Procedure to extract and purify flavones A and B
The compounds were obtained as described before . Briefly, flavone A was purified from dried flowers of Gnaphalium elegans extracted with chloroform using a silica gel chromatography column. Flavone B was purified from leaves of Achyrocline bogotensis, using chloroform, followed by crystallizations in hexane. The physical and spectroscopic properties of these compounds allowed their proper identification.
Stock solution and standards
Stock solutions of flavone A at a concentration of 100 µg/mL, prepared as described previously , and 25 µg/mL celecoxib (Toronto Research Chemicals; Toronto, ON, CA) were prepared with acetonitrile/water/acetic acid/triethylamine (60:40:0.2:0.05). Stock solutions of 100 µg/mL of flavone B, prepared as described previously , and 25 µg/mL diclofenac (MP Biomedicals, LLC; Solon, OH) were prepared with acetonitrile/water/acetic acid/trimethylamine (70:30:0.2:0.05). All stock solutions were stored protected from light at 4 °C. HPLC grade acetonitrile, acetic acid, trimethylamine, and water were purchased from Fisher Scientific (Pittsburgh, PA). Flavone A or flavone B were mixed with polyethylene glycol 400 (Electron Microscopy Sciences; Hatfield, PA) for intravenous injection.
Colon tissue was homogenized using a PowerGen 700 from Fisher Scientific (Pittsburgh, PA) in a 1:2 ratio with water (1 mg/2 mL). Serial concentrations for calibration curves (flavone A: 250–100,000 ng/g and flavone B: 1000–25,000 ng/g) were prepared. Briefly, 100 μL of blank homogenate was spiked with 100 μL flavone, 100 µL internal standard (25 µg/mL celecoxib or diclofenac), and 200 μL of organic solvent (acetonitrile). The samples were vortex mixed before being centrifuged for 15 min at 3000×g. The supernatant was removed and filtered with a PDVF filter (0.45 µm) into a clean tube and evaporated using a Labconco vacuum concentrator (Kansas City, MO). Mobile phase (200 μL) was used to reconstitute the residue and 100 μL of sample was injected into the HPLC column. Analysis was conducted in triplicate.
HPLC conditions and quantitation
HPLC assays were performed using a Shimadzu liquid chromatography system (Shimadzu Scientific Instruments Inc., Columbia, Maryland, USA) with an ACE C18 (100 × 4.6 mm) (Aberdeen, Scotland) column. Mobile phases used for HPLC contained acetonitrile/water 60:40 (flavone A) and 70:30 (flavone B) with 0.2% acetic acid and 0.05% triethylamine. Detection wavelength was at 245 nm with a temperature of 30 °C. Flow rate was 0.4 mL/min with run times of 11 and 10 min, respectively. LC solutions program was used to collect and analyze the data.
Animals and drug administration
The methods described here were used to determine the concentrations of flavone A or flavone B in colon tissue collected from male Sprague–Dawley rats (Charles River Laboratories, Raleigh, NC, USA) used in a previous study . Briefly, flavones were mixed in polyethylene glycol 400 and were administered by intravenous injection to deliver a 20 mg/kg dose of flavone A (n = 6) or flavone B (n = 6). Animals were euthanized under anesthesia 6 h post dosing. Colon tissue was collected and flash frozen using dry ice and stored at −80 °C until analyzed for the measurement of concentrations of flavones.