0.5 g of sample added to 25.0 ml methanol was sonicated for 30 min. The mixture was centrifuged at 4000 rpm and 4C for 5 min. The supernatant was filtered by a 0.22 lm pore size filter and used for HPLC analysis. For RSG commercial estrogen receptor pathway concentrated extract, 0.5 g sample was mixed with 5 ml water using a vortex mixer. Then, 20 ml methanol was added and thoroughly mixed again. The solution was centrifuged at 4000 rpm for 5 min.The supernatant was then filtered by a 0.22 lm pore size filter and used for HPLC analysis. Data analysis Data analysis was carried out by OriginPro Version 6.0 software. As recommended by State Food and Drug Administration, Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine was used for evaluating the similarities between different samples. Results and discussion HPLC optimization and HDAC constituent identification The mobile phase system of HPLC was optimized to develop the fingerprint for RSG.
The criteria of theprocedure included the number of peaks, the resolution between peaks and the separation duration. Considering the ROCK kinase presence of flavonoids in RSG, a small amount of acetic acid was added in the mobile phase to reduce the ionization and lower the polarity of these compounds. Two organic solvents, methanol and acetonitrile, with different gradient programs were compared. It was found that the system with methanol had a longer analysis duration than that with acetonitrile. Finally, an acetonitrile water system with a linear gradient program of 16 21% in 0 15 min, 21 40% in 15 40 min was chosen for its good baseline resolution and suitable analysis duration. UV spectra of each peak in the chromatogram were obtained using the DAD detector. The optimal detection wavelength was selected to be 291 nm, because most peaks showed maximum absorption at this wavelength. Figure 1a shows the chromatogram of RSG samples under optimal separation conditions. As can be seen, the separation could be completed within 40 min. Nine peaks were found and all the peaks were well separated from each other. The structural identification of each peak in the chromatogram paclitaxel was carried out by the ESI MS/MS technique. It was found that the negative ESI mode was more sensitive than the positive ESI mode in the present experiment.
Therefore, negative ESI was chosen for the analysis, and the m/z data were. In the ESI MS experiment, the molecular weight of each peak could be obtained. From MS2 spectra, information on the fragment of each peak could be provided. The detailed damage results are listed in Table 1. Peaks 1, 3, 4, 7 and 9 were unambiguously identified as 5 O caffeoylshikimic acid, astilbin, taxifolin, engeletin and trans resveratrol, respectively, by comparing the tR, UV and characteristic ESI MS/MS spectrum of those peaks with standards. The MS spectrum of 5 O caffeoylshikimic acid showed a ion at m/z 335, and its product ion at m/z 179 corresponded to the caffeoyl residue in the molecule. In the MS2 ionization mode, flavonoids combined with rhamnopyranosyl would lose the residue and show characteristic m/z data at. Astilbin and engeletin are flavone glycosides with molecular weights of 450 and 434, respectively. Their product ions at m/z of 303 and 287 resulted from the loss of rhamnosyl residues.