph with a DAD detector.

Plant Materials
The leaves of Gardneria multiflora were collected in Yangchang Town, Longli County, Guizhou province, in June 2018 and identified by Dr. Ying Zhang of Jinan University. A

General Experimental Procedures
UV and IR spectra were obtained on a JASCO V-550 spectrophotometer and a JASCO FI/IR-480 Plus Fourier transform infrared spectrometer, respectively. CD spectra were recorded on a Chirascan spectropolarimeter. Optical rotations were determined with a JASCO P-1020 Automatic Polarimeter. HR-ESI-MS data were obtained using an Agilent 6210 ESI/TOF mass spectrometer. NMR experiments were performed on Bruker AV-600 and AV-400 spectrometers. HPLC was carried out on an Agilent 1260 chromatograph and a semi-preparative chromatograph with a DAD detector.

Plant Materials
The leaves of Gardneria multiflora were collected in Yangchang Town, Longli County, Guizhou province, in June 2018 and identified by Dr. Ying Zhang of Jinan University. A voucher specimen has been deposited at the Medical College of Jiaying University (No. MCJU-021).

Extraction and Isolation
The dried, powdered leaves of G. multiflora (20 kg) were percolated with 95% ethanol (100 L×3). After evaporation of solvent in vacuum, the residue (1.5 kg) was suspended in water, and the pH was adjusted to 2-3 by 5% HCl and then partitioned with chloroform; thus, the chloroform layer and acid water layer were obtained. The acid water layer was then adjusted to pH 9-10 by ammonia water, chloroform extraction was carried out, and the crude total alkaloid (chloroform part) was obtained. The chloroform extract (31.3 g) was subjected to a silica gel column eluting with chloroform/methanol (100:0 to 0:100, v/v) to afford 8 fractions (A1-A8). Then, fraction A4 was further separated by a silica gel, ODS, Sephadex LH-20 columns, and preparative HPLC to afford 1 (2.7 mg). Fraction A6 was successively separated on Sephadex LH-20 (MeOH) a20nd purified by preparative HPLC with MeOH-H 2 O-Et 2 NH (60:40:0.0002) to afford 2 (6.6 mg) and 3 (5.9 mg).

Vasorelaxant Assay
The vasorelaxant activity of these isolates against KCl-induced contractions of rat renal artery rings was measured as described previously [20][21][22]. Renal arteries were removed rapidly out from SD rats, immediately placed into 4 • C oxygenated K-H solution, cleaned of its surrounding fat and connective tissues, and then cut into portions of about 2 mm in length. Each segment was mounted in a Multi Myograph System (Danish Myo Technology A/S, Denmark) and then bathed in K-H solution [composition (in mM): NaCl, 120; KCl, 4.6; KH 2 PO 4 , 1.2; MgSO 4 , 1.2; NaHCO 3 , 25; glucose, 10; CaCl 2 , 2.5], bubbled with 95% O 2 -5% CO 2 , and maintained at 37 • C. The isometric tension of renal artery rings was collected by four-channel physiological force transducers. All the rings were set to an optimal tension of 2 g and stabilized in normal K-H solution for 90 min. The rings were then contracted by 0.5 µM phenylephrine and challenged with 3 µM acetylcholine to confirm the integrity of the endothelium. Endothelium-intact rings contraction was evoked by a depolarizing KCl (60 mM) solution. The EC 50 values of the test compounds and the positive control (phentolamine mesylate) were calculated from cumulative concentration-tension curves by linear regression.

AChE Inhibitory Activity Assay
The AChE inhibitory activities of the isolated compounds were assayed by a modified Ellman's method [6,23]. Compounds and positive control were dissolved in 1% DMSO. The phosphate buffer (pH 8.0), tacrine, test compounds, and acetylcholinesterase (0.02 µM) were added, in sequence to 96-well plates and incubated for 20 min (30 • C). The reaction was initiated by the addition of 20 µL of 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) (0.625 mM) and 20 µL of acetylthiocholine iodide (0.625 mM) for the AChE inhibitory activity assay, respectively. The optical density was measured at 405 nm by an ELISA microplate reader. Tacrine (IC 50 0.33 µM) was used as positive control. All the reactions were performed in triplicate. The percentage inhibition (I%) was calculated as follows: I% = (1 − S)/E × 100 (S is the absorbance of the test compound-containing reaction, and E is the absorbance of the control reaction).

Conclusions
In summary, three new monoterpenoid indole alkaloids (1-3) were isolated and identified from the leaves of G. multiflora. The new compounds were elucidated by spectroscopic analyses and computational calculation. Moreover, the vasorelaxant and AChE inhibitory activities of all isolates were tested. Compound 1 exhibited potent vasorelaxant activity, with an EC 50 value of 8.7 µM, while compounds 2 and 3 exhibited AChE inhibitory activities, with IC 50 values of 26.8 and 29.2 µM, respectively. The discovery of the new alkaloids expands the family of MIAs and provides reference for further structure-activity discussions in future research.
Supplementary Materials: The following are available online. Figure S1: Leaves of G. multiflora, Tables S1 and S2: biological activities of compounds 1-3, Figures