Flavonoids from Eucommia ulmoides and their in vitro hepatoprotective activities

Abstract A phytochemical investigation of the barks of Eucommia ulmoides Oliv. resulted in the isolation of 18 flavonoids (1-18). The new compound, eucommiaflavone (1) was structurally elucidated by various spectroscopic analyses. In particular, Mo2(OAc)4-induced circular dichroism (ICD) analysis was applied to determine the absolute configuration of 1. Furthermore, five flavonoids (4, 9, 11, 13, and 15) revealed significant in vitro hepatoprotective activity against D-galactosamine-induced cytotoxicity in human hepatoma HepG2 cells. Graphical Abstract


Introduction
Eucommia ulmoides Oliv., belonging to the family of Eucommiaceae, is the sole species of the genus Eucommia (Anderson and Cronquist 1982). It is a deciduous tree which is mainly distributed in the south of China, and known as Du-Zhong in Chinese. Various parts, including the leaves, stems, barks, and flowers have been used in traditional Chinese medicine (TCM) to tonify liver and kidney and cure impotence, hypertension, hyperlipemia, diabetes, and osteoporosis (Du 2003;Guo et al. 2015;Qi et al. 2018). Previous phytochemical investigation on E. ulmoides has demonstrated the presence of flavonoids, lignans, iridoids, phenolics, steroids and terpenoids (Bianco et al. 1974(Bianco et al. , 1978(Bianco et al. , 1982He et al. 2014;Yan, Shi, et al. 2017;Yan, Zhao, et al. 2017), which possess a number of biological activities, such as anti-hypertensive, hypolipidemic, hepatoprotective, anti-aging, and anti-tumor activities. (He et al. 2014;Jiang et al. 2016;Wang et al. 2019).
During our continuous chemical studies on searching for novel hepatoprotective flavonoids from traditional Chinese medicines (Jie et al. 2012;Jiang et al. 2016;Cheng et al. 2019), a phytochemical investigation of the bark of E. ulmoides was conducted. Herein, we report the isolation and structural elucidation of 18 flavonoids (1-18), including a new compound named as eucommiaflavone (1). The in vitro hepatoprotective activities of the flavonoids against D-galactosamine-induced cytotoxicity in human hepatoma HepG2 cells were also reported.

Results and discussion
Compound 1 was obtained as a yellow powder with [a] 25 D -25.0 (c 0.04 MeOH). Its molecular formula was determined as C 20 H 18 O 6 on the basis of HR-ESI-MS at m/z 355.1185 [M þ H] þ (calcd. 355.1182), which indicated 12 degrees of unsaturation. The 1 H and 13 C NMR spectra exhibited characteristic resonances at d H 6.76 (1H, s, H-3) and d C 176.3 (C-4) for the flavone skeleton. Furthermore, a set of AA'BB' type aromatic proton resonances at d H 7.92 (2H, d, J ¼ 8.6 Hz, C-2', 6') and 6.93 (2H, d, J ¼ 8.6 Hz, C-3', 5') were assignable to ring B, and two separated aromatic proton singlets at d H 7.96 (H-5) and 7.11 (H-8) were assignable to ring A. In addition, the NMR resonances due to an isoprenoid unit were also observed, which included two coupled oxygenated methine protons at d H 4.30 (1H, d, J ¼ 4.1 Hz, H-9), 5.28 (1H, d, J ¼ 4.1 Hz, H-10), and two methyl groups at d H 1.19 and 1.15 (each 3H, s, H 3 -12 and H 3 -13), corresponding to the carbons at d C 98.4, 70.1, 25.6, and 25.4, respectively, and a quaternary carbon at d C 69.7. The structure was further elucidated by detailed interpretation of the HMBC data. Namely, the key HMBC correlations from H-5 to C-6 and C-11, and from H 3 -12 and H 3 -13 to C-11 indicated the quaternary carbon C-11 was attached to C-6 of the flavone. The hemiacetal carbon C-9 was linked to C-7 through an ether bond by the HMBC correlations from H-9 to C-7. The relative configurations of the vicinal dihydroxy moiety at C-9 and C-10 was determined as cis by the coupling constant of J H-9,10 ¼ 4.1 Hz and the NOESY correlations between H 3 -13/H-9 and H 3 -13/H-10. The absolute configurations were determined by induced ECD (ICD) method using transition metal chelate dimolybdenum tetraacetate reagent Mo 2 (OAc) 4 . According to the Snatzke rule (Bari et al. 2001). Namely, the positive cotton effect at 310 nm in the ICD spectrum indicated the dihedral angle of Mo-complex is clockwise, and thus 9S, 10R. On the basis of these data, the structure of 1 was unambiguously established and named eucommiaflavone.
The flavonoids (1-18) were evaluated for their in vitro hepatoprotective activities against D-galactosamine-induced cytotoxicity in human hepatoma HepG2 cells. As shown in Table S2 (Supplementary material), five flavonoids (4, 9, 11, 13, and 15) revealed significant in vitro hepatoprotective activity at a concentration of 10 lM, and the potency is comparable to the positive control compound, bicyclol. At the same concentration, flavonoids 11, 13 and 15 did not affect cell proliferation, whereas flavonoids 4 and 9 showed very weak cytotoxicity. Four (9, 11, 13, and 15) out of five bioactive flavonoids were classified into chalcones, suggesting the potential of this type of flavonoids as favorite hepatoprotective agents. It is noteworthy that at a high concentration, several flavonoids showed cytotoxicity against the HepG2 cells (IC 50 < 50 lM), maybe due to the isoprenoid substitutions in the structure Karel 2014).

General experimental procedures
HR-ESI-MS was performed on a Waters Xevo G2-S UPLC-Q/TOF-MS (Milford, MA). IR spectrum was recorded on a Brucker Tensor II spectrometer (Billerica, MA) using an FT-IR microscope transmission method. UV spectra was measured on a UNICO 2102PCS polarimeter (Dayton, NJ). NMR spectra was obtained on a Bruker AM-600 spectrometer (Billerica, MA), using TMS as an internal standard. Optical rotations were recorded on a Rudolph Autopol V automatic polarimeter (Hackettstown, NJ). ECD spectra was performed on a JASCO J-815 spectropolarimeter (Tokyo, Japan). A Waters e2695 HPLC system equipped with a 2998 PDA detector and a RP C 18 reversed-phase silica gel column (4.6 Â 250 mm, YMC 5 lm, Japan) was used for analytical HPLC. A Waters 2535 preparative HPLC equipped with DAD Detector and a RP C18 reversedphase silica gel column (20 Â 250 mm, YMC 5 lm, Japan) were used for sample purification. Column chromatography was performed on Silica gel (60 F254 MERCK, USA), Sephadex LH-20 gel (GE Healthcare Co. Ltd., USA), and ODS (40-75 lm, Merck Darmstadt, Germany). All solvents used were of analytical grade (Concord Technology Co. Ltd., Tianjin, China).

Plant material
The dried bark of Eucommia ulmoides Oliv. was obtained from Anguo, Hebei Province, China in August 2013, and authenticated by Prof. Lijuan Zhang (Tianjin University of Traditional Chinese Medicine). A voucher specimen (NO.201308-DZ) was deposited in the School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine.

Extraction and isolation
The dried bark (9.0 kg) was refluxed with 95% ethanol (2 h Â 3), and the concentrated extract suspended in H 2 O and successively partitioned with petroleum ether, dichloromethane, ethyl acetate (EtOAc) and n-butanol to give the corresponding extracts. 5 g of the obtained EtOAc residue was for sample preservation, and the rest part (70 g) was fractionated by column chromatography on silica gel, and eluted with a gradient of CH 2 Cl 2 :CH 3 OH (100:0-0:100) to afford nine fractions (Fr. E1-Fr. E9).

Induced ECD (ICD) of compound 1
Compound 1 (0.5 mg) was added to a DMSO solution containing Mo 2 (OAc) 4 (0.6 mg/ mL), and reacted at room temperature for 30 min. Then a Mo-complex of compound 1 and Mo 2 (OAc) 4 was formed in situ and the ICD spectrum measured on a JASCO J-815 spectropolarimeter.

Bioassay
The cytotoxic assay against HepG2 cells were measured by the MTT method (Meerloo et al. 2011) at three concentrations of 10 lM, 25 lM, 50 lM. At the selected concentration of 10 lM, the hepatoprotective bioassay was tested as follows: 100 lL of HepG2 cells (ATCC) were seeded into 96-well plates (1.2 Â 10 4 cells/well) in DMEM containing 10% FBS, and incubated at 37 C for 24 h. The test samples (10 lM) or bicyclol (positive control, 10 lM) were added into the wells and cultured for 2 h. The incubated cells were exposed to 30 mM D-galactosamine for 24 h. Then, 0.5 mg/mL MTT was added to each well and incubated for 2.5 h. After discarding the supernatant, 150 lL DMSO was added to dissolve formazan and then the absorbance was measured at 490 nm by a microplate reader. Experiments were performed in triplicate.

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

Funding
This work was financially supported by National Natural Science Foundation of China (Grant numbers: 81430095 and 81303180).