1525
J. Sep. Sci. 2014, 37, 1525–1532
Shengli Han1 Jing Huang1 Jingjing Hou1,2 Sicen Wang1 1 School
of Pharmacy, Xi’an Jiaotong University Health Science Center, Xi’an, China 2 Qinghuaderen Xi’an Xingfu Pharmaceutical Company Limited, Xi’an, China Received March 3, 2014 Revised March 28, 2014 Accepted April 1, 2014
Research Article
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 M. This two-dimensional liquid chromatography system will be a useful method in drug discovery from natural medicinal herbs for searching potential antitumor candidates. Keywords: Asarinin / Cell membrane chromatography / Epidermal growth factor receptors / High-performance liquid chromatography with mass spectrometry / Radix et Rhizoma Asari DOI 10.1002/jssc.201400236
1 Introduction Because of the high mortality, cancer is one of the most serious diseases that threaten human life and health [1]. Due to the complex mechanism of tumors and difficulty to cure them, antitumor drug discovery is one of the difficult and hot fields for cancer therapy in recent years [2]. Epidermal growth factor receptor (EGFR) is widely distributed in the surface of mammalian epithelial cells, fibroblasts, glial cells, and keratinocyte [3]. Previous studies show that EGFR is important in cell growth, proliferation and differentiation, and other physiological processes [4]. EGFR has a close association with tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of apoptosis [5]. In recent years, the extracellular and intracellular domains of EGFR screening targets have been investigated for the development of antitumor drugs and some EGFR antagonists, such as gefitinib, dasatinib, and erlotinib, which have been used clinCorrespondence: Professor Sicen Wang, School of Pharmacy, Xi’an Jiaotong University Health Science Center, 76#, Yantan Westroad, Xi’an 710061, China E-mail: [email protected] or [email protected] Fax: +86-29-82655451
Abbreviations: CMC, cell membrane chromatography; EGFR, epidermal growth factor receptor; KD , equilibrium dissociation constants; PDB, protein data bank; TCM, traditional Chinese medicine C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ically [6]. However, new antitumor drugs with lower toxicity and better therapeutic effect are still in demand. Radix et Rhizoma Asari (Xi Xin in Chinese, XX), which is a traditional Chinese medicine (TCM), is the root and rhizome of Asarum that belongs to the family Aristolochiaceae [7]. Calmness, analgesia, anti-inflammation, antiarrhythmic, antihypertensive, and antivirus effects of Radix et Rhizoma Asari have been demonstrated by previous studies [8]. But few studies have been carried out to screen the active compounds from its complex products for tumor therapy. High-throughput screening is an important method to find new compounds in new drug development. Many screening methods have been reported, such as that based on receptors or enzymes [9, 10] and virtual screening using computer-aided drug design etc. [11]. In 1996, cell membrane chromatography (CMC) was introduced by He et al. [12, 13]. Nowadays, CMC has been demonstrated as an effective method to screen active components from the complicated extract of TCMs [14, 15]. Using specific highly expressed cell lines receptor, CMC can be used to recognize active components acting on specific receptor [16–18]. But CMC can only be used to recognize active components so that techniques that can be used to identify the active components are also important. Since 1990s, HPLC–MS has been widely used in life science to analyze and identify complicated samples [19,20]. The Colour Online: See the article online to view Figs. 1, 2, 3, 4 and 5 in colour.
www.jss-journal.com
1526
S. Han et al.
multidimensional HPLC, such as the common 2D HPLC, has been established and used to separate medicinal herbs [21,22]. In this study, we combined an EGFR/CMC column with an HPLC–MS system to establish a 2D LC system. The firstdimension EGFR/CMC column was used to recognize active compounds from natural products. The second-dimension HPLC–MS system was used to separate and identify the active compound recognized by the EGFR/CMC column. The ethanol extract of Radix et Rhizoma Asari was assayed by this online 2D LC method. A competitive binding assay was performed to investigate the binding sites and affinity of active compound screened by this system with EGFR. A molecular docking assay was used to identify the active compound binding region with EGFR tyrosine kinase using SYBYL-X 1.1. Furthermore, the antitumor effect of active compounds screened by this 2D LC system was studied through a cell growth assay.
J. Sep. Sci. 2014, 37, 1525–1532
degasser, an SPD-M20A diode-array detector, an LCMS2010EV mass spectrometer, and an LCMS solution workstation. The second-dimension column was a Shimadzu Shim-pack VP-ODS column (150 × 2.0 mm id, 5 m, Shimadzu). The conditions of the first dimension were as follows: mobile phase of ammonium acetate buffer (5 mmol/L, pH 7.4) with a flow rate of 0.2 mL/min; UV detection for gefitinib 254 nm and asarinin 287 nm. The temperature of both the first-dimension column and second-dimension column was kept at 37⬚C by a CTO-20A column oven, mobile phase of acetonitrile/water (40:60, v/v) with 0.2 mL/min flow rate, diodearray detection. MS conditions were as follows: nebulizer gas (N2 , purity > 99.999%); flow rate, 1.5 L/min; drying gas (N2 , purity > 99.999%); pressure, 0.1 MPa; curved desolvation line temperature, 250⬚C; heat block temperature, 200⬚C; interface temperature, 250⬚C; detector voltage, 1.5 kV; curved desolvation line voltage, 10 V; positive ionization mode, scanning from m/z 50 to 800.
2 Materials and methods 2.1 Chemicals and reagents
2.3 Preparation of samples and standard solutions
Gefitinib (ࣙ98%) was bought from Nanjing Ange Pharmaceutical (Nanjing, China). Asarinin (ࣙ99.0%, lot#: MUST12011009) was bought from Chengdu Must Bio-tech (Chengdu, China). Atenolol, nifedipine, and tamsulosin hydrochloride were from Sigma Chemical (St. Louis, MO, USA). Silica gel (ZEX-II, 5 m, 200 Å) was obtained from Qingdao Meigao Chemical (Qingdao, China). Radix et Rhizoma Asari was bought from the TCM store (Xi’an, China), and it was authenticated by the Department of Pharmacognosy, Xi’an Jiaotong University (Xi’an, China). The EGFR highexpression cell lines were constructed with an engineering cell line named HEK293 in our laboratory. Dulbecco’s modified Eagle medium was bought from Invitrogen (Grand Island, USA). Methanol and acetonitrile (HPLC grade) were purchased from Fisher Scientific (Pittsburgh, USA). All aqueous solutions were prepared using ultrapure water produced by MK-459 Millipore Milli-Q Plus ultrapure water system.
The ethanol extract of Radix et Rhizoma Asari was prepared as follows: 20 g of Radix et Rhizoma Asari was broken into pieces and the powder was extracted with 200 mL 60% ethanol by refluxing at 60⬚C for 2 h. The refluxing was repeated and filtrates were combined in a round-bottomed flask. Then the filtrates were concentrated to dryness by distillation under reduced pressure. Sample working solution was prepared by dissolving 0.1 g dryness in 2 mL methanol followed by centrifugation (8000 × g for 10 min at 4⬚C) and filtered using a Millipore filter (0.45 m), then the sample work solution was stored at −20⬚C in the dark until use. The standard solution of gefitinib, asarinin, atenolol, nifedipine, and tamsulosin hydrochloride was prepared as follows: gefitinib, asarinin, atenolol, nifedipine, and tamsulosin hydrochloride were dissolved in methanol and the standard stock solutions of about 1 mg/mL were prepared. The standard stock solutions were stored at −20⬚C in dark and freshly prepared every week. When used, the standard stock solutions were diluted to suitable concentrations using mobile phase.
2.2 Instrument configuration and conditions The 2D online LC system was combined by a VICIAG 10G0911V 10-port 2-position valve (Valco Instrument, Houston, TX, USA), and two Shim-pack VP-ODS precolumns (10 × 2.0 mm id, 5 m, Shimadzu, Kyoto, Japan) were used as the enrichment columns. The first dimension contained an LC20AD pump, a DGU-20A3 degasser, SIL-20A autosampler, SPD-20A UV/VIS detector. The column of the first dimension was an EGFR/CMC column (10.0 × 2.0 mm id, 5 m), which was packed by the RPL-10ZD column loading machine (Dalian Replete Science and Technology, Dalian, China), and the preparation method of the EGFR/CMC column is described in Section 2.4. The second dimension (HPLC–MS system) included two LC-20AD pumps, a DGU-20A3 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.4 Preparation of EGFR/CMC column The high-expression EGFR cell line was cultured in Dulbecco’s modified Eagle medium (which contained 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin). Cells were cultured at 37⬚C in a humidified atmosphere with 5% CO2 . Cells from exponentially growing cultures were used in all experiments. When cell growth reached approximately 80% coverage, 0.25% trypsin was used to incubate for 10 min at 4⬚C. Cell counting was performed to ensure that the amount of cells was no less than 1 × 107 . The harvested EGFR cells (7 × 106 ) were washed three times with www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2014, 37, 1525–1532
physiological saline (pH 7.4) followed by centrifugation at 3000 × g for 10 min at 4⬚C. Tris-HCl (pH 7.4; 50 mmol/L) was added to produce EGFR cell suspension; the cells were then ruptured by an ultrasonic procedure for 30 min. The resulting suspension was homogenized for 3 min and clarified by centrifugation at 1000 × g for 10 min at 4⬚C. The pellet was discarded and supernatant was centrifuged at 12 000 × g for 20 min at 4⬚C. The precipitation was suspended in 10 mL physiological saline, and then the suspension was centrifuged at 12 000 × g. Then EGFR cell membrane suspension was obtained in 5 mL physiological saline. A total of 0.05 g silica was activated at 105⬚C for 30 min, and the cell membrane suspension was slowly added to silica under a vacuum and with agitation at 4⬚C. Then, the mixture was agitated for 30 min by a magnetic stirrer and allowed to stand overnight. Finally, the EGFR cell membrane stationary phase was packed into the CMC column (10 × 2.0 mm id) using a column-loading machine following a wet packing procedure.
1527
gated by injecting gefitinib into the EGFR/CMC column and the retained fraction was enriched and switched into seconddimension analytical VP-ODS column (ODS); the peak area of enriched gefitinib on ODS (AE ) was used as the indicator. Direct HPLC analysis of gefitinib was also studied and peak area (AS ) was also recorded. The enrichment ratio was calculated as AE /AS .
2.7 Application of the EGFR/CMC online HPLC–MS system The EGFR/CMC online HPLC–MS system was used to screen active components acting on EGFR from the ethanol extract of Radix et Rhizoma Asari. Gefitinib was used as a positive control to investigate this online 2D LC system. The standard solution of the active component that was recognized, analyzed, and identified by the EGFR/CMC online HPLC–MS system was also injected into this online-coupled system for further identification.
2.5 System suitability of the EGFR/CMC column The specificity of the EGFR/CMC column was investigated by injecting different drugs acting on different receptors into EGFR/CMC column. Atenolol acting on  receptor, tamsulosin hydrochloride acting on ␣1A receptor, nifedipine acting on calcium channel, and gefitinib acting on EGFR were used to investigate the specificity of the EGFR/CMC column. The reproducibility of the same EGFR/CMC column and different EGFR/CMC columns was investigated, and the retention time was used as an indicator. After the columns were packed using the column-loading machine, they were attached to the LC instrument and equilibrated for 60 min. Then for the single EGFR/CMC column, reproducibility was determined by repeated injection of 5 L of 0.1 mg/mL gefitinib, and the retention time was recorded. For different EGFR/CMC columns, 5 L of 0.1 mg/mL gefitinib was injected into the different EGFR/CMC columns, respectively, and the retention time was also recorded. The column lifetime was also investigated by repeated injections of 0.1 mg/mL gefitinib into the EGFR/CMC column.
2.6 System suitability of the EGFR/CMC online HPLC–MS system A two-position ten-port switching valve was used to combine the EGFR/CMC with HPLC–MS system. The system suitability of the EGFR/CMC online HPLC–MS system was investigated by injecting positive control drug gefitinib into the online 2D LC system. Gefitinib was recognized and retained on the EGFR/CMC column, and the retained fraction was enriched and switched into the HPLC–MS system for analysis and identification. The reproducibility of the enrichment was investigated five times repeatedly by gefitinib, and the peak area of enriched gefitinib on the second-dimension column was used as an indicator. The enrichment ratio was investi C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.8 Competitive binding assay The competitive binding assay was performed as follows [23, 24]: the competitive binding assay was studied by using gefitinib as a mobile phase additive at different concentrations changing from 6 × 10−8 to 9.6 × 10−7 mol/L, then gefitinib and asarinin were injected into the EGFR/CMC column. According to Eqs. (1) and (2), KD values of the gefitinib and asarinin binding with EGFR were calculated. k =
t − t0 t0
1 K D VM K D VM = [L ]M + k K M [R]S [R]S
(1)
(2)
KD and KM are the equilibrium dissociation constants of the analyte and mobile phase marker, respectively. [L]M is the molar concentration of the ligand in the mobile phase, [R]S is the concentration of immobilized receptors at the surface of the stationary phase, and VM is the dead volume of the column. The value k is the analyte capacity factor. When gefitinib was both used as the analyte and mobile phase additive, KD = KM , and Eq. (2) can be reduced to: VM K D VM 1 = [L ]M + k [R]S [R]S
(3)
Then [R]S /VM can be obtained from the slope, and KD of gefitinib can be determined by calculating the ratio of the intercept to the slope. In addition, the two parameters were substituted into Eq. (2), and the KD of asarinin was obtained by linear regression by plotting 1/k versus [L]M . www.jss-journal.com
1528
S. Han et al.
J. Sep. Sci. 2014, 37, 1525–1532
2.9 Molecular docking assay A molecular docking assay of gefitinib and asarinin with EGFR tyrosine kinase (PDB ID: 2ITY) was performed using SYBYL-X 1.1 to identify its binding region with protein. The substrate was constructed with SYBYL/Sketch module and optimized using Powell’s method. Energy minimization was performed using the Tripos force field with convergence criterion set at 0.005 kcal (Å mol)−1 and a maximum of 1000 iterations and Gasteiger–H¨uckel charges. A nonbonded cutoff distance of 8 Å was adopted to consider the intramolecular interaction [25].
2.10 Cell growth assay The effect of gefitinib and asarinin (which was identified by the EGFR/CMC online HPLC–MS system) on HEK293/EGFR cell viability were evaluated using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Briefly, exponentially growing cells were harvested and plated in 96-well plates at a concentration of 5 × 104 cells/well. After 24 h incubation at 37⬚C, cells were treated with gefitinib and asarinin at various concentrations for 48 h. Then, 20 L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/mL) was added to each well and incubated at 37⬚C for 4 h. After the supernatant was discarded, 150 L of DMSO was added to each well, and the optical density of cells was determined with a microplate reader (Bio-Rad instruments, USA) at 490 nm and expressed as absorbance values [26].
3 Results and discussion 3.1 System suitability of EGFR/CMC column The specificity of EGFR/CMC column was investigated by the positive control drug gefitinib. The results are shown in Fig. 1A. Figure 1A(I) showed gefitinib acting with blank silica gel column. Figure 1A(II) showed gefitinib acting with ␣1A/CMC column. Figure 1A(III) showed gefitinib acting with the RBL-2H3/CMC column. Figure 1A(IV) showed gefitinib acting with EGFR/CMC column. Only gefitinib can be retained on EGFR/CMC column. The selectivity of EGFR/CMC column was investigated by injecting different drugs acting on different receptors into EGFR/CMC column. Chromatograms of different drugs acted on EGFR/CMC column are shown in Fig. 1B. Atenolol acts on the  receptor, tamsulosin hydrochloride acts on the ␣1A receptor, nifedipine acts on the calcium channel, and gefitinib acts on EGFR. In Fig. 1B(IV), we can see that gefitinib can be retained on EGFR/CMC column, but atenolol is shown in Fig. 1B(I), tamsulosin hydrochloride shown in Fig. 1B(II), and nifedipine shown in Fig. 1B(III) cannot be retained on the EGFR/CMC column. These results suggested that gefitinib had better retention characteristics and EGFR C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. The specificity and selectivity of EGFR/CMC column. (A) Gefitinib retained on different CMC columns: (I) blank silica column, (II) ␣1A/CMC column, (III) RBL-2H3/CMC column, (IV) EGFR/CMC column. (B) Different drugs acting on the EGFR/CMC column: (I) atenolol; (II) tamsulosin hydrochloride; (III) nifedipine; (IV) gefitinib.
antagonists, and could interact with EGFR abundant in the highly expressed EGFR cell membrane. The reproducibility of different EGFR/CMC columns was tested by injecting gefitinib into different CMC columns, and the retention time of gefitinib was used as an indicator. The RSD of the retention times for different EGFR/CMC columns was 3.8% (n = 3). The reproducibility of each EGFR/CMC column was tested by repeatedly injecting gefitinib. The RSD of the retention time for each EGFR/CMC column was 1.3% (n = 5). So, the reproducibility of the EGFR/CMC column could satisfy the requirement of the experiment. As the EGFR/CMC columns have biological activity, the column lifetime was an important parameter. By the repeated injection of gefitinib, the EGFR/CMC column lifetime was investigated simultaneously. Gefitinib could be retained on the EGFR/CMC column well after the CMC column was used for 72 h. So the lifetime of the EGFR/CMC column could meet the requirements of experiment. The above results showed that the EGFR/CMC column was suitable for recognizing compounds from complex system. The specificity, selectivity, reproducibility, and the lifetime of the EGFR/CMC column met the requirements of the experiment.
3.2 System suitability of the EGFR/CMC online HPLC–MS system The EGFR/CMC column was combined with the HPLC–MS system through a ten-port column switcher, which was suitable for the qualitative analysis of active components acting on EGFR from the complex samples. The brief scheme of the 2D online LC system followed our previous studies [27–31]. Because the enrichment column used in the 2D online LC system was loaded with C18 , it had a better retentive ability than the EGFR/CMC column, so the retention fraction eluted www.jss-journal.com
J. Sep. Sci. 2014, 37, 1525–1532
Figure 2. Chromatograms of gefitinib standard solution analyzed by the EGFR/CMC online HPLC–MS system. (A) EGFR/CMC chromatogram of gefitinib standard solution including a retained fraction R (red, retention time was about 42 min); (C) HPLC–MS chromatogram of the corresponding fraction (between two dotted lines in EGFR/CMC chromatogram) extracted onto the enrichment column, and identified as gefitinib; (B) the direct HPLC–MS chromatogram of gefitinib standard solution.
from the EGFR/CMC column can be retained on the enrichment column. The retention fraction can be easily washed out and pumped into the analytical column for analysis and identification in the second dimension. Gefitinib was used to investigate the ability of EGFR/CMC online HPLC–MS system for recognition, analysis, and identification of active compound acting on EGFR. The results are shown in Fig. 2. Figure 2A showed that gefitinib can be well retained on the EGFR/CMC column (red, the retention time was about 42 min). Figure 2C shows an HPLC–MS chromatogram of the retention fraction identified as gefitinib. Figure 2B shows a direct HPLC chromatogram of gefitinib. The retention time of the retention fraction on the analytical column was consistent with the retention time of gefitinib. Results of the enrichment reproducibility and the ratio of enrichment were investigated using the peak area of the retention fraction on the analytical column (AE ), and the peak area of gefitinib was directly injected into the analytical column (AS ) as indicators. The RSD of the AE was 2.9% (n = 5). The enrichment ratio calculated by AE /AS was 78.5%. Therefore, both the reproducibility of enrichment and enrichment ratio met the requirements. So, the EGFR/CMC online HPLC–MS system is suitable for recognizing, analyzing, and identifying target components acting on EGFR.
3.3 Practical application of EGFR/CMC online HPLC–MS system Radix et Rhizoma Asari, a traditional Chinese medicine, was chosen and EGFR antagonists were screened, analyzed, and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Liquid Chromatography
1529
Figure 3. Chromatograms of the ethanol extract of Radix et Rhizoma Asari analyzed by the EGFR/CMC online HPLC–MS system. (A) The EGFR/CMC chromatogram of the ethanol extract of Radix et Rhizoma Asari including nonretained fraction R0 (yellow), and retained fraction R1 (red, retention time was about 10.2 min). (B) The direct HPLC–MS chromatograms of ethanol extracts of Radix et Rhizoma Asari. (C) The HPLC–MS chromatograms of the retained fractions. (D) The HPLC–MS chromatograms of the nonretained fractions.
identified using the EGFR/CMC online HPLC–MS system. Results of the ethanol extract of Radix et Rhizoma Asari assayed using the EGFR/CMC online HPLC–MS system are shown in Fig. 3. Figure 3A showed that there was one significant retention fraction (red, the retention time of R1 was about 9.9 min) and nonretained fraction (yellow) in the EGFR/CMC column. Then the retained fraction and nonretained fraction were assayed using the second-dimension HPLC–MS system for further separation and identification. Figure 3C shows the HPLC–MS chromatogram of the retention fractions (R1 ), which was identified as asarinin. Figure 3D shows the HPLC– MS chromatogram of the nonretained fraction (R0 ). Figure 3B shows the HPLC–MS chromatogram of the ethanol extract of Radix et Rhizoma Asari. As shown in Fig. 3C, the retained fraction (R1 ) was identified as asarinin by HPLC–MS. For further identification and screening the above result, asarinin standard solution was assayed by the EGFR/CMC online HPLC–MS system. The results are shown in Fig. 4. Figure 4A showed that the retention time of asarinin was about 10.2 min, the retained fraction R (red, between the two dotted lines) was extracted onto the enrichment column, and switched into second-dimension system for chromatographic separation and MS identification. Figure 4C shows the HPLC–MS chromatogram of the retained fraction. Figure 4B shows the direct HPLC–MS chromatogram of asarinin standard solution. This result further verified that the main retained fraction in the EGFR/CMC column was asarinin. www.jss-journal.com
1530
S. Han et al.
Figure 4. Chromatograms of asarinin standard solution analyzed by the EGFR/CMC online HPLC–MS system. (A) The EGFR/CMC chromatogram of the asarinin standard solution including retained fraction R (red, retention time was about 10.2 min). (B) The direct HPLC–MS chromatograms of the asarinin standard solution. (C) The HPLC–MS chromatograms of the retained fractions.
J. Sep. Sci. 2014, 37, 1525–1532
EGFR. The retention times of gefitinib and asarinin on the EGFR/CMC column were decreased with an increasing concentration of gefitinib in the mobile phase. The results are shown in Fig. 5. Figure 5A and B shows the elution profiles of gefitinib and asarinin on the EGFR/CMC column with different concentrations of gefitinib in the mobile phase. Figure 5C shows the regression curves achieved by plotting 1/k versus [L]M . The five concentrations of gefitinib were 6 × 10−8 , 1.2 × 10−7 , 2.4 × 10−7 , 4.8 × 10−7 , and 9.6 × 10−7 mol/L. These results indicate that gefitinib and asarinin have the same binding sites on EGFR. Besides, both the dissociation equilibrium constant of gefitinib (KD[G] ) and the dissociation equilibrium constant of asarinin (KD[A] ) were measured. The results were as follows: KD[G] = 4.02 × 10−6 M, KD[A] = 1.65 × 10−4 M. The dissociation equilibrium constant of gefitinib was smaller than that of asarinin, so the affinity of gefitinib was larger than that of asarinin. This result was corresponded with the retention time of gefitinib and asarinin on the EGFR/CMC column.
3.5 Molecular docking assay 3.4 Competitive binding assay The competitive binding assay was performed to investigate the binding sites and affinity of gefitinib and asarinin with
A molecular docking assay was often used to predict the binding sites and binding affinity between ligand and receptor. In this study, the molecular docking assay was performed using
Figure 5. The elution profiles of gefitinib (A) and asarinin (B) on the EGFR/CMC column with different concentration of gefitinib in the mobile phase. (C ) The regression cures achieved by plotting 1/k versus [L]M . The five concentrations of gefitinib were (I) 6 × 10−8 mol/L, (II) 1.2 × 10−7 mol/L, (III) 2.4 × 10−7 mol/L, (IV) 4.8 × 10−7 mol/L, and (V) 9.6 × 10−6 mol/L. D) Gefitinib docked into the active site of the EGFR (PDB ID: 2ITY). Figure 5E was the asarinin docked into the active site of the EGFR (PDB ID: 2ITY). (F ) The effect of gefitinib and asarinin on EGFR cell viability.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2014, 37, 1525–1532
the SYBYL/FlexX (Tripos) system based on the crystal structures of the EGFR (PDB ID: 2ITY) taken from the protein data bank. The results of the molecular docking assay are shown in Fig. 5. Figure 5D shows gefitinib docked into the active site of the EGFR. Figure 5E shows asarinin docked into the active site of the EGFR. In Fig. 5D and E, the results of gefitinib and asarinin binding with the EGFR at the same region can be obtained. The total score of gefitinib and asarinin binding with the EGFR was also given by the SYBYL/FlexX. The total score of gefitinib binding with the EGFR was 7.3849, and the total score of asarinin binding with the EGFR was 5.0732. This result was also corresponded with the retention time of gefitinib and asarinin on the EGFR/CMC column.
3.6 Cell growth assay Inhibitory effects of gefitinib and asarinin were tested in vitro on EGFR cell growth. The result shows that asarinin could inhibit the growth of EGFR cell, although this activity was lower than that of gefitinib, as shown in Fig. 5F. The inhibition rates of gefitinib on EGFR cell line were 7.1 ± 1.23, 16.6 ± 1.63, 19.5 ± 3.41, 20.8 ± 2.75, 22.7 ± 2.47, and 37.5 ± 2.33 at the concentrations of 0.1, 0.4, 1.6, 6.4, 25.6, and 102.4 M, respectively. For asarinin, the rates were 2.5 ± 1.22, 6.3 ± 1.25, 8.7 ± 1.47, 10.3 ± 2.31, 13.8 ± 1.83, and 23.2 ± 3.39. In the range of 0.10–102.4 M, gefitinib and asarinin showed obvious and dose-dependent inhibition activity. The inhibition activity of asarinin was weaker than that of gefitinib. The retention time of gefitinib on EGFR/CMC column was 40 min, but asarinin was only about 10 min, which indicated gefitinib had a stronger interaction with EGFR than that of asarinin. Therefore, retention behaviors of gefitinib and asarinin were correlated to their pharmacological activities.
1531
for Science & Technology Support of China (Grant number: 2012BAI29B06). The authors have declared no conflict of interest.
5 References [1] Marte, B., Nature 2013, 501, 327–372. [2] Sherr, C. J., Cell 2004, 116, 235–246. [3] Sugawa, N., Ekstrand, A., James, C. D., Collins, V., Proc. Natl. Acad. Sci. USA 1990, 87, 8602–8606. [4] Ogiso, H., Ishitani, R., Nureki, O., Fukai, S., Yamanaka, M., Kim, J., Saito, K., Sakamoto, A., Inoue, M., Shirouzu, M., Yokoyama, S., Cell 2002, 110, 775–787. [5] Richard, J., Sainsbury, C., Needham, G., Farndon, J., Malcolm, A. J., Harris, A., Lancet 1987, 329, 1398–1402. [6] Wakeling, A., Simon, G., Woodburn, J., Ashton, S., Curry, B., Barker, A., Gibson, K., Cancer Res. 2002, 62, 5749– 5754. [7] Song, N., Zhao, R., Que, L., Wan, C., Tang, Z., Chin. J. Ethnomed. Ethnopharm. 2008, 4, 50–53. [8] Li, Y., Tian, M., Yu, J., Shang, M., Cai, S., J. Nat. Med. 2010, 64, 442–451. [9] Shin, S., Anklam, K., Manning, E., Collins, M., Clin. Vaccine Immunol. 2009, 16(5), 613–620. [10] Schreiber, S., Science 2000, 287, 1964–1969. [11] Sheridan, R., Rengachari, V., Acc. Chem. Res. 1987, 20(9), 322–329. [12] He, L., Geng, X., New Prog. Biomed. Chromatogr. 1996, 3, 8–9. [13] Sun, M., Ma, W., Guo, Y., Hu, Z., He, L., J. Sep. Sci. 2013, 36, 2096–2013. [14] Liang, M., He, L., Yang, G., Life Sci. 2005, 78, 128–133.
4 Conclusions
[15] Hou, X., Wang, S., Hou, J., He, L., J. Sep. Sci. 2011, 21, 508–513.
In summary, an online 2D LC system was built successfully. Asarinin was screened, analyzed, and identified from the ethanol extract of Radix et Rhizoma Asari by this EGFR/CMC online HPLC–MS system. Results of competitive binding assay and molecular docking assay demonstrated that the binding region of asarinin was the same as that of gefitinib. A cell growth assay was carried to further verify the activity of asarinin in vitro. This 2D LC system has the potential to serve as a useful screening tool in antitumor drug discovery from natural medicinal herbs.
[16] Wang, S., Sun, M., Zhang, Y., Du, H., He, L., J. Chromatogr. A 2010, 1217, 5246–5252.
This work was supported by National Natural Science Foundation of China (Grant number: 81227802 and 81230079), National “Twelfth Five-Year” Plan for Important National Science & Technology Specific Projects of China (Grant number: 2012ZX09103201-054), and National “Twelfth Five-Year” Plan C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[17] Yue, Y., Xue, H., Wang, X., Yang, Q., Song, Y., Li, X., J. Sep. Sci. 2013, 37, 244–249. [18] Han, S., Zhang, T., Feng, L., Lv, N., Wang, S., J. Pharm. Biomed. 2013, 81–82, 133–137. [19] Li, M., Hou, X., Zhang, J., Wang, S., Fu, Q., He, L., J. Pharm. Anal. 2011, 1, 81–91. [20] Griffiths, J., Anal. Chem. 2008, 80, 5678–5683. [21] Gray, M. J., Dennis, G. R., Slonecker, P., Shalliker, R., J. Chromatogr. A 2004, 1041, 101–110. [22] Stoll, D., Li, X., Wang, X., Carr, P., Porter, S., Rutan, S., J. Chromatogr. A 2007, 1168, 3–43. [23] Du, H., He, J., Wang, S., He, L., Anal. Bioanal. Chem. 2010, 397, 1947–1953. [24] Geng, X., Regnier, F., J. Chromatogr. 1985, 332, 147–168.
www.jss-journal.com
1532
S. Han et al.
[25] Mou, J., Fang, H., Jing, F., Wang, Q., Liu, Y., Zhu, H., Shang, L., Wang, X., Xu, W., Bioorg. Med. Chem. 2009, 17, 466–473. [26] Naruse, I., Ohmori, T., Ao, Y., Fukumoto, H., Kuroki, T., Mori, M., Saijo, N., Nishio, K., Int. J. Cancer 2002, 98, 310–315. [27] He, H., Han, S., Zhang, T., Zhang, J., Wang, S., Hou, J., J. Pharm. Biomed. 2012, 62, 196–202.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2014, 37, 1525–1532
[28] Han, S., Zhang, P., Wei, F., Wang, S., Anal. Methods 2012, 4, 3351–3357. [29] Hou, X., Yuan, X., Zhang, B., Wang, S., J. Sep. Sci. 2013, 36, 706–712. [30] Han, S., Zhang, T., Huang, J., Hu, Z., Wang, S., Anal. Methods 2012, 4, 1078–1083. [31] Yang, X., Wang, Y., Zhang, X., Chang, R., Li, X., J. Sep. Sci. 2011, 34, 2586–2593.
www.jss-journal.com
J. Sep. Sci. 2014, 37, 1525–1532
Shengli Han1 Jing Huang1 Jingjing Hou1,2 Sicen Wang1 1 School
of Pharmacy, Xi’an Jiaotong University Health Science Center, Xi’an, China 2 Qinghuaderen Xi’an Xingfu Pharmaceutical Company Limited, Xi’an, China Received March 3, 2014 Revised March 28, 2014 Accepted April 1, 2014
Research Article
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 M. This two-dimensional liquid chromatography system will be a useful method in drug discovery from natural medicinal herbs for searching potential antitumor candidates. Keywords: Asarinin / Cell membrane chromatography / Epidermal growth factor receptors / High-performance liquid chromatography with mass spectrometry / Radix et Rhizoma Asari DOI 10.1002/jssc.201400236
1 Introduction Because of the high mortality, cancer is one of the most serious diseases that threaten human life and health [1]. Due to the complex mechanism of tumors and difficulty to cure them, antitumor drug discovery is one of the difficult and hot fields for cancer therapy in recent years [2]. Epidermal growth factor receptor (EGFR) is widely distributed in the surface of mammalian epithelial cells, fibroblasts, glial cells, and keratinocyte [3]. Previous studies show that EGFR is important in cell growth, proliferation and differentiation, and other physiological processes [4]. EGFR has a close association with tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of apoptosis [5]. In recent years, the extracellular and intracellular domains of EGFR screening targets have been investigated for the development of antitumor drugs and some EGFR antagonists, such as gefitinib, dasatinib, and erlotinib, which have been used clinCorrespondence: Professor Sicen Wang, School of Pharmacy, Xi’an Jiaotong University Health Science Center, 76#, Yantan Westroad, Xi’an 710061, China E-mail: [email protected] or [email protected] Fax: +86-29-82655451
Abbreviations: CMC, cell membrane chromatography; EGFR, epidermal growth factor receptor; KD , equilibrium dissociation constants; PDB, protein data bank; TCM, traditional Chinese medicine C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ically [6]. However, new antitumor drugs with lower toxicity and better therapeutic effect are still in demand. Radix et Rhizoma Asari (Xi Xin in Chinese, XX), which is a traditional Chinese medicine (TCM), is the root and rhizome of Asarum that belongs to the family Aristolochiaceae [7]. Calmness, analgesia, anti-inflammation, antiarrhythmic, antihypertensive, and antivirus effects of Radix et Rhizoma Asari have been demonstrated by previous studies [8]. But few studies have been carried out to screen the active compounds from its complex products for tumor therapy. High-throughput screening is an important method to find new compounds in new drug development. Many screening methods have been reported, such as that based on receptors or enzymes [9, 10] and virtual screening using computer-aided drug design etc. [11]. In 1996, cell membrane chromatography (CMC) was introduced by He et al. [12, 13]. Nowadays, CMC has been demonstrated as an effective method to screen active components from the complicated extract of TCMs [14, 15]. Using specific highly expressed cell lines receptor, CMC can be used to recognize active components acting on specific receptor [16–18]. But CMC can only be used to recognize active components so that techniques that can be used to identify the active components are also important. Since 1990s, HPLC–MS has been widely used in life science to analyze and identify complicated samples [19,20]. The Colour Online: See the article online to view Figs. 1, 2, 3, 4 and 5 in colour.
www.jss-journal.com
1526
S. Han et al.
multidimensional HPLC, such as the common 2D HPLC, has been established and used to separate medicinal herbs [21,22]. In this study, we combined an EGFR/CMC column with an HPLC–MS system to establish a 2D LC system. The firstdimension EGFR/CMC column was used to recognize active compounds from natural products. The second-dimension HPLC–MS system was used to separate and identify the active compound recognized by the EGFR/CMC column. The ethanol extract of Radix et Rhizoma Asari was assayed by this online 2D LC method. A competitive binding assay was performed to investigate the binding sites and affinity of active compound screened by this system with EGFR. A molecular docking assay was used to identify the active compound binding region with EGFR tyrosine kinase using SYBYL-X 1.1. Furthermore, the antitumor effect of active compounds screened by this 2D LC system was studied through a cell growth assay.
J. Sep. Sci. 2014, 37, 1525–1532
degasser, an SPD-M20A diode-array detector, an LCMS2010EV mass spectrometer, and an LCMS solution workstation. The second-dimension column was a Shimadzu Shim-pack VP-ODS column (150 × 2.0 mm id, 5 m, Shimadzu). The conditions of the first dimension were as follows: mobile phase of ammonium acetate buffer (5 mmol/L, pH 7.4) with a flow rate of 0.2 mL/min; UV detection for gefitinib 254 nm and asarinin 287 nm. The temperature of both the first-dimension column and second-dimension column was kept at 37⬚C by a CTO-20A column oven, mobile phase of acetonitrile/water (40:60, v/v) with 0.2 mL/min flow rate, diodearray detection. MS conditions were as follows: nebulizer gas (N2 , purity > 99.999%); flow rate, 1.5 L/min; drying gas (N2 , purity > 99.999%); pressure, 0.1 MPa; curved desolvation line temperature, 250⬚C; heat block temperature, 200⬚C; interface temperature, 250⬚C; detector voltage, 1.5 kV; curved desolvation line voltage, 10 V; positive ionization mode, scanning from m/z 50 to 800.
2 Materials and methods 2.1 Chemicals and reagents
2.3 Preparation of samples and standard solutions
Gefitinib (ࣙ98%) was bought from Nanjing Ange Pharmaceutical (Nanjing, China). Asarinin (ࣙ99.0%, lot#: MUST12011009) was bought from Chengdu Must Bio-tech (Chengdu, China). Atenolol, nifedipine, and tamsulosin hydrochloride were from Sigma Chemical (St. Louis, MO, USA). Silica gel (ZEX-II, 5 m, 200 Å) was obtained from Qingdao Meigao Chemical (Qingdao, China). Radix et Rhizoma Asari was bought from the TCM store (Xi’an, China), and it was authenticated by the Department of Pharmacognosy, Xi’an Jiaotong University (Xi’an, China). The EGFR highexpression cell lines were constructed with an engineering cell line named HEK293 in our laboratory. Dulbecco’s modified Eagle medium was bought from Invitrogen (Grand Island, USA). Methanol and acetonitrile (HPLC grade) were purchased from Fisher Scientific (Pittsburgh, USA). All aqueous solutions were prepared using ultrapure water produced by MK-459 Millipore Milli-Q Plus ultrapure water system.
The ethanol extract of Radix et Rhizoma Asari was prepared as follows: 20 g of Radix et Rhizoma Asari was broken into pieces and the powder was extracted with 200 mL 60% ethanol by refluxing at 60⬚C for 2 h. The refluxing was repeated and filtrates were combined in a round-bottomed flask. Then the filtrates were concentrated to dryness by distillation under reduced pressure. Sample working solution was prepared by dissolving 0.1 g dryness in 2 mL methanol followed by centrifugation (8000 × g for 10 min at 4⬚C) and filtered using a Millipore filter (0.45 m), then the sample work solution was stored at −20⬚C in the dark until use. The standard solution of gefitinib, asarinin, atenolol, nifedipine, and tamsulosin hydrochloride was prepared as follows: gefitinib, asarinin, atenolol, nifedipine, and tamsulosin hydrochloride were dissolved in methanol and the standard stock solutions of about 1 mg/mL were prepared. The standard stock solutions were stored at −20⬚C in dark and freshly prepared every week. When used, the standard stock solutions were diluted to suitable concentrations using mobile phase.
2.2 Instrument configuration and conditions The 2D online LC system was combined by a VICIAG 10G0911V 10-port 2-position valve (Valco Instrument, Houston, TX, USA), and two Shim-pack VP-ODS precolumns (10 × 2.0 mm id, 5 m, Shimadzu, Kyoto, Japan) were used as the enrichment columns. The first dimension contained an LC20AD pump, a DGU-20A3 degasser, SIL-20A autosampler, SPD-20A UV/VIS detector. The column of the first dimension was an EGFR/CMC column (10.0 × 2.0 mm id, 5 m), which was packed by the RPL-10ZD column loading machine (Dalian Replete Science and Technology, Dalian, China), and the preparation method of the EGFR/CMC column is described in Section 2.4. The second dimension (HPLC–MS system) included two LC-20AD pumps, a DGU-20A3 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.4 Preparation of EGFR/CMC column The high-expression EGFR cell line was cultured in Dulbecco’s modified Eagle medium (which contained 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin). Cells were cultured at 37⬚C in a humidified atmosphere with 5% CO2 . Cells from exponentially growing cultures were used in all experiments. When cell growth reached approximately 80% coverage, 0.25% trypsin was used to incubate for 10 min at 4⬚C. Cell counting was performed to ensure that the amount of cells was no less than 1 × 107 . The harvested EGFR cells (7 × 106 ) were washed three times with www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2014, 37, 1525–1532
physiological saline (pH 7.4) followed by centrifugation at 3000 × g for 10 min at 4⬚C. Tris-HCl (pH 7.4; 50 mmol/L) was added to produce EGFR cell suspension; the cells were then ruptured by an ultrasonic procedure for 30 min. The resulting suspension was homogenized for 3 min and clarified by centrifugation at 1000 × g for 10 min at 4⬚C. The pellet was discarded and supernatant was centrifuged at 12 000 × g for 20 min at 4⬚C. The precipitation was suspended in 10 mL physiological saline, and then the suspension was centrifuged at 12 000 × g. Then EGFR cell membrane suspension was obtained in 5 mL physiological saline. A total of 0.05 g silica was activated at 105⬚C for 30 min, and the cell membrane suspension was slowly added to silica under a vacuum and with agitation at 4⬚C. Then, the mixture was agitated for 30 min by a magnetic stirrer and allowed to stand overnight. Finally, the EGFR cell membrane stationary phase was packed into the CMC column (10 × 2.0 mm id) using a column-loading machine following a wet packing procedure.
1527
gated by injecting gefitinib into the EGFR/CMC column and the retained fraction was enriched and switched into seconddimension analytical VP-ODS column (ODS); the peak area of enriched gefitinib on ODS (AE ) was used as the indicator. Direct HPLC analysis of gefitinib was also studied and peak area (AS ) was also recorded. The enrichment ratio was calculated as AE /AS .
2.7 Application of the EGFR/CMC online HPLC–MS system The EGFR/CMC online HPLC–MS system was used to screen active components acting on EGFR from the ethanol extract of Radix et Rhizoma Asari. Gefitinib was used as a positive control to investigate this online 2D LC system. The standard solution of the active component that was recognized, analyzed, and identified by the EGFR/CMC online HPLC–MS system was also injected into this online-coupled system for further identification.
2.5 System suitability of the EGFR/CMC column The specificity of the EGFR/CMC column was investigated by injecting different drugs acting on different receptors into EGFR/CMC column. Atenolol acting on  receptor, tamsulosin hydrochloride acting on ␣1A receptor, nifedipine acting on calcium channel, and gefitinib acting on EGFR were used to investigate the specificity of the EGFR/CMC column. The reproducibility of the same EGFR/CMC column and different EGFR/CMC columns was investigated, and the retention time was used as an indicator. After the columns were packed using the column-loading machine, they were attached to the LC instrument and equilibrated for 60 min. Then for the single EGFR/CMC column, reproducibility was determined by repeated injection of 5 L of 0.1 mg/mL gefitinib, and the retention time was recorded. For different EGFR/CMC columns, 5 L of 0.1 mg/mL gefitinib was injected into the different EGFR/CMC columns, respectively, and the retention time was also recorded. The column lifetime was also investigated by repeated injections of 0.1 mg/mL gefitinib into the EGFR/CMC column.
2.6 System suitability of the EGFR/CMC online HPLC–MS system A two-position ten-port switching valve was used to combine the EGFR/CMC with HPLC–MS system. The system suitability of the EGFR/CMC online HPLC–MS system was investigated by injecting positive control drug gefitinib into the online 2D LC system. Gefitinib was recognized and retained on the EGFR/CMC column, and the retained fraction was enriched and switched into the HPLC–MS system for analysis and identification. The reproducibility of the enrichment was investigated five times repeatedly by gefitinib, and the peak area of enriched gefitinib on the second-dimension column was used as an indicator. The enrichment ratio was investi C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.8 Competitive binding assay The competitive binding assay was performed as follows [23, 24]: the competitive binding assay was studied by using gefitinib as a mobile phase additive at different concentrations changing from 6 × 10−8 to 9.6 × 10−7 mol/L, then gefitinib and asarinin were injected into the EGFR/CMC column. According to Eqs. (1) and (2), KD values of the gefitinib and asarinin binding with EGFR were calculated. k =
t − t0 t0
1 K D VM K D VM = [L ]M + k K M [R]S [R]S
(1)
(2)
KD and KM are the equilibrium dissociation constants of the analyte and mobile phase marker, respectively. [L]M is the molar concentration of the ligand in the mobile phase, [R]S is the concentration of immobilized receptors at the surface of the stationary phase, and VM is the dead volume of the column. The value k is the analyte capacity factor. When gefitinib was both used as the analyte and mobile phase additive, KD = KM , and Eq. (2) can be reduced to: VM K D VM 1 = [L ]M + k [R]S [R]S
(3)
Then [R]S /VM can be obtained from the slope, and KD of gefitinib can be determined by calculating the ratio of the intercept to the slope. In addition, the two parameters were substituted into Eq. (2), and the KD of asarinin was obtained by linear regression by plotting 1/k versus [L]M . www.jss-journal.com
1528
S. Han et al.
J. Sep. Sci. 2014, 37, 1525–1532
2.9 Molecular docking assay A molecular docking assay of gefitinib and asarinin with EGFR tyrosine kinase (PDB ID: 2ITY) was performed using SYBYL-X 1.1 to identify its binding region with protein. The substrate was constructed with SYBYL/Sketch module and optimized using Powell’s method. Energy minimization was performed using the Tripos force field with convergence criterion set at 0.005 kcal (Å mol)−1 and a maximum of 1000 iterations and Gasteiger–H¨uckel charges. A nonbonded cutoff distance of 8 Å was adopted to consider the intramolecular interaction [25].
2.10 Cell growth assay The effect of gefitinib and asarinin (which was identified by the EGFR/CMC online HPLC–MS system) on HEK293/EGFR cell viability were evaluated using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Briefly, exponentially growing cells were harvested and plated in 96-well plates at a concentration of 5 × 104 cells/well. After 24 h incubation at 37⬚C, cells were treated with gefitinib and asarinin at various concentrations for 48 h. Then, 20 L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/mL) was added to each well and incubated at 37⬚C for 4 h. After the supernatant was discarded, 150 L of DMSO was added to each well, and the optical density of cells was determined with a microplate reader (Bio-Rad instruments, USA) at 490 nm and expressed as absorbance values [26].
3 Results and discussion 3.1 System suitability of EGFR/CMC column The specificity of EGFR/CMC column was investigated by the positive control drug gefitinib. The results are shown in Fig. 1A. Figure 1A(I) showed gefitinib acting with blank silica gel column. Figure 1A(II) showed gefitinib acting with ␣1A/CMC column. Figure 1A(III) showed gefitinib acting with the RBL-2H3/CMC column. Figure 1A(IV) showed gefitinib acting with EGFR/CMC column. Only gefitinib can be retained on EGFR/CMC column. The selectivity of EGFR/CMC column was investigated by injecting different drugs acting on different receptors into EGFR/CMC column. Chromatograms of different drugs acted on EGFR/CMC column are shown in Fig. 1B. Atenolol acts on the  receptor, tamsulosin hydrochloride acts on the ␣1A receptor, nifedipine acts on the calcium channel, and gefitinib acts on EGFR. In Fig. 1B(IV), we can see that gefitinib can be retained on EGFR/CMC column, but atenolol is shown in Fig. 1B(I), tamsulosin hydrochloride shown in Fig. 1B(II), and nifedipine shown in Fig. 1B(III) cannot be retained on the EGFR/CMC column. These results suggested that gefitinib had better retention characteristics and EGFR C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. The specificity and selectivity of EGFR/CMC column. (A) Gefitinib retained on different CMC columns: (I) blank silica column, (II) ␣1A/CMC column, (III) RBL-2H3/CMC column, (IV) EGFR/CMC column. (B) Different drugs acting on the EGFR/CMC column: (I) atenolol; (II) tamsulosin hydrochloride; (III) nifedipine; (IV) gefitinib.
antagonists, and could interact with EGFR abundant in the highly expressed EGFR cell membrane. The reproducibility of different EGFR/CMC columns was tested by injecting gefitinib into different CMC columns, and the retention time of gefitinib was used as an indicator. The RSD of the retention times for different EGFR/CMC columns was 3.8% (n = 3). The reproducibility of each EGFR/CMC column was tested by repeatedly injecting gefitinib. The RSD of the retention time for each EGFR/CMC column was 1.3% (n = 5). So, the reproducibility of the EGFR/CMC column could satisfy the requirement of the experiment. As the EGFR/CMC columns have biological activity, the column lifetime was an important parameter. By the repeated injection of gefitinib, the EGFR/CMC column lifetime was investigated simultaneously. Gefitinib could be retained on the EGFR/CMC column well after the CMC column was used for 72 h. So the lifetime of the EGFR/CMC column could meet the requirements of experiment. The above results showed that the EGFR/CMC column was suitable for recognizing compounds from complex system. The specificity, selectivity, reproducibility, and the lifetime of the EGFR/CMC column met the requirements of the experiment.
3.2 System suitability of the EGFR/CMC online HPLC–MS system The EGFR/CMC column was combined with the HPLC–MS system through a ten-port column switcher, which was suitable for the qualitative analysis of active components acting on EGFR from the complex samples. The brief scheme of the 2D online LC system followed our previous studies [27–31]. Because the enrichment column used in the 2D online LC system was loaded with C18 , it had a better retentive ability than the EGFR/CMC column, so the retention fraction eluted www.jss-journal.com
J. Sep. Sci. 2014, 37, 1525–1532
Figure 2. Chromatograms of gefitinib standard solution analyzed by the EGFR/CMC online HPLC–MS system. (A) EGFR/CMC chromatogram of gefitinib standard solution including a retained fraction R (red, retention time was about 42 min); (C) HPLC–MS chromatogram of the corresponding fraction (between two dotted lines in EGFR/CMC chromatogram) extracted onto the enrichment column, and identified as gefitinib; (B) the direct HPLC–MS chromatogram of gefitinib standard solution.
from the EGFR/CMC column can be retained on the enrichment column. The retention fraction can be easily washed out and pumped into the analytical column for analysis and identification in the second dimension. Gefitinib was used to investigate the ability of EGFR/CMC online HPLC–MS system for recognition, analysis, and identification of active compound acting on EGFR. The results are shown in Fig. 2. Figure 2A showed that gefitinib can be well retained on the EGFR/CMC column (red, the retention time was about 42 min). Figure 2C shows an HPLC–MS chromatogram of the retention fraction identified as gefitinib. Figure 2B shows a direct HPLC chromatogram of gefitinib. The retention time of the retention fraction on the analytical column was consistent with the retention time of gefitinib. Results of the enrichment reproducibility and the ratio of enrichment were investigated using the peak area of the retention fraction on the analytical column (AE ), and the peak area of gefitinib was directly injected into the analytical column (AS ) as indicators. The RSD of the AE was 2.9% (n = 5). The enrichment ratio calculated by AE /AS was 78.5%. Therefore, both the reproducibility of enrichment and enrichment ratio met the requirements. So, the EGFR/CMC online HPLC–MS system is suitable for recognizing, analyzing, and identifying target components acting on EGFR.
3.3 Practical application of EGFR/CMC online HPLC–MS system Radix et Rhizoma Asari, a traditional Chinese medicine, was chosen and EGFR antagonists were screened, analyzed, and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Liquid Chromatography
1529
Figure 3. Chromatograms of the ethanol extract of Radix et Rhizoma Asari analyzed by the EGFR/CMC online HPLC–MS system. (A) The EGFR/CMC chromatogram of the ethanol extract of Radix et Rhizoma Asari including nonretained fraction R0 (yellow), and retained fraction R1 (red, retention time was about 10.2 min). (B) The direct HPLC–MS chromatograms of ethanol extracts of Radix et Rhizoma Asari. (C) The HPLC–MS chromatograms of the retained fractions. (D) The HPLC–MS chromatograms of the nonretained fractions.
identified using the EGFR/CMC online HPLC–MS system. Results of the ethanol extract of Radix et Rhizoma Asari assayed using the EGFR/CMC online HPLC–MS system are shown in Fig. 3. Figure 3A showed that there was one significant retention fraction (red, the retention time of R1 was about 9.9 min) and nonretained fraction (yellow) in the EGFR/CMC column. Then the retained fraction and nonretained fraction were assayed using the second-dimension HPLC–MS system for further separation and identification. Figure 3C shows the HPLC–MS chromatogram of the retention fractions (R1 ), which was identified as asarinin. Figure 3D shows the HPLC– MS chromatogram of the nonretained fraction (R0 ). Figure 3B shows the HPLC–MS chromatogram of the ethanol extract of Radix et Rhizoma Asari. As shown in Fig. 3C, the retained fraction (R1 ) was identified as asarinin by HPLC–MS. For further identification and screening the above result, asarinin standard solution was assayed by the EGFR/CMC online HPLC–MS system. The results are shown in Fig. 4. Figure 4A showed that the retention time of asarinin was about 10.2 min, the retained fraction R (red, between the two dotted lines) was extracted onto the enrichment column, and switched into second-dimension system for chromatographic separation and MS identification. Figure 4C shows the HPLC–MS chromatogram of the retained fraction. Figure 4B shows the direct HPLC–MS chromatogram of asarinin standard solution. This result further verified that the main retained fraction in the EGFR/CMC column was asarinin. www.jss-journal.com
1530
S. Han et al.
Figure 4. Chromatograms of asarinin standard solution analyzed by the EGFR/CMC online HPLC–MS system. (A) The EGFR/CMC chromatogram of the asarinin standard solution including retained fraction R (red, retention time was about 10.2 min). (B) The direct HPLC–MS chromatograms of the asarinin standard solution. (C) The HPLC–MS chromatograms of the retained fractions.
J. Sep. Sci. 2014, 37, 1525–1532
EGFR. The retention times of gefitinib and asarinin on the EGFR/CMC column were decreased with an increasing concentration of gefitinib in the mobile phase. The results are shown in Fig. 5. Figure 5A and B shows the elution profiles of gefitinib and asarinin on the EGFR/CMC column with different concentrations of gefitinib in the mobile phase. Figure 5C shows the regression curves achieved by plotting 1/k versus [L]M . The five concentrations of gefitinib were 6 × 10−8 , 1.2 × 10−7 , 2.4 × 10−7 , 4.8 × 10−7 , and 9.6 × 10−7 mol/L. These results indicate that gefitinib and asarinin have the same binding sites on EGFR. Besides, both the dissociation equilibrium constant of gefitinib (KD[G] ) and the dissociation equilibrium constant of asarinin (KD[A] ) were measured. The results were as follows: KD[G] = 4.02 × 10−6 M, KD[A] = 1.65 × 10−4 M. The dissociation equilibrium constant of gefitinib was smaller than that of asarinin, so the affinity of gefitinib was larger than that of asarinin. This result was corresponded with the retention time of gefitinib and asarinin on the EGFR/CMC column.
3.5 Molecular docking assay 3.4 Competitive binding assay The competitive binding assay was performed to investigate the binding sites and affinity of gefitinib and asarinin with
A molecular docking assay was often used to predict the binding sites and binding affinity between ligand and receptor. In this study, the molecular docking assay was performed using
Figure 5. The elution profiles of gefitinib (A) and asarinin (B) on the EGFR/CMC column with different concentration of gefitinib in the mobile phase. (C ) The regression cures achieved by plotting 1/k versus [L]M . The five concentrations of gefitinib were (I) 6 × 10−8 mol/L, (II) 1.2 × 10−7 mol/L, (III) 2.4 × 10−7 mol/L, (IV) 4.8 × 10−7 mol/L, and (V) 9.6 × 10−6 mol/L. D) Gefitinib docked into the active site of the EGFR (PDB ID: 2ITY). Figure 5E was the asarinin docked into the active site of the EGFR (PDB ID: 2ITY). (F ) The effect of gefitinib and asarinin on EGFR cell viability.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2014, 37, 1525–1532
the SYBYL/FlexX (Tripos) system based on the crystal structures of the EGFR (PDB ID: 2ITY) taken from the protein data bank. The results of the molecular docking assay are shown in Fig. 5. Figure 5D shows gefitinib docked into the active site of the EGFR. Figure 5E shows asarinin docked into the active site of the EGFR. In Fig. 5D and E, the results of gefitinib and asarinin binding with the EGFR at the same region can be obtained. The total score of gefitinib and asarinin binding with the EGFR was also given by the SYBYL/FlexX. The total score of gefitinib binding with the EGFR was 7.3849, and the total score of asarinin binding with the EGFR was 5.0732. This result was also corresponded with the retention time of gefitinib and asarinin on the EGFR/CMC column.
3.6 Cell growth assay Inhibitory effects of gefitinib and asarinin were tested in vitro on EGFR cell growth. The result shows that asarinin could inhibit the growth of EGFR cell, although this activity was lower than that of gefitinib, as shown in Fig. 5F. The inhibition rates of gefitinib on EGFR cell line were 7.1 ± 1.23, 16.6 ± 1.63, 19.5 ± 3.41, 20.8 ± 2.75, 22.7 ± 2.47, and 37.5 ± 2.33 at the concentrations of 0.1, 0.4, 1.6, 6.4, 25.6, and 102.4 M, respectively. For asarinin, the rates were 2.5 ± 1.22, 6.3 ± 1.25, 8.7 ± 1.47, 10.3 ± 2.31, 13.8 ± 1.83, and 23.2 ± 3.39. In the range of 0.10–102.4 M, gefitinib and asarinin showed obvious and dose-dependent inhibition activity. The inhibition activity of asarinin was weaker than that of gefitinib. The retention time of gefitinib on EGFR/CMC column was 40 min, but asarinin was only about 10 min, which indicated gefitinib had a stronger interaction with EGFR than that of asarinin. Therefore, retention behaviors of gefitinib and asarinin were correlated to their pharmacological activities.
1531
for Science & Technology Support of China (Grant number: 2012BAI29B06). The authors have declared no conflict of interest.
5 References [1] Marte, B., Nature 2013, 501, 327–372. [2] Sherr, C. J., Cell 2004, 116, 235–246. [3] Sugawa, N., Ekstrand, A., James, C. D., Collins, V., Proc. Natl. Acad. Sci. USA 1990, 87, 8602–8606. [4] Ogiso, H., Ishitani, R., Nureki, O., Fukai, S., Yamanaka, M., Kim, J., Saito, K., Sakamoto, A., Inoue, M., Shirouzu, M., Yokoyama, S., Cell 2002, 110, 775–787. [5] Richard, J., Sainsbury, C., Needham, G., Farndon, J., Malcolm, A. J., Harris, A., Lancet 1987, 329, 1398–1402. [6] Wakeling, A., Simon, G., Woodburn, J., Ashton, S., Curry, B., Barker, A., Gibson, K., Cancer Res. 2002, 62, 5749– 5754. [7] Song, N., Zhao, R., Que, L., Wan, C., Tang, Z., Chin. J. Ethnomed. Ethnopharm. 2008, 4, 50–53. [8] Li, Y., Tian, M., Yu, J., Shang, M., Cai, S., J. Nat. Med. 2010, 64, 442–451. [9] Shin, S., Anklam, K., Manning, E., Collins, M., Clin. Vaccine Immunol. 2009, 16(5), 613–620. [10] Schreiber, S., Science 2000, 287, 1964–1969. [11] Sheridan, R., Rengachari, V., Acc. Chem. Res. 1987, 20(9), 322–329. [12] He, L., Geng, X., New Prog. Biomed. Chromatogr. 1996, 3, 8–9. [13] Sun, M., Ma, W., Guo, Y., Hu, Z., He, L., J. Sep. Sci. 2013, 36, 2096–2013. [14] Liang, M., He, L., Yang, G., Life Sci. 2005, 78, 128–133.
4 Conclusions
[15] Hou, X., Wang, S., Hou, J., He, L., J. Sep. Sci. 2011, 21, 508–513.
In summary, an online 2D LC system was built successfully. Asarinin was screened, analyzed, and identified from the ethanol extract of Radix et Rhizoma Asari by this EGFR/CMC online HPLC–MS system. Results of competitive binding assay and molecular docking assay demonstrated that the binding region of asarinin was the same as that of gefitinib. A cell growth assay was carried to further verify the activity of asarinin in vitro. This 2D LC system has the potential to serve as a useful screening tool in antitumor drug discovery from natural medicinal herbs.
[16] Wang, S., Sun, M., Zhang, Y., Du, H., He, L., J. Chromatogr. A 2010, 1217, 5246–5252.
This work was supported by National Natural Science Foundation of China (Grant number: 81227802 and 81230079), National “Twelfth Five-Year” Plan for Important National Science & Technology Specific Projects of China (Grant number: 2012ZX09103201-054), and National “Twelfth Five-Year” Plan C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[17] Yue, Y., Xue, H., Wang, X., Yang, Q., Song, Y., Li, X., J. Sep. Sci. 2013, 37, 244–249. [18] Han, S., Zhang, T., Feng, L., Lv, N., Wang, S., J. Pharm. Biomed. 2013, 81–82, 133–137. [19] Li, M., Hou, X., Zhang, J., Wang, S., Fu, Q., He, L., J. Pharm. Anal. 2011, 1, 81–91. [20] Griffiths, J., Anal. Chem. 2008, 80, 5678–5683. [21] Gray, M. J., Dennis, G. R., Slonecker, P., Shalliker, R., J. Chromatogr. A 2004, 1041, 101–110. [22] Stoll, D., Li, X., Wang, X., Carr, P., Porter, S., Rutan, S., J. Chromatogr. A 2007, 1168, 3–43. [23] Du, H., He, J., Wang, S., He, L., Anal. Bioanal. Chem. 2010, 397, 1947–1953. [24] Geng, X., Regnier, F., J. Chromatogr. 1985, 332, 147–168.
www.jss-journal.com
1532
S. Han et al.
[25] Mou, J., Fang, H., Jing, F., Wang, Q., Liu, Y., Zhu, H., Shang, L., Wang, X., Xu, W., Bioorg. Med. Chem. 2009, 17, 466–473. [26] Naruse, I., Ohmori, T., Ao, Y., Fukumoto, H., Kuroki, T., Mori, M., Saijo, N., Nishio, K., Int. J. Cancer 2002, 98, 310–315. [27] He, H., Han, S., Zhang, T., Zhang, J., Wang, S., Hou, J., J. Pharm. Biomed. 2012, 62, 196–202.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2014, 37, 1525–1532
[28] Han, S., Zhang, P., Wei, F., Wang, S., Anal. Methods 2012, 4, 3351–3357. [29] Hou, X., Yuan, X., Zhang, B., Wang, S., J. Sep. Sci. 2013, 36, 706–712. [30] Han, S., Zhang, T., Huang, J., Hu, Z., Wang, S., Anal. Methods 2012, 4, 1078–1083. [31] Yang, X., Wang, Y., Zhang, X., Chang, R., Li, X., J. Sep. Sci. 2011, 34, 2586–2593.
www.jss-journal.com
