The gathered and combined filtrate was evaporated under vacuum with a Büchi Rotary Evaporator. The obtained extract was dissolved in 700 mL of water. The solution was extracted 3 times with 500 mL of water-saturated n-butanol. The mixed n-butanol phase was evaporated under vacuum and then lyophilized. Prior to pharmacological evaluation, the AG extract was analyzed using HPLC [20] and [21]. The HPLC system

was a Waters Alliance 2960 instrument (Milford, MA, USA) with a quaternary pump, an automatic injector, a photodiode array detector (Model 996), and Waters Millennium 32 software for peak identification and integration. The separation was carried out on a Prodigy ODS(2) column (250 mm × 3.2 mm inner learn more diameter) with a guard column (3.0 mm × 4.0 mm inner diameter) BMS 754807 (Phenomenex, Torrance, CA, USA). For HPLC analysis, a 20-μL sample was injected into the column and eluted at room temperature with a constant flow rate of 1.0 mL/min. For the mobile phase, acetonitrile (solvent A) and water (solvent B) were

used. Gradient elution started with 17.5% solvent A and 82.5% solvent B. Elution solvents were then changed to 21% A for 20 min, then to 26% A for 3 min and held for 19 min, at 36% A for 13 min, at 50% A for 9 min, at 95% A for 2 min, and held for 3 min. Lastly, eluting solvents were changed to 17.5% A for 3 min and held for 8 min. The detection wavelength was set at 202 nm. All sample solutions were filtered through a membrane filter (0.2 μm pore size). The content of the constituents were calculated using the standard curves of 13 ginsenosides. The measurement for the content analysis of the AG was performed in triplicate. The experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Chicago, Chicago, IL, USA. All experiments were carried out in male A/J mice, aged approximately 6 weeks, weighing 18–22 g, obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were maintained under enough controlled room temperature,

humidity, and light (12/12 h light/dark cycle) and allowed ad libitum access to standard mouse chow and tap water. The mice were allowed to acclimate to these conditions for at least 7 days prior to inclusion in the experiments. As shown in Fig. 1, animals were separated into three groups (n = 12 per group): control (or negative control), model, and AG groups. All animals initially received a single intraperitoneal injection of AOM (7.5 mg/kg). One week after the AOM injection (set as Day 1), the animals began to receive 2.5% DSS in drinking water for 8 consecutive days. The animals in AG group also received AG extract 0.15 mg/mL in drinking water for up to 90 consecutive days. We calculated that the daily dose of American ginseng was approximately 30 mg/kg. For the acute phase observation, six animals per group were sacrificed on Day 14. The remaining animals were kept in the chronic phase and were sacrificed on Day 90.