Anticancer Phenolics from Dryopteris fragrans (L.) Schott

Cancer is one of the most major diseases that threatens human health and life. The aim of this work was to obtain novel anticancer molecules from D. fragrans, a kind of medicinal plant. The structure of the new compound was identified using spectroscopic data (1H-NMR, 13C-NMR and two dimensions NMR). Its anticancer properties were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay against four human cells including lung cancer cells (A549), breast cancer cells (MCF-7), gastric cancer cells (SGC7901) and noncancerous human umbilical vein endothelial cells (HUVEC). A new phenylpropanoid—(E)-caffeic acid-9-O-β-d-xylpyranosyl-(1→2)-β-d-glucopyranosyl ester (1), with seven known compounds (2–8)—was isolated. The IC50 value of compound 1 against MCF-7 cells was 2.65 ± 0.14 µM, and the IC50 values of compound 8 against three cancer cells were below 20 µM.


Introduction
D. fragrans (Figure 1), from the Dryopteris genus of the Dryopteris family, is a deciduous perennial herb that is mostly distributed in Northeast China, Korea Japan, Russia and North America. [1,2]. The chemical components isolated from D. fragrans have exerted many biological effects [3][4][5][6]. In recent years, there have been many studies regarding the anticancer components of D. fragrans. Zhao [7] isolated a new coumarin-Dryofracoumarin A-from D. fragrans, which has cytotoxic activity. Su [8] reported that Dryofragin, a phloroglucinol derivative from D. fragrans, could stop human osteosarcoma U2OS cells from migrating and invading by lowering MMP-2 and MMP-9 expression and up-regulating the expression of TIMP-2 and TIMP-1 through the p38 MAPK and PI3K/AKT signal pathways. Zhong [9] obtained a novel sesquiterpene-Dryofraterpene A-from D. fragrans, which could significantly inhibit the proliferation of five kinds of cancer cell lines.
Thus, to discover new anticancer molecules, the chemical components of D. fragrans and its anticancer bioactivity were studied. This work can provide new resources for research and the development of new drugs against cancer. Thus, to discover new anticancer molecules, the chemical components of D. fragrans and its anticancer bioactivity were studied. This work can provide new resources for research and the development of new drugs against cancer.
In the 13 C-NMR spectrum and DEPT spectrum, it showed an ester carbonyl carbon (δ 167.3) and a pair of trans-double-bond carbons at δ 148.2 and δ 114.5.

Identification of Isolated Compounds
Compound 1 (Figure 2) was puce powder. Its acid hydrolysis yielded D-glucose and D-xylose [10]. The molecular formula-C 20 H 26 O 13 -was deduced from High Resolution Electrospray Ionization Mass Spectrometry (HR-ESI-MS) with an [M + Na] + peak at 497.1274 (calcd. for 497.1271). In the infrared spectrum, it displayed a hydroxyl group (3412 cm −1 ), a carbonyl group (1706 cm −1 ), a double-bond group (1680 cm −1 ) and a benzene ring group (1602 cm −1 ). Thus, to discover new anticancer molecules, the chemical components of D. fragrans and its anticancer bioactivity were studied. This work can provide new resources for research and the development of new drugs against cancer.
In the 13 C-NMR spectrum and DEPT spectrum, it showed an ester carbonyl carbon (δ 167.3) and a pair of trans-double-bond carbons at δ 148.2 and δ 114.5.   In the 13 C-NMR spectrum and DEPT spectrum, it showed an ester carbonyl carbon (δ 167.3) and a pair of trans-double-bond carbons at δ 148.2 and δ 114.5.
In the 1 H Detected Heteronuclear Multiple Bond Correlation (HMBC) spectrum (Figure 3), the correlation of the anomeric proton signal of β-D-glucose at δ 5.68 with the ester carbonyl carbon at δ 167.3 indicated that the ester carbonyl group was located at C-1 . The linkage between C-1 from β-D-xylose and C-2 from β-D-glucose by oxygen was revealed through the HMBC correlations of H-1 /C-2 . On the basis of nuclear magnetic resonance (NMR) data and the relevant literature [12], compound 1 was identified as (E)-caffeic acid-9-O-β-D-xylpyranosyl-(1→2)-β-D-glucopyranosyl ester. The new compound was trivially named Fragranoside B. In the 1 H Detected Heteronuclear Multiple Bond Correlation (HMBC) spectrum (Figure 3), the correlation of the anomeric proton signal of β-D-glucose at δ 5.68 with the ester carbonyl carbon at δ 167.3 indicated that the ester carbonyl group was located at C-1′. The linkage between C-1′′ from β-D-xylose and C-2′ from β-D-glucose by oxygen was revealed through the HMBC correlations of H-1′′/C-2′. On the basis of nuclear magnetic resonance (NMR) data and the relevant literature [12], compound 1 was identified as (E)-caffeic acid-9-O-β-D-xylpyranosyl-(1→2)-β-D-glucopyranosyl ester. The new compound was trivially named Fragranoside B.

In Vitro Cytotoxicity Assay
A549, MCF-7, SGC7901 and human umbilical vein endothelial (HUVEC) cells were measured for cytotoxicity using the MTT assay with taxol as the positive control. As shown in Table 2, compound 1 showed significant inhibitory activity against MCF-7 cells with an IC50 value of 2.65 ± 0.14 µM. Compound 8 showed good cytotoxic activity against A549, MCF-7 and SGC7901 cells with IC50 values of 8.96-19.44 µM. However, the others were not active (IC50 > 50 µM). Notably, compound 1 exhibited no cytotoxic activities against A549, SGC7901 and noncancerous HUVEC cells. It showed good selectivity toward MCF-7. Thus, compound 1 can be considered as a promising lead compound and its anticancer mechanism should be further studied.  (7) [18] and caffeic acid (8) [19] through a comparison with the NMR data in the literature.

In Vitro Cytotoxicity Assay
A549, MCF-7, SGC7901 and human umbilical vein endothelial (HUVEC) cells were measured for cytotoxicity using the MTT assay with taxol as the positive control. As shown in Table 2, compound 1 showed significant inhibitory activity against MCF-7 cells with an IC 50 value of 2.65 ± 0.14 µM. Compound 8 showed good cytotoxic activity against A549, MCF-7 and SGC7901 cells with IC 50 values of 8.96-19.44 µM. However, the others were not active (IC 50 > 50 µM). Notably, compound 1 exhibited no cytotoxic activities against A549, SGC7901 and noncancerous HUVEC cells. It showed good selectivity toward MCF-7. Thus, compound 1 can be considered as a promising lead compound and its anticancer mechanism should be further studied.

Acid Hydrolysis
Compound 1 (5 mg) was hydrolyzed with 10 mL of 0.01 M H 2 SO 4 for 4 h at 100 • C. After cooling, the hydrolysate was neutralized by 0.02 M KOH then extracted by CH 2 Cl 2 . The sugars in the aquatic layer were monitored by Thin-Layer Chromatography (TLC) with BuOH-H 2 O-AcOH (40:10:50, v/v/v, upper BuOH layer) as a developing system when compared with authentic sugars. The TLC plate was sprayed with a vanillin-H 2 SO 4 solvent [20].

Cell Culture
Human A549, MCF-7, SGC7901 and HUVEC cells were obtained from the Cell Library of Committee on Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured at 37 • C, 5% CO 2 in the Roswell Park Memorial Institute (RPMI) medium containing 10% FBS, 100 U/mL penicillin and 100 U/mL streptomycin.

MTT Assay
Anticancer activity was evaluated by the MTT assay. Compounds 1-8 were dissolved in dimethyl sulfoxide (DMSO) and diluted with RPMI medium for appropriate concentrations (0 µM, 0.08 µM, 0.4 µM, 2 µM, 10 µM and 50 µM). A549, MCF-7, SGC7901 and HUVEC cells were seeded in 96-well microtiter plates (100 µL, 5000 cells/well). After 24 h, the medium was removed and 100 µL of tested compounds with various concentrations were added into 96-well microtiter plates for 48 h. Next, 10 µL of MTT was added and the 96-well microtiter plates were incubated for another 4 h. The medium was removed and 150 µL DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured by microplate spectrophotometer (Molecular Devices, Palo Alto, CA, USA) at 570 nm. Taxol was used as the positive control (0 µM, 0.01 µM, 0.04 µM, 0.16 µM, 0.64 µM, and 2.56 µM for 48 h). Half Maximal Inhibitory Concentration (IC 50 ) values were calculated by GraphPad Prism. Data were obtained from three independent assays.