Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2018, 66, 653−660
Polydopamine-Coated Magnetic Molecularly Imprinted Polymers with Fragment Template for Identification of Pulsatilla Saponin Metabolites in Rat Feces with UPLC-Q-TOF-MS Yu-Zhen Zhang,† Jia-Wei Zhang,† Chong-Zhi Wang,‡ Lian-Di Zhou,*,§ Qi-Hui Zhang,*,†,‡ and Chun-Su Yuan‡ †
School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China Basic Medical College, Chongqing Medical University, Chongqing 400016, China ‡ Tang Center for Herbal Medicine Research and Department of Anesthesia & Critical Care, University of Chicago, Chicago, Illinois 60637, United States §
S Supporting Information *
ABSTRACT: In this work, a modified pretreatment method using magnetic molecularly imprinted polymers (MMIPs) was successfully applied to study the metabolites of an important botanical with ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The MMIPs for glucoside-specific adsorption was used to identify metabolites of Pulsatilla chinensis in rat feces. Polymers were prepared by using Fe3O4 nanoparticles as the supporting matrix, D-glucose as fragment template, and dopamine as the functional monomer and cross-linker. Results showed that MMIPs exhibited excellent extraction performance, large adsorption capacity (5.65 mg/g), fast kinetics (60 min), and magnetic separation. Furthermore, the MMIPs coupled with UPLC-Q-TOF-MS were successfully utilized for the identification of 17 compounds including 15 metabolites from the Pulsatilla saponin metabolic pool. This study provides a reliable protocol for the separation and identification of saponin metabolites in a complex biological sample, including those from herbal medicines. KEYWORDS: magnetic molecularly imprinted polymers, metabolites study, P. chinensis, fragment template
1. INTRODUCTION Pulsatilla chinensis (Bunge) Regel is a medical and edible botanical, which has been used for thousands of years in Asia. Everdwindling numbers of the wild plant have forced the price of P. chinensis higher and higher, with the shortage replaced through artificial cultivation.1 As an edible tonic tea, P. chinensis is widely consumed alone or in combination with other botanicals for preventing viral infection.1,2 In addition, P. chinensis is commonly used for treating intestinal amebiasis, malaria, vaginal trichomoniasis, bacterial infections, and malignant tumors.3,4 The triterpenoid saponins have been demonstrated to be the main chemical constituents and major active ingredients of P. chinensis.3 Anemoside B4 (AB4), the most important bioactive triterpenoid saponin of P. chinensis, has been reported to possess antiinflammatory activity.5 It can significantly inhibit the proliferation of Hep G2 cells6 and has prominent effects on the antitumor activities associated with cell proliferation, apoptosis, and oxidative stress.7,8 Recently, we reported the metabolic profile of AB4 associated with the microflora in rat small and large intestines. Ten metabolites were detected and identified with ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS).8 However, metabolic studies in complex biological samples are still challenging analytically because of the low concentration of analytes in complex biological matrices.9 Thus, developing powerful enrichment and high selectivity techniques for the extraction of target components from complex samples remains an important role. © 2017 American Chemical Society
Molecularly imprinted polymers (MIPs), with three-dimensional complementary cavities engineered through templates, has attracted great attention in the fields of complex matrices,10−13 drug delivery,9,14,15 biosensor,16−18 selective photocatalytic degradation,19,20 and chromatographic separation.21,22 Furthermore, magnetic surface imprinted materials can be rapidly and conveniently separated from sample under external magnetic fields without centrifugation or filtration procedures23 and can also improve mass transfer and reduce permanent entrapment.24 Thus, the novel magnetic MIPs (MMIPs) technique has widespread application in the pretreatment of samples. Dopamine (DA) is a small molecule mimicking adhesive proteins, which contains catechol and amine functional groups. At a weak alkaline pH, it can produce an adherent polydopamine (PDA) by self-polymerization.25 Recently, it has been reported that the self-polymerization of the functional monomer dopamine is used in the preparation of imprinted nanoparticles.26,27 Thus, we innovatively developed a facile, simple, and green approach to imprint templates on the surface coating of magnetic nanoparticles with a thin layer of PDA polymers. However, some template molecules, such as purified natural products, are so expensive that they limit the application of the MMIPs technique. Imprinted polymers prepared with fragment Received: Revised: Accepted: Published: 653
December December December December
7, 2017 19, 2017 20, 2017 20, 2017 DOI: 10.1021/acs.jafc.7b05747 J. Agric. Food Chem. 2018, 66, 653−660
Article
Journal of Agricultural and Food Chemistry
magnetically stirred at room temperature until forming a clear solution. Subsequently, 3.1 g of sodium acetate anhydrous and 0.4 g of sodium citrate were added. After that, the mixtures were vigorously stirred at 150 °C for 30 min, and then the homogeneous black solution was transferred to a Teflon-lined stainless steel autoclave. The autoclave was maintained at 200 °C for 8 h. Finally, the obtained black materials were rinsed with anhydrous ethanol and ultrapure water, and then dried under vacuum at 50 °C for 8 h. After that, 75 mg of synthesized magnetic nanoparticles was dispersed into 30 mL of Tris−HCl buffer solution (pH = 8.5, 10 mM) under ultrasonic vibration. Then 75 mg of D-glucose was added and the mixture solution was shaken at room temperature for 30 min, followed by the addition of 75 mg of DA·HCl. The resulting mixtures were reacted by self-polymerization in the dark and mechanically stirred at room temperature for 12 h. The harvested products were collected using a magnet and washed with acetonitrile−acetic acid (9:1, v/v) several times to remove D-glucose and then dried under vacuum for further use. As a control, the corresponding MNIPs were prepared in the identical manner without D-glucose. 2.4. Adsorption Experiments. The adsorption capacity of MMIPs/MNIPs was evaluated using AB4 as the test compound. The static adsorption experiments were carried out at 30 °C. 15 mg of MMIPs/MNIPs was added to 3 mL of different concentrations (10 μg/mL to 100 μg/mL) of AB4, which dissolved in Tris−HCl buffer solution (pH = 8.5, 10 mM). Then, the solution was shaken on an oscillator at 90 rpm for 4 h. The adsorption capacity of MMIPs/MNIPs (Q, mg/g) was calculated on the basis of the following equation:
templates (partial structures of templates) could solve the problem of these expensive and rare templates.13 Moreover, MMIPs using fragment templates can avoid template leakage28 and adsorb a number of active ingredients.29 Thus, it is of importance to extract the saponin metabolites of Pulsatilla and generate comprehensive metabolite information. Herein, we describe the synthesis of MMIPs based PDA polymer using candidate fragment templates, which derive from the four components of AB4, D-glucose, L-rhamnose, L-arabinose, and oleanolic acid. In addition, we describe the application of MMIPs in the isolation of metabolites of triterpenoid saponins in rat feces after oral administration of P. chinensis. In this present study, we incorporate aspects of green chemistry in the synthesis of MMIPs and determine the suitability and sensitivity of our technique as a new option for identifying metabolites from a metabolic pool in complex biological systems.
2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. Anemoside B4 standard (>98%) was obtained from Sichuan Vic’s Biological Technology Co., Ltd. (Sichuan, China). D-Glucose, L-rhamnose, L-arabinose, oleanolic acid, and dopamine hydrochloride (DA·HCl) were acquired from Adamas Reagent Co., Ltd. (Basel, Switzerland). Iron(III) chloride hexahydrate (FeCl3·6H2O), sodium acetate anhydrous (NaAc), ethylene glycol (CH2OH)2, sodium citrate (Na3C6H5O7·2H2O), hydrochloric acid (HCl), methanol (MeOH), anhydrous ethanol (EtOH), and acetic acid (AcOH) were purchased from Chengdu ke Long chemical reagent factory (Chengdu, China). Acetonitrile (ACN) and formic acid of HPLC grade were from Merck (Darmstadt, Germany). The ultrapure water used in experiments was prepared by a Milli-Q system (Millipore, Milford, MA, USA). Other reagents were of analytical grade. 2.2. Instruments. Scanning electron microscope (SEM) and transmission electron microscopy (TEM) images were adopted by MERLIN Compact (ZEISS, Germany) and JEM 2100F (JEOL, Japan). FT-IR spectra were obtained by an IR Affinity-1 Fourier transform near IR spectrometer (Shimadzu, Japan). The thermogravimetric analysis was performed by TGA/DSL1/1600LF (Mettler Toledo, Switzerland). UPLC analysis was carried out on a Nexera UPLC LC-30A system (Shimadzu, Kyoto, Japan) equipped with a binary pump, an online degasser, an autoinjector, and a thermostatically controlled column compartment. The Agilent Zorbax Eclipse plus C18 column (2.1 × 100 mm, 1.8 μm) was used for chromatographic separation. 2.3. Synthesis of MMIPs Nanoparticles. The synthetic routine of MMIPs is shown in Figure 1. First, magnetic Fe3O4 nanoparticles
Q = (C0 − Ce)V /m where C0 (μg/mL) is the concentration of the initial solution, Ce (μg/mL) is the equilibrium concentration of AB4 solution, V (mL) is the volume of AB4 solution, and m means the mass of MIPs/NIPs (mg). To further estimate the adsorption capacity of MMIPs/MNIPs, kinetic adsorption experiments were also done at 30 °C. 15 mg of MMIPs/MNIPs was mixed with 3 mL of 80 μg/mL Tris−HCl (pH = 8.5, 10 mM) AB4 solution. Afterward, the mixtures were shaken on an oscillator at 90 rpm for 5, 10, 20, 30, 40, 60, and 90 min. The equilibrium adsorption capacity of MMIPs/MNIPs (Q, mg/g) at different times was calculated with the following equation: Q = (C0 − Ct )V /m Here, Ct (μg/mL) is equilibrium concentration of AB4 solution at different times. 2.5. UPLC-MS Analysis. UPLC analysis was operated on a Nexera UPLC-LC-30A system (Shimadzu, Kyoto, Japan). Chromatographic separation was carried out on an Agilent Zorbax Eclipse plus C18 column (2.1 × 100 mm, 1.8 μm) with flow rate of 0.3 mL/min at 30 °C. The gradient mobile phase consisting of A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) was as follows: 10−25% B at 0−2 min, 25−70% B at 3−20 min, 70−95% B at 21−25 min, 95−95% B at 26−27 min, 95−10% B at 28−30 min. The sample volume injected was set at 2 μL, while spectra were acquired at 203 nm. An electrospray ionization (ESI) source was used for the ionization on AB Sciex Triple TOF 5600+ system. The following MS/MS conditions were optimized as follows: ion spray voltage, 4.5 kV; the turbo spray temperature, 550 °C; collision energy, −55 eV. The scan range of m/z 100−1500 was chosen in negative modes. Nitrogen was used as the nebulizer gas, and the nebulizer gas (gas 1) and the heater gas (gas 2) were set to 60 and 60 psi, respectively. A real time multiple mass defect filter and dynamic background subtraction performed on AB Sciex software (Analyst TF 1.6 software) was used to screen the profile and provide a whole production scan to avoid the omission of minor metabolites. In addition, an automated calibration delivery system was used to regulate MS and MS/MS behavior in the experiment. Data were evaluated by PeakView 1.2 and Metabolite Pilot 1.5 software (AB Sciex, Foster City, CA, USA). The theoretical mass tolerance was set at the mass accuracy of
