Shengli Han

Screening active compounds acting on the epidermal growth factor receptor from Radix scutellariae via cell membrane chromatography online coupled with HPLC/MS

Radix scutellariae is a traditional Chinese medicine (TCM) and has many pharmacological effects, including antiviral, antibacterial, antifungal, antipyretic, hypotensive, anti-inflammatory and anti-anaphylaxis effects. However, few studies have screened the active compounds in this complex product for tumor therapy. In this study, a two-dimensional online method was developed to screen the active compounds from Radix scutellariae acting on the epidermal growth factor receptor. The screening results showed that wogonin from Radix scutellariae was the targeted component which acted on epidermal growth factor receptor specificity. The in vitro inhibitory activity of wogonin on the viability of cells with high epidermal growth factor receptor expression was tested using the MTT assay. In the dosage range of 0.40-50.0 μM, inhibition of HEK293/EGFR by wogonin was 8.94 ± 0.2, 20.64 ± 5.10, 34.16 ± 5.90 and 69.03 ± 7.80 at the concentrations of 0.4 × 10(-6), 2 × 10(-6), 10 × 10(-6) and 50 × 10(-6) molL(-1), respectively. These results showed that wogonin inhibited the growth of cells with high epidermal growth factor receptor expression in a dose-dependent manner.

New method of screening allergenic components from Shuanghuanglian injection: with RBL-2H3/CMC model online HPLC/MS system.

Shuanghuanglian (SHL) injection is a traditional Chinese medicine (TCM) injection widely used in China to treat influenza, tonsillitis, bronchitis, and faucitis. Adverse drug reactions (ADR) of SHL injection, majorly manifested as allergic reactions, were among the leading causes of death from TCM injection. In this study, an RBL-2H3/CMC online LC/MS system was established to screen and identify allergenic components in SHL injection, by which Baicalin was identified as a potential allergenic component. Allergenic activities of baicalin were investigated in RBL-2H3 cell degranulation and β-hexosaminidase release tests in vitro. Our results showed that baicalin dose-dependently induced RBL-2H3 cell degranulation and β-hexosaminidase release in the range of 0-50μg/ml. The RBL-2H3/CMC online LC/MS system developed in this study may potentially be used to screen allergenic components in other TCM injections.

Combined fibroblast growth factor receptor 4 cell membrane chromatography online with high performance liquid chromatography/mass spectrometry to screen active compounds in Brassica albla

We investigated an analytical method for the recognition separation, and identification of active components from the traditional Chinese medicinal plant Brassica albla L. using fibroblast growth factor receptor 4 cell membrane chromatography (FGFR4/CMC) with high performance liquid chromatography/mass spectrometry (HPLC/MS). HEK293-FGFR4 cells were obtained by stable transfection of the HEK293 cell line with pcDNA3.1 vector containing the FGFR4 gene. Crude extracts of B. albla L. were firstly subjected to FGFR4/CMC column, and the retain contents on the FGFR4/CMC column were then transferred and enriched using a pre-column, and a ten port column switcher were used between FGFR4/CMC column and HPLC. The retained components on FGFR4/CMC column were then directly delivered to the HPLC/MS system for separation and identification. Gefitinib, nicotine, atenolol, and nimodipine were used in order to verify FGFR4/CMC-HPLC/MS system specificity. Subsequently, we investigated the reproducibility and reliability of the FGFR4/CMC-HPLC/MS system. Finally, sinapine was identified as an active component of B. albla L. The MTT colorimetric assay revealed sinapine could inhibit the proliferation of HEK293-FGFR4 cells with dose dependence. Competitive displacement assay approved getitinib could occupy binding site of sinapine with competition way. And FleX dock simulation further exhibited sinapine and gefitinib could bind with the FGFR4 tyrosine active domain. Thus, sinapine is a potential tumor antagonist that acts on the tyrosine kinase domain.

Screening of target compounds from Fructus Piperis using high α1A adrenoreceptor expression cell membrane chromatography online coupled with high performance liquid chromatography tandem mass spectrometry

Benign prostatic hyperplasia (BPH) is a common disease in elderly men. The main treatment for BPH is α-adrenergic antagonists and 5α-reductase inhibitors. In this study, a two-dimensional (2D) α₁A cell membrane chromatography (CMC) online liquid chromatography/mass spectrometry system was built. Fructus Piperis, a traditional Chinese medicine and food homolog, was assayed with this 2D system. Piperine was identified as the active compound acting on α₁A receptors. A competitive binding assay and molecular docking assay were performed to investigate the binding sites and the affinity of piperine for the α₁A receptor. The results of the competitive binding assay (dissociation equilibrium constant of tamsulosin was 1.43 × 10(-6)M and piperine was 2.13 × 10(-6)M) and molecular docking assay (total score for tamsulosin binding with the α₁A receptor was 6.9719, and for piperine it was 4.4891) corresponded with the retention time of tamsulosin and piperine on the α₁A/CMC column.

Screening active components acting on α1A adrenergic receptors from agrimony using a Sprague-Dawley rat prostate cell membrane chromatography online coupled HPLC/MS method

Agrimony is a traditional Chinese medicine and a herb of Agrimonia pilosa Ledeb belonging to the Rosaceae family. Many pharmacological effects of agrimony have been demonstrated in previous studies. Few studies have screened the active compounds in this complex product for chronic prostatitis. In this study, a two dimensional online method was established. A Sprague-Dawley (SD) rat prostate CMC-online-HPLC/MS method was used to screen, analyze, and identify active compounds acting on α1 adrenergic receptor from agrimony. Via this online method agrimonolide was screened, analyzed, and identified as the active compound acting on α1 adrenergic receptor. Thus, agrimonolide is a potential α1 adrenergic receptor antagonist and requires further study.

Screening antiallergic components from Carthamus tinctorius using rat basophilic leukemia 2H3 cell membrane chromatography combined with high‐performance liquid chromatography and tandem mass spectrometry

Carthamus tinctorius, used in traditional Chinese medicine, has many pharmacological effects, such as anticoagulant effects, antioxidant effects, antiaging effects, regulation of gene expression, and antitumor effects. However, there is no report on the antiallergic effects of the components in C. tinctorius. In the present study, we investigated the antiallergic components of C. tinctorius and its mechanism of action. A rat basophilic leukemia 2H3/cell membrane chromatography coupled online with high-performance liquid chromatography and tandem mass spectrometry method was developed to screen antiallergic components from C. tinctorius. The screening results showed that Hydroxysafflor yellow A, from C. tinctorius, was the targeted component that retained on the rat basophilic leukemia 2H3/cell membrane chromatography column. We measured the amount of β-hexosaminidase and histamine released in mast cells and the key markers of degranulation. The release assays showed that Hydroxysafflor yellow A could attenuate the immunoglobulin E induced release of allergic cytokines without affecting cell viability from 1.0 to 50.0 μM. In conclusion, the established rat basophilic leukemia 2H3 cell membrane chromatography coupled with online high-performance liquid chromatography and tandem mass spectrometry method successfully screened and identified Hydroxysafflor yellow A from C. tinctorius as a potential antiallergic component. Pharmacological analysis elucidated that Hydroxysafflor yellow A is an effective natural component for inhibiting immunoglobulin E-antigen-mediated degranulation.

Cell membrane chromatography coupled with UHPLC-ESI-MS/MS method to screen target components from Peucedanum praeruptorum Dunn acting on α1A adrenergic receptor.

Peucedanum praeruptorum Dunn (BaiHuaQianHu in Chinese) is a traditional Chinese medicine that has a long history of use in China. In this study, HEK 293 α1A adrenergic cell membrane chromatography was coupled with UHPLC-ESI-MS/MS and successfully used to identify active components from Peucedanum praeruptorum Dunn. Paeruptorin A, paeruptorin B, and paeruptorin C were identified with α1A adrenergic receptor activity. Pharmacological assays showed that tamsulosin hydrochloride, paeruptorin A, paeruptorin B, and paeruptorin C in concentrations of 1×10(-8) to 1×10(-4)mol/mL could relax prostate strips pre-contracted with adrenalin in a concentration dependent manner. Therefore, the HEK293 α1A cell membrane chromatography coupled UHPLC-ESI-MS/MS system may be a potentially useful drug discovery method for screening for medicinal herbal components with α1A adrenergic receptor inhibitory activity.

Screening target components from Radix salviae miltiorrhiae using an EGFR/CMC-online-HPLC/MS method

Radix salviae miltiorrhizae is a widely used traditional Chinese medicine, it is the root and rhizome of Salvia miltiorrhiza Bge and belongs to the family Labiatae. Many of its pharmacological effects have been reported in a previous study. In this study, a two-dimension online analytical method of epidermal growth factor receptor cell membrane chromatography combined with liquid chromatography/mass spectrometry was built for recognition, separation and identification of active components from traditional Chinese herb Radix salviae miltiorrhizae. Gefitinib was used as positive control, while atenolol, nicotine, and nimodipine were used as negative controls to investigate the selectivity of the EGFR/CMC column and system suitability of this online analytical method. Cryptotanshinone was screened from Radix salviae miltiorrhizae by this online analytical method. Through in vitro pharmacological trials, cryptotanshinone showed obvious and dose-dependent inhibition activity in the range 0.40 to 50.0 μM. Molecular docking assay showed that the cryptotanshinone binding region was in very good agreement with gefitinib. The EGFR/CMC-online-LC/MS two-dimension analytical method was demonstrated as an effective screening method that rapidly recognizes and enriches active components acting on the epidermal growth factor receptor from Radix salviae miltiorrhizae and separates and identifies them.

Prostate Cell Membrane Chromatography -Liquid Chromatography -Mass Spectrometry for Screening of Active Constituents from Uncaria rhynchophylla

Uncaria rhynchophylla is a traditional Chinese medicinal herb used to treat hypertension and convulsive disorders such as epilepsy. Rat prostate cell membrane chromatography combined with liquid chromatography-mass spectrometry (LC-MS) was used to identify active constituents from U. rhynchophylla extracts. Four compounds (corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline) were discovered. Competitive binding assay results indicated that the four compounds were in direct competition at a single common binding site and interacted with α1A adrenergic receptors (α1A-AR) in a manner similar to tamsulosin. Affinity constant values of the four compounds binding with α1A-AR were also measured using rat prostate cell membrane chromatography (CMC). Finally, their pharmacodynamic effects were tested on rat caudal arteries. This CMC combined LC-MS system offers a means of drug discovery by screening natural medicinal herbs for new pharmacologically active molecules targeting specific receptors.

Screening epidermal growth factor receptor antagonists from Radix et Rhizoma Asari by two‐dimensional liquid chromatography

Radix et Rhizoma Asari is a traditional Chinese medicine, and has many pharmacological effects, such as calming, analgesia, anti-inflammation, antiarrhythmic, antihypertensive, antivirus, etc. But few studies have screened the active compounds from extracts of Radix et Rhizoma Asari for tumor therapy. In this study, a two-dimensional liquid chromatography system was built to screen active compounds acting on epidermal growth factor receptor (EGFR) from Radix et Rhizoma Asari. The screening result showed that asarinin from Radix et Rhizoma Asari was the targeted component that could act on EGFR specificity. The competitive binding assay and molecular docking assay results showed asarinin binding with EGFR in similar manner as with gefitinib, which was used as a positive control drug. Then the antitumor effect of asarinin was studied through cell growth assay in vitro. The results showed that gefitinib and asarinin could inhibit highly expressed EGFR cell growth in a dose-dependent manner in the range of dose from 0.10 to 102.4 μM. This two-dimensional liquid chromatography system will be a useful method in drug discovery from natural medicinal herbs for searching potential antitumor candidates.