Discovery and antitumor activities of constituents from Cyrtomium fortumei (J.) Smith rhizomes

Background Cyrtomium fortumei (J.) Smith is an important Chinese herbal medicine because of its biological functions. However, systematic and comprehensive studies on the phytochemicals from Cyrtomium fortumei (J.) Smith and their bioactivity are limited. Results Using the bioassay-guided technique, the ethyl acetate and n-BuOH extracts of the rhizomes of Cyrtomium fortumei (J.) Smith were shown to exhibit good antitumor activities, consequently leading to the isolation of 23 compounds. All compounds were isolated from the plant for the first time. The inhibitory activities of these compounds were investigated on tumor cells MGC-803, PC3, and A375 in vitro by MTT (thiazolyl blue tetrazolium bromide) assay, and the results showed that pimpinellin (3) had potent cytotoxic activities against the three cell lines, with the IC50 values of 14.4 ± 0.3 μM, 20.4 ± 0.5 μM, and 29.2 ± 0.6 μM, respectively. The mechanism of the antitumor action indicated that pimpinellin inhibited the growth of MGC-803 cells via the induction of tumor cell apoptosis, with apoptosis ratio of 27.44% after 72 h of treatment at 20 μM. Conclusions This study suggests that most of the compounds from the roots of Cyrtomium fortumei (J.) Smith could inhibit the growth of human carcinoma cells. Moreover, pimpinellin inhibited the growth of tumor cells via the induction of tumor cell apoptosis.


Background
Cyrtomium fortumei (J.) Smith belongs to the Dryopteridaceae family which comprises approximately 14 genera and 1700 species throughout the world, and is widely spread in tropical and subtropical regions. The Chinese Pharmacopoeia (2005 edition) listed Cyrtomium fortumei (J.) Smith as an official drug, which showed that the plant could be used as anticancer herbs [1]. In the past, people used the rhizomes and extracts from the plant as vermifuges [2]. The plant can be used to cure many diseases, such as influenza, acute and chronic pharyngitis, cancer, and migraine [3]. In addition, the herb was used as antiviral agents to cure severe acute respiratory syndrome, a life-threatening viral respiratory illness believed to be caused by a coronavirus [4]. Furthermore, phoroglucinols and flavonoids are known to display a wide array of pharmacological and biochemical actions, and have been isolated from many species of the Dryopteridaceae family [5]. However, so far the constituents with anticancer activity of the plant remain unclear. Thus it is necessary to identify the potent antitumor compounds from this plant.

Isolation and identification
Dried rhizomes of Cyrtomium fortumei (J.) Smith (10 kg) were cut into pieces and extracted with 80% EtOH (3 × 40 L) under reflux, 6 h for the first time, 3 h for the second time, and 1 h for the last time. The combined EtOH extracts were evaporated to dryness to yield a dried EtOH extract (424 g). The extract was suspended in water and then extracted with petroleum ether (10 L × 3 times), ethyl acetate (10 L × 3 times), and n-BuOH (10 L × 3 times), respectively. Different extracts solvent were then concentrated using evaporator under vacuum at 50°C to afford the petroleum ether extract (46 g), ethyl acetate extract (95 g), and n-BuOH extract (110 g). MTT assay was used to evaluate their antitumor activities. The results suggested that the antitumor agents were mainly contained in ethyl acetate and n-BuOH extracts. Further experiments were performed on ethyl acetate and n-BuOH extracts to separate antitumor compounds. The ethyl acetate and n-BuOH extracts were subjected to column chromatography (CC) to yield 16 fractions. An activity-directed isolation process was used to get compounds, and 23 compounds were obtained from these fractions.
The isolated compounds were identified via spectroscopic analyses, including 1 H-and 13 C-NMR spectroscopy. The results of the analyses were compared with the NMR and IR data reported in literatures to identify the chemical structures. All compounds were isolated from Cyrtomium fortumei (J.) Smith for the first time.

Biological evaluation
The potential effect of the extracts from Cyrtomium fortumei (J.) Smith was investigated on the viability of MGC-803, PC3, A375, and NIH3T3 cells using MTT assay at the concentration of 20 μM, with ADM (Adriamycin) being used as the positive control and DMSO being used as the negative control. The inhibitory percentage of cells was treated with 20 μM or 50 μg/mL of each compound or extract for 72 h ( Table 1). The results showed that the ethyl acetate and n-BuOH extracts had great antitumor activity, and PPI had good activities against the three human cancer cell lines tested than other compounds. The inhibitory ratios of PPI at 72 h after treatment were 67.1% on MGC-803 cells, 57.2% on PC3 cells, 45.8% on A375 cells, and 24.8% on NIH3T3 cells. It could be seen that PPI had good antitumor activities and low cytotoxic effect on normal cell line NIH3T3. Further experiments found that proliferation of these three carcinoma cells were significantly inhibited by PPI in a concentration-dependent manner, as shown in Figure 1. The IC 50 values of PPI against MGC-803, PC3, and A375 cells were determined to be 14.4 ± 0.3 μM, 20.4 ± 0.5 μM, and 29.2 ± 0.6 μM, respectively, which were lower than that on NIH3T3 cells (IC 50 > 100 μM).
To determine whether the growth inhibitory activity of PPI was related to the induction of apoptosis, the morphological character changes of MGC-803 cells were investigated using the AO/EB staining, Hoechst 33258 staining, and TUNEL assay.
The cell morphologic changes after PPI treatment were assessed by fluorescence microscopy after AO/EB staining [6]. AO is a vital dye that could penetrate the normal cell membrane [7], whereas EB will stain only those cells that have lost their membrane integrity [8]. The stained cells revealed four different types under a fluorescence microscope: the chromatin of living cells was green with normal structure; the chromatin of nonapoptotic dead cells was red with normal structure; yellow coloration of early apoptotic cells with morphous in the form of pycnosis; and orange coloration of late apoptotic cells with morphous in the form of pycnosis [9][10][11][12][13]. With hydroxycamptothecine (HCPT) as positive control, the cytotoxicity of PPI at a concentration of 20 μM against MGC-803 cells for 24 h was detected via AO/EB staining.
As can be seen in Figure 2A, green live MGC-803 cells with normal morphology were seen in the negative control group. Green yellow or orange dots were detected in the HCPT after 24 h. After cells were treated with PPI for 24 h, the nuclei stained as yellow green or orange, and the morphology showed pycnosis, membrane blebbing and cell budding. These phenomena indicate PPI could induce apoptosis without any significant cytotoxicity.
As can be seen in Figure 2B, the cells of the negative group (DMSO) were normal blue. However, the cells of HCPT treatment appeared to be compact and condensed. After treatment with PPI for 24 h, most of cell nuclei appeared to be highly condensed and crescentshaped, indicating that PPI induced apoptosis against   MGC-803 cell lines. These results were consistent with AO/EB double staining. In addition, TUNEL, one of the popular methods, identified apoptotic cells in situ via the detection of DNA fragmentation [23,24]. Under a biological microscope, the cells were observed that where brown precipitate was the result of positive apoptosis [25][26][27]. MGC-803 cells were treated PPI and HCPT 20 μM at with for 24 h.
As can be seen in Figure 2C, the cells of the negative group (DMSO) did not appear as brown precipitates, whereas both PPI and HCPT appeared as brown precipitates. Therefore, it can be further concluded that PPI induced apoptosis against MGC-803 cells. The results were identical with the previous experiment.
The apoptosis ratios induced by PPI in tumor cells was quantitatively assessed by FCM [28]. In early apoptotic cells, phosphatidylserine (PS) which distributed inside the lipid bilayer in the normal cells was transferred from the inside of the cell membrane to the outside. Annexin V, a Ca 2+ dependent phospholipid-binding protein with a high affinity for PS, was used to detect early apoptotic cells. PI (Propidine Iodide) was a red fluorescent dye and stained cells that had lost membrane integrity. So, the different periods of apoptotic cells could be distinguished when Annexin V matched with PI: necrotic cells (the upper left quadrant, Annexin -/PI + ), late apoptotic cells (the upper right quadrant, Annexin + /PI + ), intact cells (the lower left quadrant, Annexin -/PI -) and early apoptotic cells (the lower right quadrant, Annexin + /PI -) [29,30]. As shown in Figure 3, with HCPT as positive control, PPI could induce apoptosis of MGC-803 cells, with the highest apoptosis ratio of 27.44% at 72 h after treatment at 20 μM. Furthermore, as shown in Figure 4 the apoptosis of MGC-803 which treated with PPI increased gradually in a time-dependent manner.

General procedures and reagents
The melting points of the products were determined using an XT-4 binocular microscope (Beijing Tech Instrument Co. Ltd., Beijing, China). Infrared spectra were recorded on a Bruker VECTOR22 spectrometer in KBr disks. 1 H-NMR and 13 C-NMR were recorded using a JEOL-ECX500 spectrometer at 22°C, with tetramethylsilane as the internal standard and CDCl 3 , DMSO-d 6 , CD 3 COCD 3 , or CD 3 OD as the solvent. Column chromatography was performed using silica gel (200-300 meshes) (Qingdao Marine Chemistry Co., Qingdao, China) and silica gel H (Qingdao Marine Chemistry Co., Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), HP-20 (Mitsubishi Chemical Corp., Toukyu Met, Japan), YMC RP-18 (YMC Corp., Kyoto, Japan) and MCI-gel CHP 20P (Mitsubishi Chemical Corp., Toukyu Met, Japan). All other chemicals were of analytical reagent grade and used without further purification.

Plant materials
Fresh samples of Cyrtomium fortumei were collected from Longli, Guizhou Province in China, in August 2011. Prof. Qingde Long, Department of Medicine, Guiyang Medical University, identified the plant material. A voucher specimen was deposited at Guiyang Medical University, Guiyang, China.
All spots on TLC were visualized by heating silica gel plates sprayed with 10% Phosphomolybdic acid hydrate in EtOH and 1% FeCl 3 in EtOH.

MTT assay
All tested extracts and compounds were dissolved in DMSO and subsequently diluted in the culture medium before treatment of the cultured cells. When the cells were 80-90% confluent, they were harvested by treatment with a solution containing 0.25% trypsin, thoroughly washed and resuspended in supplemented growth medium. Cells (2 × 10 3 /well) were plated in 100 μL of medium/well in 96well plate. After incubations overnight, the cells were treated with different concentrations of extracts in RPMI 1640 with 10% FBS for 72 h. In parallel, the cells treated with 0.1% DMSO served as negative control and ADM (Adriamycin) as positive control. Finally, 100 μL of MTT (Beyotime Co., Jiangsu, China) was added, and the cells were incubated for 4 h. The MTT-formazan formed by metabolically viable cells was dissolved in 100 μL of SDS for 12 h. The absorbance was then measured at 595 nm with a microplate reader (BIO-RAD, model 680), which is directly proportional to the number of living cells in culture [51].

AO/EB staining
The cells were seeded at a concentration of 5 × 10 4 cell/ mL in a volume of 0.6 mL on a sterile cover slip in 6well tissue culture plates. Following incubation, the medium was removed and replaced with fresh medium plus 10% FBS and then supplemented with compounds. After the treatment period, the cover slip with monolayer cells was inverted on the glass slide with 20 μL of AO/EB (Beyotime Co., Shanghai, China) stain (100 μg/ mL). The fluorescence was read using an IX71SIF-3 fluorescence microscope (OLYMPUS Co., Toukyu Met, Japan).

Hoechst 33258 staining
The cells grown on the sterile cover slip in 6-well tissue culture plates were treated with compounds for a certain range of treatment time. The culture medium containing compounds was removed, and the cells were fixed in 4% paraformaldehyde for 10 min. The cells were washed twice with PBS, and were consequently stained with 0.5 mL of Hoechst 33258 staining (Beyotime Co., Jiangsu, China) for 5 min. The stained nuclei were washed twice with PBS, and were consequently observed under an IX71SIF-3 fluorescence microscope at 350 nm excitation and 460 nm emissions.

TUNEL assay
TUNEL assays were performed using a colorimetric TUNEL apoptosis assay kit according to the manufacturer's instructions (Beyotime). The cells grown in 6-well culture clusters were treated as mentioned in mitochondrial depolarization assay. The MGC-803 cells grown in 6-well tissue culture plates were washed with PBS and fixed in 4% paraformaldehyde for 40 min. The cells were washed once with PBS, and were consequently permeabilized with immunol staining wash buffer (Beyotime) for 2 min on ice. The cells were rewashed once with PBS, and were consequently incubated in 0.3% H 2 O 2 in methanol at room temperature for 20 min to inactivate the endogenous peroxidases, after which the cells were washed thrice with PBS. Thereafter, the cells were incubated with 2 μL of TdT-enzyme and 48 μL of Biotin-dUTP per specimen for 60 min at 37°C. The cells were terminated for 10 min, and were consequently incubated with streptavidin-HRP (50 μL per specimen) conjugate diluted at 1:50 in sample diluent for 30 min. The cells were washed three times with PBS, and were consequently incubated with diaminobenzidine solution (200 μL per specimen) for 10 min. Thereafter, the cells were rewashed twice with PBS, and were consequently imaged under an XDS-1B inverted biological microscope (Chongqing Photoelectric Devices Co. Chongqing, China).

Flow cytometry analysis
Prepared MGC-803 cells (1 × 10 6 /mL) were washed twice with cold PBS and then re-suspended gently in 500 μL binding buffer. Thereafter, cells were stained in 5 μL Annexin V-FITC and shaked well. Finally, 5 μL PI was added to these cells and incubated for 20 min in a dark place, analyzed by FACS Calibur, Becton Dickinson.

Statistical analysis
All statistical analyses were performed using SPSS 10.0, and the data were analyzed using one-way ANOVA. The mean separations were performed using the least significant difference method. Each experiment was performed in triplicate, and all experiments were run thrice and yielded similar results. Measurements from all the replicates were combined, and the treatment effects were analyzed.