Two new compounds from Verbena bonariensis

Abstract Two new compounds verboncin A (1) and verboncin B (4) and 14 known compounds (2–3 and 5–16) were isolated from Verbena bonariensis, and these 14 compounds were first obtained from this plant. Their chemical structures were established by one and two-dimensional NMR and HRESIMS analysis and the results were compared with literature values. The absolute configuration of 1 was determined by calculating electronic circular dichroism (ECD). The cytotoxicity of some of the compounds against MCF-7, HCT-116, MDA-MB-231, and SW620 human cancer cell lines were evaluated, in which compound 4 showed negligible cytotoxic activity with an IC50 value of 68.08 ± 0.35 µM against the MCF-7 cell line.  


Introduction
Verbena bonariensis belongs to the genus Verbena of the family Verbenaceae. There are roughly 250 species of Verbena in the world. In addition, two to three species were discovered outside of the Eastern Hemisphere, and they are all native to tropical to temperate America. In recent years, studies found that Verbena plants mainly contained volatile oils, flavonoids, triterpenes, iridoids, and other components [1]. Pharmacological studies reported that the crude extracts of the plants in this genus showed anti-inflammatory [2], antibacterial [3], and antioxidant [4]. The active substances were mainly iridoids, which have good anti-cancer and immunomodulatory effects [5]. To further develop the medicinal value of V. bonariensis, phytochemical experiments were carried out, and two new (1 and 4) and 14 known compounds

General experimental procedures
Optical rotations were measured on a Rudolph Autopol VI polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer (Shimadzu, Kyoto, Japan). Circular dichroism (CD) spectra were measured on an Applied Photophysics V100 Chirascan instrument (Applied Photophysics, London, UK). IR spectra were obtained on a Thermo Scientific Nicolet IS10 spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) with KBr pellets. HRESIMS was performed on an Agilent 1100 HPLC-TOF spectrometer (Thermo Fisher Scientific, Massachusetts, USA). One-dimensional (1 D) and two-dimensional (2 D) NMR spectra were recorded on a Bruker AV-400 spectrometer (Bruker, Zurich, Switzerland) using tetramethylsilane as the internal standard. Chemical shifts (d) are expressed in parts per million with reference to the solvent signals. Column chromatography (CC) was performed using silica gel (80-100 mesh and 200-300 mesh; Qingdao Marine Chemical Inc, Qingdao, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Silica gel plates (GF254, Qingdao Marine Chemical Inc. Qingdao, China) were used for analytical thin-layer chromatography (TLC), and spots were visualized by ultraviolet (UV) light (254 nm) and sprayed with Dragendorff's reagent. Semi-preparative high-performance liquid chromatography (HPLC) was performed on an Agilent 1260 liquid chromatography system with a Zorbax SB-C18 column (9.4 mm Â 250 mm, 5 mm), eluted by isocratic acetonitrile-water (85:15, v/v) at 3 ml/min flow rate, and the signal was detected by a UV detector at 254 nm.

Plant material
The leaves of Verbena bonariensis were collected from Xingyi, Guizhou Province, China, in August 2016 and identified by Professor Yu Chen. A voucher specimen (XWL 2016010) was deposited at the Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Yunnan University.

Extraction and isolation
Verbena bonariensis (10 kg) was powdered and then extracted with 90% ethanol (EtOH) three times (three days for each time) at room temperature. The solvent was removed by evaporation under reduced pressure to yield the crude extract, which was then suspended in water and partitioned with petroleum ether (PE) and ethyl acetate (EtOAc). The EtOAc fraction (470 g) was subjected to macroporous adsorbent resin (D-101) with a gradient elution of MeOH/H 2 O (40:60-90:10) to yield five fractions (fractions A-E).

Quantum chemical ECD calculations
The equilibrium population at 298.15 K for each conformation was calculated using the Boltzmann statistic based on the relative free energy. Conformational searches were originally performed using the Spartan'14 program. The main conformation was then optimized by Gaussian 09 at the B3LYP/6-31þþG (d,p) level using density functional theory (DFT) calculations [21]. The theoretical calculation of ECD was performed using time dependent Density Functional Theory (TDDFT) at the B3LYP/ 6-31G level in MeOH. The calculated ECD curve was generated using SpecDis software [22]. The SpecDis program was used to compare the calculated curves with the experimental ECD spectra. The results showed that the theoretically calculated ECD spectrum of 1 was in good agreement with the experimental ECD spectrum of 1 in the region of 200-400 nm.

Cytotoxicity assay in vitro
The anti-proliferative effects of the partially isolated compounds were evaluated. The MCF-7 cell line was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) cell bank. The HCT-116 cell line was purchased from the American Type Culture Collection (ATCC) cell bank. The MDA-MB-231 cell line and SW620 cell line were purchased from the DSMZ cell bank. Cell viability was analyzed using a CCK-8 assay kit [23]. The four cell lines were plated in 96-well plates at a concentration of 5 Â 10 3 cells/well and cultured with the appropriate media (100 ml/ well) for 24 h. Some of these compounds were dissolved in dimethyl sulfoxide (DMSO) and then diluted with the cell culture medium to the desired concentrations (0, 4, 8, 16, 32, and 64 mM). The cells were treated with varying concentrations of compounds for 24 h at 37 C and 5% CO 2 . Cell viability was evaluated using the CCK-8 kit according to the manufacturer's instructions. The culture medium was removed and CCK-8 kit solution (10 ml of CCK-8 kit reagent and 100 ml cell culture medium/well) was added to each well. Then, the plate was incubated for 1.5 h at 37 C and 5% CO 2 . The optical density (OD) was read at 450 nm using a microplate reader.