Effect of claycosin and formononetin on 16HBE cell proliferation.

<p>(A) Effect of claycosin and formononetin on 16HBE cell proliferation for 12 h. (B) Effect of claycosin and formononetin on 16HBE cell proliferation for 24 h. (mean±SD, n = 3). All the experiments were performed in triplicates.</p

Comparative chromatograms of fingerprint of YPFS extract, final wash eluate, blank desorption eluate (in which YPFS extract was replaced by RPMI 1640) and 16HBE-binding desorption eluate.

<p>Detection was at 254 nm. All the experiments were performed in triplicates.</p

Effect of calycosin and formononetin administered at the initial stage of sensitization on ear thickness and histopathological changes in ACD mice.

<p>(A) Flow charts of compound administration in ACD murine model. (B) Effect of calycosin and formononetin on ear thickness. (mean±SD, n = 8, *p<0.05, **p<0.01, ***p<0.001). (B) Histopathological changes of dermatitis examined by H&E staining. All the experiments were performed in triplicates.</p

Effect of claycosin and formononetin on the production of TSLP at the initial stage in a murine model of ACD <i>in vivo</i>.

<p>TSLP in ear homogenates was analyzed by ELISA and total protein was determined by BCA kit. TSLP level was assessed with the formula: concentration of TSLP in homogenate/total protein (pg/mg). (mean±SD, n = 8, *p<0.05, **p<0.01, ***p<0.001). All the experiments were performed in triplicates.</p

Peak assignment for the components from desorption eluate.

<p>Peak assignment for the components from desorption eluate.</p

RRT and RPA of each peak in fingerprint of YPFS extract.

<p>RRT and RPA of each peak in fingerprint of YPFS extract.</p

Effect of claycosin and formononetin on NF-κB translocation.

<p>NF-κB translocation in 16HBE cells was examined by immunofluorescence assay. n = 3. All the experiments were performed in triplicates.</p

Determination of components in serum from YPFS-treated mice and identification of YPFS extract chemicals binding to 16HBE cells by HPLC-MS.

<p>(A) UV visible spectrum of YPFS components I–V in the 16HBE-binding desorption eluate corresponding to the standard materials at 254 nm. (B) ESI-MS negative ionization spectrum of compound I and its structural formula (inset). Calycosin-7-glucoside, (+)ESI-MS m/z was 447[M+H]⁺(m/z). (C) ESI-MS negative ionization spectrum of compound II and its structural formula (inset). Ononin, (+)ESI-MS m/z was 431[M+H]⁺(m/z). (D) ESI-MS negative ionization spectrum of compound III and its structural formula (inset). Claycosin, (+)ESI-MS m/z was 285[M+H]⁺(m/z). (E) ESI-MS negative ionization spectrum of compound IV and its structural formula (inset). Sec-o-glucosylhamaudol, (+)ESI-MS m/z was 439[M+H]⁺(m/z). (F) ESI-MS negative ionization spectrum of compound V and its structural formula (inset). Formononetin, (+)ESI-MS m/z was 269[M+H]⁺(m/z). (G) UV spectrum of serum from YPFS-treated mice, compared with fingerprint of YPFS extract and control serum at 254 nm. (H) UV spectrum of components a–c detected in serum of YPFS-treated mice corresponding to the standard materials at 254 nm. (I) ESI-MS negative ionization spectrum of compound “a” and its structural formula (inset). Cimifugin, (+)ESI-MS m/z was 307[M+H]⁺(m/z). All the experiments were performed in triplicates.</p

Effect of claycosin and formononetin on the transcriptional activation of NF-κB induced by TNF-α.

<p>16HBE cells transfected with pNFκB-TA-luc were pretreated with claycosin or formononetin for 2 h or treated with medium only as a control, then stimulated with TNF-α(100 ng/mL) for 1 h. Transcriptional activation of NF-κB was measured in the cellular extracts using a dual luciferase reported gene assay kit. (A) Effect of claycosin on the transcriptional activation of NF-κB. (B) Effect of formononetin on the transcriptional activation of NF-κB. (mean±SD, n = 3, ***p<0.001). All the experiments were performed in triplicates.</p

HPLC fingerprint of YPFS extract at 254 nm.

<p>YPFS was detected by HPLC-MS with an optimal condition for obtaining maximum peaks. There were 32 main peaks in YPFS extract. All the experiments were performed in triplicates.</p