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706 Xiaofang Hou1 Xilong Yuan1 Bing Zhang1 Sicen Wang1 Qinhua Chen2

1 School

J. Sep. Sci. 2013, 36, 706712

Research Article

of Medicine, Xian Jiaotong University, Xian, P . R. China 2 Dongfeng Hospital, Hubei University of Medicine, Shiyan, P . R. China

Screening active anti-breast cancer compounds from Cortex Magnolia ofcinalis by 2D LCMS
Most of the anti-breast cancer drugs are often limited owing to drug resistance and serious adverse reactions. Therefore, development of more targeted and low toxic drugs from traditional Chinese medicines for breast cancer are needed. At the same time, establishment of fast and effective drug screening methods are urgently required. We describe here a 2D LC method of MDA-MB-231 cell membrane chromatography combined with HPLC/MS for recognition, separation, and identication of target components from traditional Chinese medicine Cortex Magnolia ofcinalis. The MDA-MB-231 cells membrane was used to prepare the chromatographic stationary phase in the rst dimension. The active compounds had a retention characteristic on the cell membrane chromatography model (10 2.0 mm, 5 m). The retention fractions were enriched using an online C18 column (10 1.0 mm, 5 m) and were analyzed by the second dimension RP chromatography. Finally, the activity of the retention fractions was tested through in vitro experiments. Results showed that the retention fractions were honokiol and magnolol and the inhibition rate on MDA-MB-231 cell growth were 23 and 64 M, respectively. These results support the conclusion that this coupled analytical technique could be an efcient method in drug discovery. Keywords: Active screening / Breast cancer cell / Cell membrane chromatography / Cortex Magnolia ofcinalis / Coupled analytical techniques DOI 10.1002/jssc.201200896

Received October 7, 2012 Revised October 25, 2012 Accepted November 1, 2012

1 Introduction
Breast cancer is the most frequently occurring cancer among women in the world. According to the cancer statistics 2012, there are 226 870 new cancer cases worldwide and more than 39 510 deaths [1]. Until now, some drugs have been clinically used in monotherapy or in a combinatorial therapy with other chemotherapeutic agents in the treatment of breast cancer such as monoclonal antibodies (tanstuzumab, ertumaxomab, and pertuzumab) [2], small molecule tyrosine kinase inhibitors (lapatinib, neratinib, and afatinib) [3], chemotherapeutic agents (taxanes and epothilones) [4], and targeting signaling pathways (temsirolimus, everolimus, and deforolimus). However, most of these drugs are often limited by the development of drug resistance and serious adverse reactions [5, 6]. Therefore, development of more targeted and low toxic drugs for breast cancer are urgently required.
Correspondence: Dr. Sicen Wang, School of Medicine, Xian Jiaotong University, 76 Yanta West Road, Xian 710061, Shaanxi, P . R. China E-mail: wangsc@mail.xjtu.edu.cn Fax: +86-29-82655451

Abbreviations: CMC, cell membrane chromatography; CMO, Cortex Magnolia ofcinalis; ER, estrogen receptor; ER36, estrogen receptor-alpha 36; TCM, traditional Chinese medicine

As we know, traditional Chinese medicines (TCMs) commonly play a dominant role in the discovery of leading compounds [7]. Recently, many screening methods have been developed. Among these, in vitro drug screening methods based on the model of diseases are very tedious and difcult because a large amount of pure compounds have to be prepared from TCMs before the screening experiments [8]. In recent years, drug screening methods based on target receptors and enzymes have been applied widely [911]. But the articial membranes, proteins, or enzymes cannot represent the real cell membrane receptors on the membrane surface. Little research can give a solution for direct screening and discovering active compounds from TCMs. To solve these problems, He and Geng proposed a bioafnity chromatography cell membrane chromatography (CMC) in 1996 [12], which could easily reect the real interaction between drugs and membrane receptor [13, 14]. In our previous studies, CMC has been used to screen the target components from medicinal herbs [1519]. They used tissue cell or macrophages as CMC. And they combined CMC with GC/MS to screen volatile compounds from TCMs. In order to screen highboiling compounds, in recent years, combining CMC with HPLC/MS system has been successfully developed to recognize, separate, and identify the active compounds from TCMs [20, 21]. However, using MDA-MB-231 cell to screen antibreast cancer active compounds from TCMs has not yet been reported.

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Magnolia ofcinalis Rehdet Wils is a species of Magnolia native to the mountains and valleys of China at altitudes of 3001500 m. The bark of Cortex M. ofcinalis (CMO) (known as Houpu) is stripped from the stems, branches, and roots and is used in TCM. The bark has been used in traditional formulas such as Banxia Houpu Tang [22], Xiaozhengai Tang, and Pingwei San [23] in modern clinical practice. According to the literature, it shows many pharmacological effects such as anti-inammatory [24], anticancer [25], antioxidant [26], antidepressant [27], and antispasmodic effects [28]. And many studies have shown that it could inhibit proliferation and induce apoptosis in many kinds of cancerous cells [29, 30]. Therefore, it is expected to screen the active components against breast cancer from CMO. Breast cancer was rst recognized to be an estrogendependent disease in 1896 [31]. Estrogen receptor (ER) expression is an important prognostic and predictive factor of breast cancer and has been become endocrine therapies target [32]. Therefore, ER is one of the most important targets in drug discovery. ER alpha 36 (ER-36) is a variant of ER- [33]. Studies have shown that ER-36 is highly expressed on the MDA-MB-231 cell membrane [34, 35]. Therefore, in this work, we chose this cell membrane as CMC stationary phase and developed the coupled chromatography method (MDA-MB-231/CMC-HPLC/MS) to screen and identify the potential active compounds from CMO. In addition, the inhibitory effect of the potential active compounds on the growth of MDA-MB-231 cells was also studied.

Factory (Tianjin, China). HPLC-grade water was purchased from Tedia (OH, USA).

2.2 Instruments The apparatus used was an HPLC-MS system (Shimadzu, Kyoto, Japan), which includes three LC-20AD pumps, an SIL-20A autosampler, a CTO-20A column oven, a DGU-20A degasser, an SPD-M20A diode array detector, LCMS2010EV MS, and LC-MS solution software. The one-dimensional column is MDA-MB-231/CMC column (10 mm 2.0 mm id, 5 m). The stationary phase is packed through an RPLZD10 pump (Dalian Replete Technology Corporation, Dalian, China). A VICIAG 10G-0911V 10 port 2-pos valve (Valco Instrument, Houston, TX, USA) was taken as the column switcher and two Shimpack VP-ODS pre-columns (10 mm 1.0 mm id, 5m) as the enrichment columns. The twodimensional column is a Shimadzu shim-pack VP-ODS column (150 mm 2.0 mm id, 5 m). Cell proliferation assay is quantied by a Model 680 plate reader (Bio-rad, Richmond, CA, USA). Ultrapure water is prepared by an UPT-IV purication system (UP Corporation, Chengdu, China).

2.3 Preparation of standard and sample solutions Standards were prepared separately with methanol in concentrations as soranib (2 mg/mL), nifedipine (1 mg/mL), dexamethasone acetate (1 mg/mL), and mixed standards (0.4 mg/mL). Honokiol and magnolol were prepared with methanol as 0.2 mg/mL. The CMO extract was obtained by ultrasonic extraction procedure. A total of 0.5 g coarse powder was extracted with 25 mL 95% methanol for 45 min at 30C. Supernatant was collected and the insoluble was extracted again according to the previse situation. The merged supernatant was ltered and then concentrated with rotary evaporator. Finally, the concentrated extract was dissolved with 50 mL methanol and stored at 4C in the dark.

2 Materials and methods


2.1 Materials was purchased The Silica gel (ZEX-II, 5 m, 100 A) from Qingdao Meigao Chemical Co. (Qingdao, China) and was activated at 105C before using. Soranib (purity 98%) was obtained from Nanjing Ange Pharmaceutical Co. (Nanjing, China). CMO was purchased from TCM store (Xian, China) and was identied by Professor Xiaofeng Niu, who is an authenticator of pharmacognosy lab of medical school of Xian Jiaotong University. And the voucher specimens have been deposited at Herbarium of Natural Medicine (No: 1131), Institute of Materia Medica, School of Medicine, Xian Jiaotong University, Shaanxi, China. In this experiment, Cortex of bark of M. ofcinalis was used. Magnolol and honokiol (purity 98%) were purchased from Chengdu Mansite Pharmaceutical Co. (Chengdu, China). The MDA-MB-231 cell line was purchased from the China Centre for Type Culture Collection (CCTCC, Wuhan, China). Dulbecco Minimum Essential Medium (DMEM) was purchased from Invitrogen (Grand Island, NY, USA) and the fetal bovine serum was ordered from Lanzhou Minhai Co. (Lanzhou, China). DMSO, 3-(4,5)-dimethylthiazol(2-yl)-3,5diphenyltetrazoliumbromide (MTT), and trypsin were obtained from Sigma (St. Louis, MO, USA). Ammonium acetate was purchased from Tianjin Fuchen Chemical Reagent
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2.4 Establishment of MDA-MB-231/CMC model The MDA-MB-231 cells were cultured in DMEM (with 10% fetal bovine serum) at 37C in a humidied atmosphere with 5% CO2 . Cells in the exponential growth phase were harvested by trypsin and were incubated in medium at 4C for 10 min. MDA-MB-231 cell membrane stationary phase was prepared as following steps: harvested cells (about 7 106 ) were washed with PBS (pH 7.4) three times by centrifuging at 210 g for 10 min at 4C. The cells were suspended with 50 mM Tris-HCl (pH 7.4) and ruptured by ultrasonic procedure for 30 min in icy water. The homogenate was centrifuged at 1000 g for 10 min at 4C. The pellet was discarded and the supernatant was centrifuged at 12 000 g for 20 min at 4C. The supernatant was then discarded and the
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Figure 1. The schematic diagram of MDAMB-231/CMC-HPLC/MS system (where CMC is cell membrane chromatography). UV, ultraviolet detector; DAD/MS, diode array detector/mass spectrometer detector; 1D CMC column: the MDA-MB-231/CMC column. 2D analytical column: the VP-ODS column.

cell membrane pellet was obtained. The cell membrane pellet was suspended with distilled water. Then, the cell membrane was immobilized according to reported method [16]. Briey, 0.05 g silica was activated at 105C for 30 min and mixed with cell membrane suspension under vacuum and agitation conditions at 4C. Then, the MDA-MB-231 cell membrane stationary phase was packed into the column through the column loading pump in 2.0 L/min distilled water owing. The MDA-MB-231/CMC column was loaded onto the chromatography system and equilibrated with 0.2 mL/min ammonium acetate buffer solution (pH 7.4, 10 mM) at 37C.

2.6 Validation of MDA-MB-231/CMC-HPLC/MS system In this section, soranib, nifedipine, and dexamethasone acetate were taken as controls to validate the specication of the MDA-MB-231/CMC-HPLC/MS system. First, soranib, nifedipine, and dexamethasone acetate were injected into the MDA-MB-231/CMC to investigate the CMC selectivity. The lifetime of the column and the precision of different patches of columns were also investigated (n = 3). Second, the mixed standards were injected into the 1D MDA-MB-231/CMC column. The nonretention fraction (0.662.0 min) and the retention fraction (45.070.0 min) were captured online and analyzed by the 2D HPLC/MS system. The 1D mobile phase was 50 mM NH4 Ac with a ow rate of 0.2 mL/min. The 2D mobile phase was methanol/aqueous formic acid (0.5%, v/v; 70:30, v/v) with a ow rate of 0.2 mL/min. The column temperature was 37C. The wavelength was set at 254 nm. The injection volume was 5 L.

2.5 Interface of MDA-MB-231/CMC and HPLC/MS system Figure 1 shows that the MDA-MB-231/CMC (1D) and HPLC/MS (2D) were combined through a 10-port valve and two enrichment columns. In position A, the sample was screened by MDA-MB-231/CMC model and the VP-ODS column was preequilibrated with mobile phase. The retention fraction was captured by the enrichment column through switching the 10-port valve to position B. After enrichment, the retention fraction was taken to the 2D HPLC/MS system to be analyzed through switching valve to position A.
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2.7 Application of the MDA-MB-231/CMC-HPLC/MS system The total extract of CMO was injected into the combined MDA-MB-231/CMC-HPLC/MS system. The retention
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fractions (3.015.0 min) in MDA-MB-231/CMC module were captured and analyzed with HPLC/MS system. The 1D chromatographic conditions were as same as that in Section 2.6. The 2D mobile phase was water (solvent A) and methanol (solvent B), and the gradient program was as follows: 020 min, 2535% B; 2045 min, 3575% B. MS conditions were as follows: ionization mode, ESI; nebulizer gas, N2 (purity 99.999%); ow rate 1.5 L/min; drying gas, N2 (99.999%); pressure, 0.1 MPa; interface temperature, 250C; heat block temperature, 200C; curved desolvation line temperature, 260C; curve dissolution line voltage, 10 V; detector voltage, 1.5 kV; negative ionization mode; scanning from m/z 200 to 800.

2.8 Validation of the active components The honokiol and magnolol standards were injected and analyzed in the MDA-MB-231/CMC-HPLC/MS system with the identical chromatography procedures in Section 2.7.
Figure 2. Chromatograms of soranib, nifedipine, and dexamesone acetate on the MDA-MB-231/CMC module (where CMC is cell membrane chromatography). (A) soranib, (B) nifedipine, and (C) dexamesone acetate.

2.9 Cell proliferation assay The effects of soranib, honokiol, and magnolol on MDAMB-231 were evaluated using the MTT assay. Briey, exponentially growing cells were harvested and plated in 96well plates at a centration of 5000 cells per well with 200 L DMEM medium. After 24-h incubation at 37C, cells were treated with the soranib (0, 15, 20, 25, 30, 35 M), honokiol (0, 40, 45, 50, 55, 60 M), and magnolol (0, 75, 80, 85, 90, 100 M) for 48h incubation, respectively. A total of 20 L of MTT (5 mg/mL) was added to each well and the plates were incubated at 37C 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 plate reader at 490 nm and expressed as absorbance values.

positive drug had an obvious retention characteristic, while nifedipine and dexamesone acetate had no retention characteristic. Figure 3A shows that their mixed standards had an obvious retention characteristic. The CMC chromatogram was divided as R0 and R1 fractions. R0 is represented as nonretention fraction and R1 is represented as retention fraction. Figure 3B shows the RP chromatograms of R0 and R1 fractions. Figure 3C shows the RP chromatograms of the mixed standards. It was found that dexamesone acetate (a) and nifedipine (b) were in R0 fraction and soranib (c) was in R1 fraction. 3.2 Practical application Figure 4A shows that the total extract of CMO had two obvious retention characteristics. The CMC chromatogram was divided as R0 and R1 fractions. Figure 4B shows the RP chromatograms of R0 and R1 fractions. R0 is represented as nonretention fraction and R1 is represented as retention fraction. Figure 4C shows the RP chromatograms of the extract. It was found that there were two chromatographic peaks in the R1 fraction. The retention time of peak a and b was 30.19 and 33.26 min, respectively, which was corresponding to the retention time of two components in the total extract, i.e. 30.26 and 33.29 min (Fig. 4C). The two peaks (a, b) were determined by the following MS. Figure 5 shows the mass spectrum of the peak a (honokiol) and peak b (magnolol), both of the massto-charge ratio were 265 (m/z). According to [29], the two potential active components might be the isomers: honokiol and magnolol. 3.3 Validation of the target components In order to verify the surmise, honokiol, and magnolol were tested on the MDA-MB-231/CMC-HPLC/MS system.
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3 Results and discussion


3.1 Suitability and reliability of the MDA-MB-231/CMC-HPLC/MS method According to the literature, tumor growth in the MDA-MB231 mammary tumor was inhibited by sorafenib, but not by getinib [36]. Therefore, we chose sorafenib as positive drug to study MDA-MB-231/CMC model. The reproducibility of the different MDA-MB-231/CMC columns was tested by the soranib standard solutions. The results showed that the RSD (%) of retention time (tR ) of soranib peak was 5.90% when changing MDA-MB-231/CMC columns (n = 3). The precision between the columns was to meet the assay requirements. The lifetime of the column was 3 days. Soranib, nifedipine, and dexamesone acetate and their mixed standards were used to test the suitability and reliability of the MDA-MB-231/CMC. Figure 2 shows that soranib as a
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J. Sep. Sci. 2013, 36, 706712

Figure 3. Chromatograms of mixed standards using the combined MDA-MB-231/CMC-HPLC/MS method (where CMC is cell membrane chromatography). (A) MDA-MB231/CMC chromatogram of the mixed standards including R0 , and R1 fractions (between two dotted lines), (B) HPLC/MS chromatograms of the R0 fraction, and (C) HPLC/MS chromatograms of the R1 fraction. a, dexamesone acetate; b, nifedipine; c, soranib.

Figure 4. Chromatograms of the total extract of CMO using the combined MDA-MB-231/CMC-HPLC/MS method (where CMC is cell membrane chromatography). (A) MDA-MB-231/CMC chromatogram of the total extract of CMO including R0 and R1 fractions (between two dotted lines), (B) HPLC/MS chromatograms of the R0 fraction. a: honokiol; b: magnolol, and (C) HPLC chromatogram of the total extract of CMO.

Figure 6A and C shows that honokiol and magnolol had very strong retention characteristics on the MDA-MB-231/CMC. Rh is represented as a retention fraction of honokioland and Rm is represented as a retention fraction of magnolol. The retention time of honokiol (Rh ) and magnolol (Rm ) were 4.48 and 5.28 min, respectively. Their retention time on the 2D chromatogram of Rh and Rm fractions was 30.15 min and 33.24 min, respectively, which corresponds to the retention time of peak a (30.26 min) and b (33.29 min) in Fig. 6E. The

results showed that honokiol and magnolol were the target components.

3.4 Effect of honokiol and magnolol on the growth of MDA-MB-231 cells The inhibitory effects of honokiol and magnolol on MDAMB-231 cell growth were tested by MTT. As shown in Fig. 7B,

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Figure 5. Mass spectrum of honokiol and magnolol. a, honokiol; b, magnolol.

Figure 6. Chromatograms of honokiol and magnolol using the combined MDA-MB-231/CMCHPLC/MS method (where CMC is cell membrane chromatography). (A and C) MDA-MB231/CMC chromatograms of honokiol and magnolol including Rh and Rm fraction (between two dotted lines), respectively; (B and D) HPLC chromatograms of the Rh and Rm fraction, h, honokiol; m, magnolol; and (E) HPLC chromatogram of the total extract of Cortex Magnolia ofcinalis.

Figure 7. Inhibiting effects of honokiol and magnolol on MDA-MB-231 cell growth. Data represent the means SD (n = 10) with * P < 0.05 and ** P < 0.01 versus the control. Ch , concentration of honokiol; Cm , concentration of magnolol.

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magnolol showed dose-dependent manner on MDA-MB-231 cell growth. The IC50 of magnolol was 64 M after 24 h. There were signicant differences from 55 to 75 M concentrations of magnolol compared with the control group (P < 0.05). Honokiol showed dose-independent manner on MDAMB-231 cell growth. The IC50 of honokiol was 23 M after 24 h. The inhibitory rate increased from 20 to 80% when honokiol concentration varied from 20 to 25 M. These results indicated that both honokiol and magnolol could inhibit the growth of MDA-MB-231 cells. The in vitro experimental results showed the feasibility and effectiveness of the coupled chromatography methods for fast screening active compounds from CMO.

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4 Conclusions
A coupled method MDA-MB-231/CMC-HPLC/MS was developed for screening honokiol and magnolol from the TCM Cortex M. ofcinalis. The in vitro results indicated that both of honokiol and magnolol could inhibit the growth of MDA-MB231 cells. The detailed interaction of honokiol and magnolol with ER-36 receptor will be further studied in the future. In summary, this coupled method will enable screening procedures to be more efcient to some extent. The authors thank Professor Langchong He for the valuable directions and assistance with the experiments. This work was also supported by National Science Foundation for Distinguished Young Scholars of China (No:81102414) and Hubei National Scientic Foundation of China (Nos. D20122401). The authors have declared no conict of interest.

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