Phosphospecific mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases (ERK)1/2, c-Jun NH2-terminal kinases (JNK), and p38 were purchased from Cell Signaling Technology (Beverly, MA). ERK1/2, JNK, p38, inhibitory kappa-Ba heme oxygenase-1 (HO-1), and ��-actin were purchased from Santa Cruz Biotechnology (Santa kinase inhibitor Vorinostat Cruz, CA). Plant materials The roots of NJ were purchased from a standard commercial source (Omni Herb, Seoul, South Korea). The herb��s identity was confirmed at Wonkwang University. Voucher specimens were deposited at the College of Oriental Medicine Herbarium of Wonkwang University. The NJ roots were prepared by decocting the dried prescription of herbs (100 g) with boiling distilled water (1 L). The decoction time was approximately 2 h.

The water extract was frozen at -80 ��C and then freeze-dried to be powdered (7.35 g, 7.35 w/w%). Preparation of NJ4 fraction The water extract (3.8 g) was subjected to octadecyl functionalized silica gel flash column (5 cm �� 20 cm; 63-200 ��m particle size) chromatography (Figure (Figure1).1). The column was eluted with a stepwise gradient with 500 mL aliquots of MeOH in H2O (starting from 10% and followed by 20%, up to 100% at 20% increments), affording 6 fractions (NJ1: 1.24 g; NJ2: 79.1 mg; NJ3: 490.7 mg; NJ4: 416.2 mg; NJ5: 273.1 mg; NJ6: 67.8 mg). NJ4 (60% MeOH) in saline was used as the main fraction (Figure (Figure11). Figure 1 Fractionation of the aqueous extract of Nardostachys jatamansi. NJ: Nardostachys jatamansi; HPLC: High-performance liquid chromatography. Preparation of NJ4 fraction A portion (9.

0 mg) of the fraction eluted with 60% MeOH inH2O (NJ4-2) was then purified by semi-preparative reversed-phase high-performance liquid chromatography (HPLC) [Shiseido Capcell Pak C18 column (10 mm �� 250 mm; 5 ��m particle size); 2 mL/min; detection at 254 nm] eluting with a gradient from 40% to 70% MeOH in H2O (0.1% formic acid) over 30 min to yield NJ4-2 (3.0 mg, tR = 28.0 min) (Figure (Figure11). HPLC sample preparation and HPLC conditions For HPLC analysis, the chromatographic system consisted of a pump (3000 HPLC pump; Dionex Association, United States), a ultraviolet detector (Photodiode array detector; Dionex Association), and an autosampler (Waters Association, United States). A hydrosphere C18 column (4.6 mm �� 250 mm, 5 ��m) was used.

Water-methanol glacial (50:50) was used as the mobile phase. Detection of the peaks was made at 254 nm and the sensitivity was set at 0.5 absorbance units full scales. The injection volume was 10 ��L and the flow rate was 1.0 mL/min. A standard solution was prepared Entinostat by dissolving in distilled methanol (10 ��g/10 mL). The solution was filtered through a 0.45 ��m membrane filter and applied to HPLC (Figure (Figure22). Figure 2 High-performance liquid chromatography findings of the aqueous extract of Nardostachys jatamansi (A) and the 4th fraction of Nardostachys jatamansi (B).