Identification and Growth Inhibitory Activity of the Chemical Constituents from Imperata Cylindrica Aerial Part Ethyl Acetate Extract

Imperata cylindrica (L.) Raeusch. (IMP) aerial part ethyl acetate extract has anti-proliferative, pro-apoptotic, and pro-oxidative effects towards colorectal cancer in vitro. The chemical constituents of IMP aerial part ethyl acetate extract were isolated using high-performance liquid chromatography (HPLC) and identified with tandem mass spectrometry (ESI-MS/MS) in combination with ultraviolet-visible spectrophotometry and 400 MHz NMR. The growth inhibitory effects of each identified component on BT-549 (breast) and HT-29 (colon) cancer cell lines were evaluated after 48/72 h treatment by MTT assay. Four isolated compounds were identified as trans-p-Coumaric acid (1); 2-Methoxyestrone (2); 11, 16-Dihydroxypregn-4-ene-3, 20-dione (3); and Tricin (4). Compounds (2), (3), and (4) exhibited considerable growth inhibitory activities against BT-549 and HT-29 cancer cell lines. Compounds (2), (3), and (4) are potential candidates for novel anti-cancer agents against breast and colorectal cancers.

Cancer is one of the leading causes of death worldwide. Herbal medicines are commonly used as both complementary ingredients and alternative therapies in cancer treatments. Potential bio-active components from herbal medicines can be isolated and purified using a high-performance liquid chromatography (HPLC) system. A tandem MS/MS detection system providing fragmentation information of the targets is one of the best choices adopted in chemical structural characterization and drug discovery [8,9].
Our previous study demonstrated that IMP aerial part ethyl acetate extract had growth-inhibiting, pro-apoptotic, and pro-oxidative effects on a colorectal cancer cell line HT-29 in vitro [10]. The present study aims to isolate the chemical constituents from IMP aerial part ethyl acetate extract and identify the bio-active compounds with considerable growth inhibitory activity against cancers.   Chemical structures of four compounds isolated from IMP aerial part ethyl acetate extract are shown in Figure 1.  The MS/MS fragmentation pattern of compound (1) accurately matched with the MS 2 spectrum from the NIST14 mass spectral database and the trans-p-Coumaric standard ( Figure 3).

Figure 2.
HPLC-DAD chromatogram of IMP aerial part ethyl acetate extract at 323 nm. IMP aerial part ethyl acetate extract solution (10 mg/mL in methanol, 20 µL) was analyzed in the 60 min HPLC gradient program. (A) The retention time of the trans-p-Coumaric acid standard (0.125 mg/mL in methanol, 20 µL) purchased from Sigma (B) was consistent with compound (1) in IMP aerial part ethyl acetate extract fingerprint.
The MS/MS fragmentation pattern of compound (1) accurately matched with the MS 2 spectrum from the NIST14 mass spectral database and the trans-p-Coumaric standard ( Figure 3).

Compound (4): Tricin
The molecular formula of compound (4), C 17 H 14 O 7 , was identified by comparing the MS/MS spectrum with the published literature [11] (Figure 6A,B). The UV spectrum ( Figure 6C), obtained using λ max at 351 nm, was consistent with the previous description [12].  . DEPT spectra revealed that there were two primary carbons, five tertiary carbons, and ten quaternary carbons. A signal at δ3.887 (s, 6H) observed in the 1H-NMR spectrum and a signal at δ 56.32 observed in the 13C-NMR spectrum indicated that there were two equivalent methoxy groups. The NMR results (Figure 7) were consistent with the published data [13,14]. . DEPT spectra revealed that there were two primary carbons, five tertiary carbons, and ten quaternary carbons. A signal at δ3.887 (s, 6H) observed in the 1H-NMR spectrum and a signal at δ 56.32 observed in the 13C-NMR spectrum indicated that there were two equivalent methoxy groups. The NMR results ( Figure 7) were consistent with the published data [13,14]. Compounds (1)-(4) identified by tandem mass spectrometry (MS 2 ) are listed in Table 1. Quantitative analysis of each isolated and purified compound in IMP aerial part ethyl acetate extract was determined by HPLC-DAD. The linearity of the calibration curve, limit of detection (LOD), and limit of quantification (LOQ) are listed in Table 2.  11.02 0.59 ± 0.041 a y, the value of peak area (by HPLC-DAD at 323 nm); x, the value of concentration (mg/mL); b R 2 , correlation coefficient for six points on the calibration curves (n = 3); c LOD, limit of detection (S/N = 3); d LOQ, limit of quantification (S/N = 10).

Growth Inhibitory Evaluation of Compounds (1)-(4) on Breast Cancer and Colorectal Cancer In Vitro
The purified dried powder of each compound was dissolved in DMSO with a gradient of concentrations (µM). The growth inhibitory effects of compounds (1)-(4) on BT-549 (breast cancer cell line) were evaluated after 48/72 h treatment by MTT assay (Figure 8). Data are presented as mean values ±SD from three independent studies (n = 3). Compounds (1)-(4) identified by tandem mass spectrometry (MS 2 ) are listed in Table 1. Quantitative analysis of each isolated and purified compound in IMP aerial part ethyl acetate extract was determined by HPLC-DAD. The linearity of the calibration curve, limit of detection (LOD), and limit of quantification (LOQ) are listed in Table 2.  11.02 0.59 ± 0.041 a y, the value of peak area (by HPLC-DAD at 323 nm); x, the value of concentration (mg/mL); b R 2 , correlation coefficient for six points on the calibration curves (n = 3); c LOD, limit of detection (S/N = 3); d LOQ, limit of quantification (S/N = 10).

Growth Inhibitory Evaluation of Compounds (1)-(4) on Breast Cancer and Colorectal Cancer In Vitro
The purified dried powder of each compound was dissolved in DMSO with a gradient of concentrations (µM). The growth inhibitory effects of compounds (1)-(4) on BT-549 (breast cancer cell line) were evaluated after 48/72 h treatment by MTT assay (Figure 8). Data are presented as mean values ±SD from three independent studies (n = 3). The growth inhibitory effects of compounds (1)-(4) on HT-29 (colon cancer cell line) are shown in Figure 9.  Table 3). The growth inhibitory effects of compounds (1)-(4) on HT-29 (colon cancer cell line) are shown in Figure 9. The growth inhibitory effects of compounds (1)-(4) on HT-29 (colon cancer cell line) are shown in Figure 9.  Table 3).  Table 3).
Compound (1), trans-p-Coumaric acid, was able to induce apoptosis of HCT-15 colon cancer cells through a ROS-mitochondrial pathway with an IC 50 value of 1400 µM [15]. Natural trans-p-Coumaric acid exists in a wide variety of edible plants. The phenolic components from flaxseed oil was reported to have cytotoxic and pro-oxidant effects on MCF-7 human breast cancer cells [16]. The high gastric absorption efficiency of p-Coumaric acid was observed in rats, which makes it a potential bio-active compound in vivo [17]. Compound (2), 2-Methoxyestrone, is one kind of metabolite of estrone and estradiol. It is worth mentioning that 2-Methoxyestradiol was under a phase II clinical trial and expected to be a novel oral drug against multiple human melanoma, including breast cancer and ovarian cancer [18,19]. Metabolic inter-conversion between 2-Methoxyestrone and 2-Methoxyestradiol are based on the enzymatic catalyze reactions. Reductive activity promotes 2-Methoxyestrone conversion to 2-Methoxyestradiol. 2-Methoxyestrone can be formed by the enzymatic oxidation of 2-Methoxyestradiol [20]. Our study first reported the growth inhibitory activities of compound (3), 11, 16-Dihydroxypregn-4-ene-3, 20-dione, against BT-549 and HT-29 cancer cell lines. The structure of 11, 16-Dihydroxypregn-4-ene-3, 20-dione is similar to the well-known endogenous steroid (11α-Hydroxyprogesterone, C 21 H 30 O 3 ). Transformations of 11α-Hydroxyprogesterone generate a series of metabolites. Amounts of metabolites with different isoforms were identified as novel candidates of steroid drugs [21]. The molecular mechanisms of 11, 16-Dihydroxypregn-4-ene-3, 20-dione against cancers can be further investigated. Compound (4), Tricin, a well-studied bio-active flavonoid, is widely distributed in rice bran and bamboo leaves [22,23]. A previous study also isolated Tricin from the aerial part of Imperata cylindrica (L.) Beauv. [5]. Tricin was reported to have remarkable anti-cancer potential against SW-480 colon cancer cells and MDA-MB-468 breast cancer cells, and is safe for clinical development as a cancer preventive agent [24][25][26][27][28].

Cells, Chemicals and Reagents
BT-549 and HT-29 cell lines were obtained from ATCC (Manassas, VA, USA). BT-549 and HT-29 cells were cultured at 37 • C in a humidified atmosphere of 5% CO 2 in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA). Acetonitrile (ACN) (E. Merck, Darmstadt, Germany), Methanol (E. Merck, Darmstadt, Germany) and trifluoroacetic acid (TFA) (Sigma Aldrich, St. Louis, MO, USA) were of HPLC grade, and distilled and deionized water (ddH 2 O) was prepared using a Millipore water purification system (Millipore, Milford, MA, USA). All other reagents used in this study were of analytical reagent grade or higher and purchased from Sigma Aldrich.

Preparation of Powder Extract of IMP Aerial Part
The extraction method was described previously [10].

HPLC Analysis
The HPLC fingerprint was analyzed on a HP1100 series system (Santa Clara, CA, USA) equipped with a diode-array detector. An extract solution of 50 mg/mL (dissolved in methanol) was filtered with a 0.22 µm polytetrafluoroethylene (PTFE) membrane. A 15 µL sample was injected to a semi-preparative HPLC column (ALLTIMA C18, 5 µm, 250 mm × 10 mm i.d. Hichrom, Searle, UK) and detected at 323 nm. The initial mobile phase composed of solvent A (0.1% TFA in ddH 2 O) and solvent B (100% methanol). The gradient for the HPLC analysis was programmed as follows: 0-5 min, 65% B; 5-15 min, 70% B; at a flow rate of 1.5 mL/min; 15-25 min, 80% B, at a flow rate of 1.0 mL/min; 25-40 min, 85% B, at a flow rate of 0.8 mL/min; 40-50 min, 100% B, at a flow rate of 2.0 mL/min, and then was held for additional 5 min.

Mass Spectrometry
The identification of each purified component was performed on a tandem mass spectrometer equipped with an electrospray ionization source. Each purified compound was dissolved in methanol at an appropriate concentration and was infused into the QTRAP 5500 mass spectrometer system (AB SCIEX, Framingham, MA, USA) equipped with a Turbo VTM Spray ion source. Multiple reaction monitoring (MRM) in both positive and negative mode was used to enhance the selectivity of detection. The source-dependent parameters for the mass spectrometer (MS) were set as follows: ion spray voltage (IS) = ±5500 V; curtain gas (CUR) = 20 psi; collision gas (CAD) = 10 psi; nebulizer gas (GS1) = 12 psi, heater gas (GS2) = 0 psi, and source temperature (TEM) = 0 • C. The fraction-dependent parameters were set as follows: declustering potential (DP) = +120.0 V/−130.0 V; entrance potential (EP) = ±10.0 V; collision cell exit potential (CXP) = ±13.0 V. The MS/MS optimized collision energy applied to compounds (1)-(4) was given as follows: 25 V in positive mode, 25 V in negative mode, 25 V in negative mode, and 40 V in positive mode, respectively. For trans-p-Coumaric acid standard, the collision energy applied was 10 V in positive mode. Raw data and images of spectra were generated by Analyst ® Software (Redwood, CA, USA) and modified using Excel ® (Redmond, WA, USA).

Ultraviolet-Visible Spectrophotometry
The UV spectrum of compound (4) was measured using a Shimadzu UV-3600 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Each absorption spectrum was recorded from 200.00 nm to 400.00 nm. Profiles were generated by UVProbe 2.21 Software (Shimadzu Corporation, Kyoto, Japan). A control of 100% methanol was set and auto-zeroed automatically by software.

Quantitative Analysis
The content of each identified compound in IMP aerial part ethyl acetate extract was determined using a HPLC-DAD system. The linearity and range was evaluated by constructing a calibration curve (peak area vs concentration). Quantification was performed upon six levels of external standards. The limit of detection (LOD) was determined as the concentration with a signal-to-noise ratio of three, and the limit of quantification (LOQ) was determined as the concentration with a signal-to-noise ratio of ten.

MTT Assay
The growth inhibitory effects of compounds (1)-(4) on HT-29 (colon) and BT-549 (breast) cancer cell lines were evaluated. Cells were seeded at 4 × 10 3 cells per 96-well and incubated for 24 h. The cells were then treated by 0.5% DMSO (as solvent control) or various concentrations of compounds (as treatment group) and incubated at 37 • C for 48 and 72 h. The MTT assay and data analysis were performed as previously described [29].

Data Analysis
All statistics were calculated with SPSS 17.0 software and data were expressed as mean ± standard deviation (SD) for each analyte. For MS/MS spectrometry analysis, each mass spectrum shown was the average spectra of each sample detected with ten repetitions in each analysis. Compounds were identified by comparing the tandem mass (MS/MS) fragmentation patterns with those in the literatures, NIST14 mass spectral database, and the MS Search Program v.2.2 (National Institute of Standards and Technology, Gaithersburg, MD, USA). For the viability assay, a nonlinear regression test was applied to obtain a fit curve (R 2 > 0.98). Analysis of the differences between the 48/72 h treatment groups was carried out by one-way ANOVA (coupled with a post-test, Dunnett's test) with * p < 0.05.
Author Contributions: Y.W. and W.S.H. designed the experiments and wrote the paper; Y.W. and J.Z.S. purified each compound by HPLC system; Y.W.C. conducted the tandem MS analysis and analyzed the data; Y.W. identified each compound; J.Z.S. performed the MTT assay and evaluated the growth inhibitory activities of each purified compound against breast cancer and colon cancer cell lines. All authors approved the final manuscript.