, Hong Kong); then extracted solutions were filtered. This procedure was repeated three times. Thus we employed this solvent to standardize the extraction and to focus on screening. Approximately 300 mL of each extracted solution was collected http://www.selleckchem.com/products/Bicalutamide(Casodex).html and, then concentrated by rotary evaporation (EYELA, Tokyo Rikakikai Co., Ltd., Japan) under vacuum in a 60��C water bath. All the extracts were finally subjected to lyophilization (LABCONCO, Laboratory Construction Company, MO, USA) at ?40��C under vacuum of 105 ��bar. Each yield of plants was powdered and mixed until uniform, and then stored at 4��C for later use. Dimethyl sulfoxide (DMSO) was used as the solvent to dissolve the extract, and it was loaded as the vehicle control for all cell cultures.

Although the aqueous extract of HLJDT have been used in some studies, we found that the ethanol extract was ideal to isolate most of the bioactive compounds with higher quantity from HLJDT when compared to aqueous extract (data not shown). It was reported that ethanol can extract higher concentrations of flavonoid, polyphenols and more alkaloid compounds as compared to aqueous extract [17],[18]. Thus we employed this solvent to standardize the extraction and to focus on screening. Chromatographic Conditions HPLC was carried out on an Agilent 1100 series with a G1315A diode array detector (California, USA). An Alltima? C18 column (250��4.60 mm, particle size 5 ��m) was used for separations. HPLC conditions were as follows: eluent A, 0.1% formic acid in H2O; eluent B, methanol with a linear gradient elution (0 min, 15% B; 0~15 min, 15%��38% B; 15~30 min, 38%��90% B; 30~30.

1 min, 90%��100% B; 30.1~33 min, 100% B; 33~33.1 min, 100%��15% B; 33.1~36 min, 15% B) at a flow of 1 mL/min. Peaks were assigned by matching their retention times with that of each reference compound eluted in parallel with the same mobile phase. The concentrations of the analytes were determined from representative calibration curves. From the HPLC profiles of ethanolic extract of HLJDT and its individual herbs, we have found that, in comparing the concentrations of six known components (geniposide, berberine, palmatine, baicalein, baicalin and wogonin) that can be found in ethanolic extract, berberine is most abundant (6.02%), followed by geniposide (4.01%), baicalin (2.67%), baicalein (1.31%), palmatine (0.867%) and wogonin (0.

615%) (Figure 1B; Table S1). Based on the four herbal components of HLJDT, we know that geniposide is contributed by FG; berberine and palmatine are mainly found in RC and CP, respectively; and baicalin, baicalein and wogonin are contributed by RS. Recent studies have shown that berberine, baicalein GSK-3 and geniposide have neuroprotective effects in Alzheimer��s disease models [15],[19],[20]. Therefore, these three compounds (Figure 1A) were used for quantitative analysis of ethanolic extract of HLJDT. Figure 1 Selected components of Huang-Liang-Jie-Du-Tang (HLJDT).