# COX2-Inhibitory and Cytotoxic Activities of Phytoconstituents of _Matricaria chamomilla_ L.
## 2 Materials and Methods  
### Phytochemical Analysis  
Chemicals  
Nine standards were used as reference compounds. The compounds were _cis_-GMCA [(_Z_)-2-\(\beta\)-D-glucopyranosyloxy-4-methoxycinnamicacid] (**2**), _trans_-GMCA [(_E_)-2-\(\beta\)-D-glucopyranosyloxy-4-methoxycinnamic acid] (**4**), quercetin-7-O-\(\beta\)-D-glucopyranoside (**3**), apigenin-7-O-\(\beta\)-D-glucose (**6**), apigenin 7-O-(6\({}^{\prime\prime}\)-O-acetyl-\(\beta\)-D-glucopyranoside) (7), tonghosu [2-(2\({}^{\prime}A^{\prime}\)-hexadiynylidene)-1,6-dioxaspiro [4; 4]-non-3-ene] (**9**) were isolated at the National Center for Natural Products Research (NCNPR), University of Mississippi, Oxford, MS, USA. The identity and purity of these compounds are confirmed by chromatographic (TLC, HPLC) methods, and the analysis of the spectral data (IR, 1D- and 2D-NMR, ESI-HRMS) in comparison with published spectral data is also confirmed. The purity of these isolated compounds was found to be greater than 95%. Chlorogenic acid (**1**) and apigenin (**8**) were purchased from Chromadex (Santa Ana, CA, USA, purity greater than 97%). Luteolin-7-O-\(\beta\)-D-glucose (**5**) was purchased from Indofine Chemical Company, Inc. (Hillsborough, NJ, USA, purity greater than 97%). Acetonitrile and formic acid were of HPLC grade and purchased from Fisher Scientific (Fair Lawn, NJ, USA). Water for the HPLC mobile phase was purified using a Milli-Q system (Millipore) [29].  
Instrumentation and Chromatographic Conditions of Liquid Chromatography with a Diode Array Detector-Quadrupole Time-of-Flight Mass Spectrometry (LC-DAD-QToF)  
The phytochemical analysis using liquid chromatography-diode array detector-quadrupole time-of-flight mass spectrometry (LC-DAD-QToF) was performed according to recently published protocols [29; 30]. An Agilent 1290 Series liquid chromatographic system was used (Agilent Technologies, Santa Clara, CA, USA), comprising a binary pump, a vacuum solvent degasser, an autosampler with 108-vial well-plate trays, a thermostatically controlled column compartment, and a diode array detector. The separation was achieved on an Agilent Poroshell 120 EC-C18 column (150 mm \(\times\) 2.1 mm I.D., 2.7 um). The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) at a flow rate of 0.25 mL/min. Analysis was performed using the following gradient elution: 90% A/10% B to 60%A/40%B in 15 min; in the next 15 min to 100% B. Each run was followed by a 5-min wash with 100% B and an equilibration period of 5 min with initial conditions of 90% A/10% B. One microliter of sample was injected, and the column temperature was maintained at 40 \({}^{\circ}\)C. The mass spectrometric analysis was performed with a QToF-MS (Model #G6545B, Agilent Technologies, Santa Clara, CA, USA) equipped with an ESI source using the following parameters: drying gas (N\({}_{2}\)) flow rate, 13 L/min; drying gas temperature, 325 \({}^{\circ}\)C; nebulizer pressure, 35 psig; sheath gas temperature, 300 \({}^{\circ}\)C; sheath gas flow, 11 L/min; capillary voltage, 3500 V; nozzle voltage, 0 V; skimmer, 65 V; Oct RF V, 750 V; and fragmentor voltage, 175 V. All the operations, acquisition, and analysis of data were controlled using Agilent MassHunter Acquisition Software Ver. A.10.1 and processed with MassHunter Qualitative Analysis Software Ver. B.7.00. Each sample was analyzed in positive and negative modes over the range of \(m/z=100\)-1700 and extended dynamic range (flight time to \(m/z=1700\) at a 2 GHz acquisition rate). Accurate mass measurements were obtained by means of reference ion correction using reference masses at \(m/z=121.0509\) (protonated purine) and 922.0098 [protonated hexakis (1H, 1H, 3H-tetrafluoropropoxy) phosphazine, or HP-921] in positive ion mode. The UV detection wavelength was in the range of 200-500 nm. The phytochemical analysis with UHPLC-DAD-QToF was performed according to recently published protocols [29; 30].