Flavonoids from the leaves of Epimedium Koreanum Nakai and their potential cytotoxic activities

Abstract Phytochemical studies on the leaves of Epimedium koreanum Nakai have resulted in the discovery of two new flavonol glycosides, koreanoside F (1) and koreanoside G (2), along with six known flavonoids. Their structures were elucidated on the basis of HRESIMS, UV, IR, 1 D NMR and 2 D NMR data. Absolute configurations of 1 and 2 was further determined by 13C-NMR spectra with gate decoupling (GD). All of the compounds were evaluated for cytotoxic activities by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay. The results indicated that compounds 3, 5, 6, 7 and 8 inhibited the proliferation of A549 and NCI-292 cells with IC50 values of 5.7–23.5 μM. Real-time monitoring in three kinds of lung cancer cells and a kind of human bronchial epithelial cells treated with compound 6 was also assessed. Graphical Abstract


Introduction
Genus Epimedium, belonging to the family of Berberidaceae, is widely cultivated in Eastern Asia. In Chinese Pharmacopoeia, Epimedium brevicornum Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim., Epimedium wushanense Ying and Epimedium koreanum Nakai have been recorded as traditional medicinal plants (Pharmacopoeia Commission of PRC 2015). The aerial parts of those plants have been used for alleviating a variety of disorders. Previous phytochemical investigations on genus Epimedium revealed the occurrence of two mainly types of compounds, flavonoid and dihydro-phenanthrene derivative (Chen et al. 2008;Liang HR et al. 1997;Sun et al. 1995;1998). Notably, a great many of flavonoids from E. koreanum (Epimedium koreanum Nakai) were substituted with prenyl groups. And the prenylated compounds gained increasing attention because of their diverse biological activities, including antimicrobial (Tantry et al. 2012), anti-osteoporosis (Liang et al. 2012;Zeng et al. 2014), anti-inflammation (Chi et al. 2001), anti-cancer (Liang et al. 2012;Tong et al. 2011) and so on. In our ongoing program, two new flavonol glycosides, koreanoside F (1) and koreanoside G (2), together with six known flavonoids have been isolated from the leaves of E. koreanum (Figure 1). Cytotoxic activities were evaluated by using MTT assay. Herein, we report the isolation, structural elucidation and bioassay of these compounds. In addition, in order to further investigate the cytotoxic of compound 6, real-time monitoring was also measured. Herein, we report the isolation, structural elucidation and bioassay of these compounds.

Results and discussion
2.1. Structure elucidation of compounds 1 and 2 Compound 1 was isolated as pale-yellow amorphous powder. Its molecular formula was determined as C 27 H 28 O 11 by the analysis of HRESIMS spectrum (m/z 529.1704 [M þ H] þ , calcd. for 529.1710). The UV spectrum showed maxima at 270, 300, and 350 nm. The IR spectrum showed bands due to hydroxyl (3339 cm À1 ) and benzyl (2980, 1657 and 1454 cm À1 ) functions. The 1 H NMR spectrum of 1 revealed the presence of a flavone skeleton, which was suggested by two aromatic protons (A ring) at d H 6.87 (1 H, s, H-5) and 6.85 (1 H, s, H-8), a 1,4-disubstituted aromatic system observed for the four B ring protons at d H 7.13 (2 H, d, J ¼ 8.5 Hz, H-3 0 , H-5 0 ) and 7.95 (2 H, d, J ¼ 8.5 Hz, H-2 0 , H-6 0 ). With the aid of HSQC and HMBC experiments, 15 carbon resonances were assigned to the flavone skeleton. The 1 H and 13 C NMR spectra also revealed the presence of a 2-(1-hydroxy-1-methylethyl)-3-hydroxy furan ring, which showed methyl proton signals at d H 1.64 (6 H, s, H-14, H-15) with carbon signals at d C 160.4 (C-11), 164.9 (C-12), 69.6 (C-13) and 28.9 (C-14, C-15) ( Table S1). The location of this furan ring was assigned to C-7 and C-8 according to the HMBC correlations from H-5 to C-6 (d C 110.6), C-11 (d C 160.4), and C-12 (d C 164.9) ( Figure S1). A methoxy substitution was revealed by the signals at d H 3.91 (3 H, s, OMe-4 0 ) and 56.0 (OMe-4 0 ). The HMBC correlation from OMe-4 0 to C-4 0 (d C 163.6) assigned the methoxy group to be at C-4 0 ( Figure S1). d H 0.92 (3 H, d, J ¼ 6.0 Hz, H-6 00 ) is characteristic for L-rhamnopyranose. The HMBC experiment showed the correlation between H-1 00 (d H 5.46, 1 H, d, J ¼ 0.8 Hz) and C-3 (d C 137.2), indicating the sugar residue was assigned to C-3. Complete assignment of the remaining resonances in the 13 C NMR spectrum of 1 was discerned by analysis of the HSQC and HMBC data. On the basis of above analyses, the relative configuration of compound 1 was identified.
However, the absolute configuration determination of this compound was not conducted. L-rhamnopyranose's dominant energy conformation is ' 1 C 4 ', means the C-2 00 OH of L-rhamnopyranose is located on the 'a' bond. Therefore, the absolute configuration of glycoside bond cannot be simply deduced by the coupling constant of the anomeric proton due to the same dihedral angle between H-1 00 and H-2 00 in two kinds of configurations. But there are differences in chemical shift of C-3 00 , C-5 00 and 1 J C-H of C-1 00 between a-L-rhamnopyranose and b-L-rhamnopyranose. To confirm the configuration, a kind of 13 C-NMR, GD spectrum was applied to measure the C-H coupling constant of the C-1 00 . The comparation of the positions of C-3 00 (d C 72.3), C-5 00 (d C 68.2) and coupling constant (J ¼ 174.3 Hz) with literature measured in same solvent indicated this sugar to be a-L-rhamnopyranose (Pei et al. 2011;Tanaka 1985). Thus, the structure of 1 was elucidated and named as koreanoside F.

Cytotoxic activities
Compounds 1-8 were tested for their cytotoxicity against lung cancer A549 and NCI-H292 cells by using MTT method (Table S2). The results showed that compound 6 was the most potent among the 8 compounds, while compound 1 and 2 were almost nontoxic. Compared the structures of these compounds, it suggested that the prenyl, hydroxyl and glycosyl groups within the flavonoids were critical for the lung cancer cells viability inhibitory effects. Glycosylation of the flavonoid and hydroxylation of the prenyl decreased the cytotoxicity of the compounds, this may be related to the increasing of hydrophilicity of the compounds. Compound 6 is an aglycone with two intact prenyls, this may be the reason that it possesses the most potent effect in these 8 compounds. Compound 5 and 7 were structurally similar with 6, only one hydroxyl substitution in one prenyl decreased the cytotoxicity. To further determine the viability inhibitory effects of 6, A549, Calu1 and H1299 were treated with 6 at doses of 3.13, 6.25, 12.5 and 25.0 lM. BEAS-2B cells were treated at doses of 6.25, 12.5 and 25.0 lM. The viability of cells was monitored using xCELLigence system, which allows for dynamic monitoring of cell growth in response to treatment in real-time. Both 12.5 and 25.0 lM of 6 showed significate inhibitory effect on proliferation against lung cancer cell A549, Calu1 and H1299. 12.5 lM of 6 displayed no toxic on human bronchial epithelial cells BEAS-2B and inhibited proliferation of three kinds of cancer cells at the same time ( Figure S21).

Plant material
The leaves of E. koreanum were purchased from Jianlian Traditional Chinese Medicine herbs shop in August 2017 and authenticated by one of the co-authors Tao Shen. A voucher specimen (No. 20170810-10) has been deposited in the School of Pharmaceutical Sciences, Shandong University.

Cell lines and cell culture
The human lung cancer cell lines A549, NCI-H292, Calu1, H1299 and human bronchial epithelial cells BEAS-2B were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI 1640 or DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/mL penicillin and 100 U/mL streptomycin at 37 C in a humidified incubator containing 5% CO 2 .

Measurement of cell viability
The cytotoxicity of compounds 1-8 were evaluated by MTT method. Lung cancer NCI-H292 and A549 cells were used for the assay. In brief, 1 Â 10 4 cells per well were seeded in a 96-well plate and incubated overnight. After treating with several concentrations of compounds for 48 h, 20 L of 2.0 mg/mL MTT solution was added and incubated for an additional 4 h, the plate was centrifuged and the medium was removed. For each well, 100 L of DMSO was added and crystals were dissolved by shaking the plate at room temperature. Absorbance was measured at 570 nm by a microplate reader. Triplicate wells were used for each sample and the experiments were repeated at least three times to get means and standard deviations. Cisplatin (CDDP) was used as positive control (Zhao et al. 2016).

xCELLigence system
Real-time cell monitoring was conducted using the xCELLigence system (ACEA Biosciences, San Diego, CA, USA). The system use impedance as readout. Cells grow on top of electrodes so that the impedance varies based on the number of cells attached and the quality of cell-electrode interaction. Electrode impedance, which is displayed as Cell Index (CI), can be used to monitor cell viability, number, morphology, and cell adhesion (Urcan et al. 2010). In this experiment, A549, Calu1, H1299 and BEAS-2B cells were seeded onto E-plates with the number of 8000 per well, after an overnight incubation, cells were exposed to compound 6 (3.13, 6.25, 12.5, and 25.0 lM). The impedances were measured per 30 min for 3 days and expressed as CI values (Hu et al. 2012;Huang et al. 2017).

Supplementary material
Supplementary material relating to this paper is available online: key HMBC and 1 H-1 H correlations of 1 and 2 ( Figure S1), IR, UV, HRESIMS, 1D and 2D NMR spectra of 1 and 2 ( Figure S2-S20), Real-time monitoring of cell viability treated with compound 6 ( Figure S21), 1 H and 13 C NMR data of compounds 1 and 2 (Table S1) and MTT data (Table S2).

Disclosure statement
The authors declared no conflict of interest.

Funding
This work was financially supported by the Key Research and Development Program of Shandong Province (No. 2018GSF118085 and2017GSF218049)