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Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures
Accepted Manuscript Title: Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures Author: Haibo Wang Xiaoping Zhao Shufang Wang Shan Tao Ni Ai Yi Wang PII: DOI: Reference:
S0021-9673(15)00369-6 http://dx.doi.org/doi:10.1016/j.chroma.2015.03.002 CHROMA 356335
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
18-12-2014 28-2-2015 2-3-2015
Please cite this article as: H. Wang, X. Zhao, S. Wang, S. Tao, N. Ai, Y. Wang, Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures, Journal of Chromatography A (2015), http://dx.doi.org/10.1016/j.chroma.2015.03.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fabrication of enzyme-immobilized halloysite nanotubes
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for affinity enrichment of lipase inhibitors from complex
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mixtures
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Haibo Wang1†, Xiaoping Zhao2†, Shufang Wang1, Shan Tao1, Ni Ai1, Yi Wang*1
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University, Hangzhou, 310058, China
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College of Preclinical Medicine, Zhejiang Chinese Medical University, Hangzhou
310053, P. R. China
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These authors contributed equally
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Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang
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University, Tel/Fax: +86-571-88208426, e-mail: [email protected];
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Corresponding Author: Dr. Yi Wang, College of Pharmaceutical Sciences, Zhejiang
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Highlights
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> Lipase was immobilized into nanotubes by electrostatic interaction.
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> A nanoscale affinity selection strategy for ligand fishing from mixture.
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>A new type of biphenyl-type neolignans were identified as lipase inhibitors.
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ABSTRACT:
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Lipase is the key enzyme for catalyzing triglyceride hydrolysis in vivo, and lipase
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inhibitors have been used in the management of obesity. We present the first report on
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the use of lipase-adsorbed halloysite nanotubes as an efficient medium for the
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selective enrichment of lipase inhibitors from natural products. A simple and rapid
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approach was proposed to fabricate lipase-adsorbed nanotubes through electrostatic
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interaction. Results showed that more than 85% lipase was adsorbed into nanotubes in
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90 min, and approximately 80% of the catalytic activity was maintained compared with free lipase. The specificity and reproducibility of the proposed approach was validated by screening a known lipase inhibitor (i.e., orlistat) from a mixture that contains active and inactive compounds. Moreover, we applied this approach with
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high performance liquid chromatography-mass spectrometry technique to screen
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lipase inhibitors from the Magnoliae Cortex extract, a medicinal plant used for
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treating obesity. Two novel biphenyl-type natural lipase inhibitors magnotriol A and
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magnaldehyde B were identified, and their IC50 values were determined as 213.03 and 2
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96.96 μM, respectively. The ligand–enzyme interactions of magnaldehyde B were
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further investigated by molecular docking. Our findings proved that enzyme-adsorbed
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nanotube could be used as a feasible and selective affinity medium for the rapid
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screening of enzyme inhibitors from complex mixtures.
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KEYWORDS. Affinity selection mass spectrometry; enzyme inhibitor; rapid
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screening; microextraction; traditional Chinese medicine
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1. INTRODUCTION
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Triglyceride hydrolysis is catalyzed by lipase in vivo, and this process can lead to the
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accumulation of free fatty acids and monoacylglycerol in the body. The re-absorption
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of these constituents by the intestinal system may induce obesity and other relevant
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diseases [1,2]. Lipase inhibitors, such as orlistat, which forms covalent binding to the
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enzyme (lipase) and hence inhibits the triglyceride hydrolysis [3,4], have become an
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efficient way to control or treat obesity [5,6]. Natural products are promising
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resources of lipase inhibitors. Various naturally derived lipase inhibitors have been
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reported in recent years and have been shown to have potentials for drug development
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[7-9]. However, the screening and identification of lipase inhibitors from natural
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products are time-consuming and labor-intensive works that often involve repeated
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bioassay guided isolation and identification [8,10]. Thus, the rapid screening of lipase
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inhibitor from complex mixture of natural products is of great demand to discover novel therapeutics for diseases related to abnormal fat accumulation. During the last decade, various nanomaterials, such as carbon and TiO2 nanotubes have been used as microextraction mediums for the selective enrichment of specific
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compounds [11,12]. Furthermore, a number of affinity mass spectrometry- based
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approaches, such as ultrafiltration [13-15], enzyme-immobilized magnetic particles,
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enzyme reactors [16-18], and cell membrane chromatography [19], have been
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developed to explore potential ligands from complex mixtures. Recently, we have 4
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demonstrated that enzyme-immobilized magnetic beads can be used for the screening
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of active compounds from herbal extracts [20]. Compared with commonly used
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immobilization techniques based on covalent attachment or cross-linking, the
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adsorption of enzymes onto the carriers offers advantages, including (1) the retention
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of active conformation of free enzyme and (2) avoidance of complicated pretreatment
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and chemical modification processes. Meanwhile, the physical/chemical property of
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the carriers (e.g. surface area) is critical to the efficacy of ligand screening and to the
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extent of non-specific binding, which may confound the interpretation of results.
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Halloysite, a naturally occurring aluminosilicate nanotube, has attracted increasing
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interest because of its outstanding physical characteristics. The inner diameter,
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external diameter and length of halloysite nanotubes (HNTs) are 20–30 nm, 30–50 nm,
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and 1–2 µm, respectively [21]. HNTs provide an ideal nanometer-scale entrapment
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system for the storage of drugs, enzymes, and biocides [22,23]. More importantly, the
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external (mainly composed of O-Si-O groups) and inner surfaces (consist of Al2O3) of halloysite nanotubes provide more selective enzyme binding and hence reduce the non-specific adsorption of ligands onto HNTs. Therefore, HNTs may be used as a novel medium for ligands screening.
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The aim of this study is to develop a rapid and effective method to screen and
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characterize lipase inhibitors from Magnoliae cortex by lipase-adsorbed nanotube
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combined with HPLC-MS analysis. Magnoliae cortex, the bark of Magnoliae
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officinalis L. (Magnoliaceae), is a medicinal herb commonly used for digestive
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diseases. Many other biological activities have been associated with the extracts and 5
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ingredients of Magnoliae cortex, such as anti-inflammatory[24-28], anti-oxidant
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[29,30], anti-microbial [31], anti-clastogenic activities [32], and modulating the
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proliferation and migration of vascular smooth muscle cells [33]. To date, limited
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studies have been reported to identify the active compounds responsible for its
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efficacy in treating digestive diseases. In this study, four neolignan compounds were
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found to be potential ligands of lipase. Two of them were identified, and their
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structures were confirmed by nuclear magnetic resonance (NMR). Furthermore, their
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lipase inhibitory activities were verified by a functional assay. This new type of lipase
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inhibitors from Magnoliae cortex was reported for the first time.
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2. Experimental
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2.1. Materials and reagents
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The halloysite nanotubes were obtained from Wenzhou XinchengShenfei Aluminum
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Alloy Co., Ltd., China. Cortex Magnolia officinalis was obtained from the Hangzhou
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traditional Chinese herbal medicine factory (Hangzhou, China). Lipase (from porcine pancreas), 4-methylumbelliferyl oleate, orlistat and 4-nitrophenyl palmitate (pNPP), were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, Missouri, USA). Schisandrin was purchased from Ronghe Pharmaceutical Technology Development
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Co. Ltd. (Shanghai, China). HPLC-grade methanol and acetonitrile were purchased
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from Thermo Fisher Scientific (New Jersey, USA). Formic acid (HPLC-grade) was
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purchased from Roe Scientific Inc. (Newark, DE, USA).
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2.2. Characterization of lipase immobilized halloysite nanotubes 6
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The morphological characteristics of the halloysite nanotubes and lipase-immobilized
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HNTs were obtained by transmission electron microscopy (TEM). Samples were
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prepared by drying drops of a diluted aqueous solution of empty HNTs or
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enzyme-immobilized HNTs onto a carbon-coated copper grid. TEM analysis was
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performed on a JEM-3010 TEM at 80 kV. The Fourier-transform infrared (FT-IR)
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spectra of these samples were recorded by a Thermo Nicolet Avatar 370 FT-IR
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spectrometer in transmission mode between 400 and 4000 cm−1 at a resolution of 4
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cm−1.s
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2.3. Optimization of operative parameters for lipase immobilization
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To improve the binding efficiency of lipase on HNTs, parameters including lipase
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concentration, HNTs-to-enzyme ratio, pH of Tris buffer, incubation time, and
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incubation temperature were optimized. Different amounts of HNTs (5, 10, 20, 40, 60,
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and 80 mg) were mixed with lipase (1mL, 2 mg mL−1) at various time intervals (30,
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60, 90, 120, 180, and 240 minutes). After incubation at a specific time, the mixture was washed three times with 1mL of Tris buffer, centrifuged at 13,000 rpm for 5 min to collect the supernatant, and stored at 4 °C until use. The amount of immobilized lipase on HNTs was calculated by obtaining the difference in the amount of protein contents in the supernatant before and after adsorption. The catalytic activity of
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immobilized lipase was also determined and compared with free lipase by measuring
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the
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spectrophotometrical method [34]. Moreover, different concentrations of lipase (1mL,
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0.5, 1, 1.5, 2, 3, and 4 mg mL−1) were incubated with a constant amount of HNTs (40
hydrolysis
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mg) to optimize the enzyme concentrations during the adsorption process. In addition,
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different pH values (5.6, 6.2, 6.8, 7.4, and 8.0) and temperatures (4, 25, and 37 °C) of
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the incubation solution were used to investigate the extent of enzyme adsorption. The
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stability of lipase-immobilized nanotube reactor was evaluated by comparing its
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catalytic capability with free lipase after 2 weeks’ storage.
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2.4. Optimization of operative parameters for lipase immobilized-HNTs based ligand
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screening
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To test the specificity and to select the best conditions for ligand screening of the
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lipase-immobilized HNTs, a self-made test mixture containing orlistat (a lipase
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inhibitor with IC50 of 0.423 μM) and schisandrin (a phytochemical without lipase
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inhibitory activity) was used. Briefly, lipase-immobilized HNTs were incubated with
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the mixture (1mL, containing 2mg mL−1 orlistat and 0.2 mg mL−1 schisandrin) for 2 h,
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followed by three times of washing with 1mL of Tris buffer (ionic strength varied
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from 150mM to 1000mM; pH varied from 5.6 to 8.0). Degeneration solvent (10% to 100% acetonitrile) was added to release the potential ligands from the enzyme-loaded HNTs, whereas empty HNTs were operated in the same manner to exclude non-specific adsorbed compounds. The specificity of ligands screening was determined by the binding degree which was calculated by the following formula:
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binding degree = (A–A0)/Atotal, where A and A0 are the peak areas of the compound in
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the degeneration solution of lipase-coated HNTs and the empty HNTs, respectively.
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Atotal represents the peak area of the compound in the original test mixture. The
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detailed experimental conditions on the analysis of the mixture are found in 8
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Supporting Information S1.
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2.5. Screening potential ligands from Magnoliae cortex using lipase immobilized
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HNTs
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The screening of potential lipase ligands from the extract of Magnoliae cortex was
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performed as follows. A 1mL extract solution (5 mg mL−1) was incubated with
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lipase-immobilized HNTs (40 mg, dissolved in 1mL pH 6.8 Tris buffer) for 2 h at
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room temperature. After washing three times with 1mL of Tris buffer (to remove
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unbound compounds), the potential ligands were then released by 1mL of 50%
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acetonitrile and followed by centrifuging for 5 min. The supernatants were injected
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into the HPLC-MS. Two groups were designed for this experiment, namely, the
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sample group using the lipase-immobilized HNTs and the control group using the
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blank HNTs, to distinguish the non-specific binding compounds.
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The HPLC-MS analysis of degenerated samples were performed on an Agilent
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1100 series HPLC system connected to the LCQ DecaXPplus mass spectrometer via an ESI source. Agilent Zorbax SB-C18 column (4.6 mm, 250 mm, 5μm) was used for separation. The flow rate was 0.8 mL min−1, and the column temperature was set at 30 °C. The mobile phase was 0.05% formic acid–water (A) and acetonitrile (B). The
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linear gradient program was as follows: 0/20, 45/95, 50/95 (min/ B%). The MS
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signals were acquired in both the positive and negative ionization modes. The
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instrument settings were as follows: 60 arb sheath gas (N2), 10 arb auxiliary gas (N2),
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capillary temperature 350 °C, ESI spray voltages –3.5 kV. 9
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2.6. Identification and characterization of active compounds
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By using the preparative HPLC (Shimadzu LC-8A system with a photodiode array
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detector) and Agilent Zorbax SB-C18 column (250 × 21.2 mm, 7 μm), two identified
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compounds, namely, magnotriol A and magnaldehyde B, were isolated, and their 1H
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NMR (500 MHz) and
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AVANCE DMX 500 NMR spectrometer. The dose-dependent inhibitory activities of
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two compounds were determined by the conventional lipase inhibitory assay reported
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in our previous study[35]. The IC50 values of the compounds were calculated by the
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GraphPad Prism.
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2.7. Molecular docking of lipase and identified active compounds
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To investigate how the identified active compounds interact with lipase, we employed
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a molecular docking approach in the study. The high resolution crystal structure of
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lipase was retrieved from the Protein Data Bank (PDB id = 1LPB). Three-dimensional
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C NMR (125 MHz) spectra were obtained by a Bruker
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structures of the compounds were built, and energy was minimized using Molecular Operating Environment (MOE) (Montreal, CA). These structures were docked into the putative binding site of lipase by the MOE docking module. Each compound was docked 50 times, and the conformation with the highest docking score was kept for
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further evaluation.
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3. Results
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3.1. Characterization of natural halloysite nanotubes and lipase-immobilized
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halloysite nanotubes 10
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As shown in Figure 1A, HNTs form clear hollow tubular structure with 50 nm i.d., 80
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nm o.d., and ~1.5 μm in length and open spaces on the surfaces. The nanotubes were
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also open at the ends with an inner diameter of around 50 nm. After
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incubation/fabrication, the enzyme was adsorbed on the surfaces of the HNTs (the
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empty spaces were occupied, Figure 1B). The immobilization of lipase in HNTs is
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also evidenced by the presence of characteristic bands at 1659 cm−1 (representing the
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–C=O (amide I band) stretching vibration) and 1538 cm−1 (–NH (amide II band)
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bending vibration from the peptide group), as observed in the adsorbed lipase (Figure
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1C). The results suggest that the proposed incubation approach is feasible for
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incorporating lipase into HNTs.
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3.2. Optimization of operative conditions for lipase immobilization
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To select the best parameters for lipase immobilization, the effects of lipase
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concentration, the HNT-to-enzyme ratio, pH of buffer, incubation time, and
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incubation temperature were investigated. As shown in Figure 2A and 2B, the amount of adsorbed lipase and the relative enzyme activity increased with the increase in added lipase concentration, but then reached a plateau when the added lipase concentration was approximately 2 mg mL−1. The amount of adsorbed lipase and the relative enzyme activity also exhibited an increasing trend as the HNT-to-enzyme
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ratio increased from 20 to 40 mg/mg and remained unchanged at higher ratio (Figure
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2C and 2D). To minimize the oversaturated HNTs that may cause non-favorable
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non-specific adsorption of chemicals during screening, a lower HNTs-to-enzyme ratio
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should be chosen. Furthermore, no significant difference (p >0.05) was observed 11
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between the percentage of adsorbed lipase (82.78% to 84.02%, Figure 2E) and the
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relative enzyme activity (70.9% to 72.7%, Figure 2F) at various buffer pH values
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(from pH 5.6 to 8.0), though the maximum activity was observed at the physiological
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pH (i.e., pH 6.8).
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The amount of loaded lipase and relative enzyme activity increased as the
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incubation time increased from 30 min to 90 min, and then reached an equilibrium
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(Figure 3A and 3B). Moreover, the highest amount of adsorbed lipase and the optimal
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enzyme activity were achieved when the incubation temperature was set to 37°C
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(Figure 3C and 3D). After incubation, the sample mixture was rinsed three times with
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Tris buffer, and negligible protein was detected in the washes, suggesting that the
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enzyme was strongly adsorbed onto HNTs. Furthermore, the hydrolytic activity of
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immobilized lipase (after the washes) remained at a relatively high level (about 80%
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of the activity of free lipase), which was in consistent with the previous finding that
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the catalytic activity of adsorbed lipase can be retained/maintained on hydrophilic
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that the immobilized lipase could maintain more than 80% of the original activity for
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14 days, whereas the stored free lipase rapidly loses its efficacy after 2 weeks’ storage
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(Figure 3E). The reusability of the lipase-immobilized nanotube reactor was also
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tested by the repeated use of the same sample for ligand screening. Figure 3F shows
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carriers [36].
To test the stability of immobilized lipase at long term storage, the immobilized
lipase samples were stored at 4 °C for 2 weeks storage, and the catalytic activity was compared with those free lipase to examine the storage stability. The results showed
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that the immobilized lipase maintained over 50% of the original activity after seven
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cycles. The immobilization of lipase on various types of carriers has been vastly
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investigated in the past decades [34,37]. Compared with previous reports, the loading
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amount (12.5 mg g−1) and stability of lipase on HNTs were satisfied because of the
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quick and simple procedure of adsorption-based immobilization.
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3.3. Optimization of operative conditions for lipase-immobilized HNTs based
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screening
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A mixture of orlistat (lipase inhibitor) and schisandrin (non-lipase inhibitor, used as
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negative control) was used to evaluate the screening selectivity/feasibility of
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lipase-immobilized HNTs. As shown in Figure 4A, an increase in the amount (peak
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area) of orlistat in the dissociation solution after incubated with lipase-adsorbed HNTs
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(when compared with those using unloaded HNTs) was observed, indicating that the
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adsorption of lipase was highly selectively. No change in the amount of schisandrin, was observed in both the lipase-adsorbed and unloaded HNTs. The washing steps after incubation play important roles in ligand fishing because
unbound or nonspecific compounds may lead to false positive results. Investigations
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on different washing times (one to three times) were carried out. The results indicated
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that all nonspecific binding orlistat were completely removed after three times of
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rising (Figure 4B). The fourth wash was performed by different proportions of
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acetonitrile–water solutions to release the bound ligands from the HNTs. We found 13
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that 50% acetonitrile–water (v/v) was the optimal degeneration solvent (Figure 4C).
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As shown in Figure 4D, no apparent difference in the binding efficiency was observed
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when the pH of the buffer varied from 6.8 to 8.0. Thus, buffer with pH 6.8 was
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adopted in the screening steps. Figure 4E shows that an increase in the ionic strength
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of the buffer medium reduced the amount of orlistat. This result may be explained by
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the fact that the surface charge of lipase was neutralized by the excess ions in the
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buffer solution and hence led to a significant decrease in the electrostatic interaction
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between the orlistat and the lipase [38]. In summary, Tris buffer (150mM, pH6.8) for
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washing three times and 50% acetonitrile-water(v/v) were found to be the optimal
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operation conditions. After optimization, the screening procedure was repeated, and
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the results were found to be reproducible (peak area of orlistat within 3.1% RSD,
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n=6). The binding capacity of orlistat was calculated as 16.5% from the linearity
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curve (Figure 4F). Those results indicated that the proposed lipase-adsorbed nanotube
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reactor was stable, selective, reproducible, and convenient for ligand screening.
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remain unclear. To further investigate on this issue, a screening test was conducted for
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this crude herbal extract. After the incubation of the crude extract with
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lipase-adsorbed HNTs and subsequent sample washings and LC/MS analysis, four
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abundant signals (peaks) were identified (Figure 5). The chromatographic portions of
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3.4. Screening and identification of lipase inhibitor from extract of Magnoliae cortex
Our preliminary study demonstrated that the crude extract of Magnoliae cortex exhibits anti-lipase activity (IC50=14.46 μg mL−1). However, its active compounds
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peaks 1 and 4 were further isolated and identified (based on their NMR data,
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Supporting Information Tables S-1 and S-2) as magnotriol A and magnaldehyde B,
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respectively. Peaks 2 and 3 were tentatively identified as magnaldehyde A and
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magnaldehyde D according to multistage tandem MS analysis. The binding degree of
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magnotriol A (1), magnaldehyde A (2), magnaldehyde D (3), and magnaldehyde B (4)
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were calculated as 4.5%, 5.0%, 7.0%, and 7.4%, respectively. Validation assay
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showed that the IC50 values of magnotriol A (1) and magnaldehyde B (4) were 213
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and 96.96 μM, respectively. It is obvious that magnaldehyde B with a higher binding
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degree exerted better anti-lipase activity than magnotriol A, suggesting that the extent
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of binding determined by this method was consistent with the lipase inhibitory
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activity.
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3.5. Molecular docking of lipase and magnaldehyde B
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Figure 6A illustrates the complex of magnaldehyde B–lipase predicted by molecular
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docking. As shown in Figure 6B, docking simulations revealed significant binding
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may inhibit lipase activity by occupying the catalytic site, therefore blocking the
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lipolytic activity of the enzyme (Figure 6).
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4. Discussion
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interactions between magnaldehyde B and key amino acids in the catalytic pocket of the pancreatic lipase, such as the hydrogen bonding of the hydroxyl group with His263. In addition to the polar interactions with the enzyme, several hydrophobic contacts were observed for this compound. Our results suggested that this compound
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Nanomaterials, in particular inorganic nanomaterials with excellent physical
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properties, including high surface area, excellent chemical and thermal stability, high
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mechanical strength, and ultra-light weight, are important advanced materials
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commonly used in biomedical research (e.g., biosensors, bioanalysis and biocatalysis)
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[39,40]. For example, Hua et al. immobilized the enzyme on multi-walled carbon
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nanotubes for glucose detection[41]. Miyahata and co-workers developed a graphene
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oxide-based DNA sensing platform by using fluorescence resonance energy transfer
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for DNA analysis[42]. However, application of nanomaterials in ligand screening is
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restricted by the relatively high proportion of non-specific adsorption from
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non-covalent hydrophobic and π-stacking interactions between the carriers and the
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small molecules. Thus, the development of nanomaterials with limited non-specific
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adsorption is of great importance to maximize their potential as carrier system for
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ligand screening.
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In this study, we described a novel approach in identifying potential active
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HNTs exhibited high selectivity for the solid-phase extraction of trace Pd(II) [47]. To
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the best of our knowledge, HNTs have never been explored as a carrier for drug
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screening. This technique was applied to the crude extract of Magnoliae cortex and
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two novel biphenyl-type neolignans with lipase inhibitory effects were identified.
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ligands from complex mixtures by using enzyme-immobilized nanomaterials. Loading or immobilization of the enzymes in halloysite nanotubes has been reported in several literatures[43,44]. However most of them used HNTs as carriers for drug delivery [45] and catalytic reaction[46]. Li et.al. also reported that functionalized
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Moreover, our docking results indicated that magnaldehyde B can be bound to several
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key amino acids in the catalytic pocket of the pancreatic lipase, suggesting that this
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adsorption-immobilized enzyme reactor is a useful tool for screening inhibitors from
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complex mixture.
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Our results showed that enzyme-immobilized HNTs can be utilized as an
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alternative affinity based screening technique for ligand fishing from natural products.
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Compared with magnetic materials based ligand fishing assays[48,49], which need
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multi-step reaction to immobilize enzymes on carriers through covalent binding or
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crosslinking, the proposed adsorption-based immobilization strategy is much quicker
335
and simpler. Meanwhile, in comparison with our previous proposed hollow
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fiber-based immobilization, the loading amount and efficiency of enzyme adsorbed
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onto the nanotube are much higher because of the large area-to-volume ratio of HNTs.
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Furthermore, since HNTs can be packed in column for on-line solid phase extraction,
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it can be applied as an automated immobilization devices and can be coupled with
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LC-MS for on-line screening and identification of ligands, which would dramatically reduce the screening time and hence improve productivity.
5. Conclusion
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In this work, we demonstrated an adsorption immobilization of lipase on
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nanotubes and its application in ligand fishing from natural products. It is a rapid,
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convenient, and efficient technique for screening enzyme inhibitors when combined
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with LC-MS. Two neolignans with lipase inhibitory effects were identified. Future 17
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work will focus on improving the adsorption capacity of the nanomaterials and
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simplifying the operating procedure by incorporating these nanotubes with an on-line
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analytical apparatus.
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ACKNOWLEDGEMENTS
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This study was supported by the National Key Scientific and Technological Project of
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China (No. 2012ZX09304007).
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Figure Captions
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Figure 1. Characterization of HNTs and lipase immobilized HNTs. (A) TEM images
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of blank HNTs. (B) TEM images of lipase loaded HNTs. (C) FT-IR spectra of lipase
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(green), HNTs (blue), and lipase loaded HNTs (red).
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Figure 2. Optimization of operative parameters for lipase immobilization. Effects of
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lipase concentration on (A) binding efficacy and (B) relative activity, effects of ratio
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of HNTs to lipase on (C) binding efficacy and (D) relative activity, and effects of
498
buffer pH on (E) binding efficacy and (F) relative activity.
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Figure 3. Effects of incubation time on (A) binding efficacy and (B) relative activity,
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and effects of incubation temperature on (C) binding efficacy and (D) relative activity.
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(E) Storage stability of free and immobilized lipase and (F) reusability of immobilized
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lipase.
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Figure 4. (A) Representative chromatograms of mixture incubated with
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lipase-adsorbed HNTs and blank HNTs in negative ion mode.
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Figure 6. Magnaldehyde B–lipase complex predicted by molecular docking (A) 3D
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view of docking results. Magnaldehyde B was colored by atom type (Carbon: grey,
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Oxygen: red) and the lipase is shown in cartoon representation. Interaction surface
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lipase-adsorbed HNTs and blank HNTs. Effects of (B) wash times, (C) acetonitrile proportion, (D) pH, and (E) ionic strength on the binding efficiency of orlistat. (F) Linearity of gradient concentrations of orlistat incubation with immobilized lipase. Figure 5. HPLC-MS chromatograms of Magnoliae cortex extract incubated with
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between ligand and protein is displayed and colored according to atom polarity (polar:
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magenta, hydrophobic: green, mild polar: blue). (B) Two–dimensional view of
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binding interaction of magnaldehyde B with key amino acids.
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S0021-9673(15)00369-6 http://dx.doi.org/doi:10.1016/j.chroma.2015.03.002 CHROMA 356335
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
18-12-2014 28-2-2015 2-3-2015
Please cite this article as: H. Wang, X. Zhao, S. Wang, S. Tao, N. Ai, Y. Wang, Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures, Journal of Chromatography A (2015), http://dx.doi.org/10.1016/j.chroma.2015.03.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fabrication of enzyme-immobilized halloysite nanotubes
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for affinity enrichment of lipase inhibitors from complex
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mixtures
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Haibo Wang1†, Xiaoping Zhao2†, Shufang Wang1, Shan Tao1, Ni Ai1, Yi Wang*1
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University, Hangzhou, 310058, China
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2
College of Preclinical Medicine, Zhejiang Chinese Medical University, Hangzhou
310053, P. R. China
†
These authors contributed equally
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Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang
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*
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University, Tel/Fax: +86-571-88208426, e-mail: [email protected];
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Corresponding Author: Dr. Yi Wang, College of Pharmaceutical Sciences, Zhejiang
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Highlights
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> Lipase was immobilized into nanotubes by electrostatic interaction.
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> A nanoscale affinity selection strategy for ligand fishing from mixture.
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>A new type of biphenyl-type neolignans were identified as lipase inhibitors.
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ABSTRACT:
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Lipase is the key enzyme for catalyzing triglyceride hydrolysis in vivo, and lipase
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inhibitors have been used in the management of obesity. We present the first report on
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the use of lipase-adsorbed halloysite nanotubes as an efficient medium for the
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selective enrichment of lipase inhibitors from natural products. A simple and rapid
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approach was proposed to fabricate lipase-adsorbed nanotubes through electrostatic
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interaction. Results showed that more than 85% lipase was adsorbed into nanotubes in
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90 min, and approximately 80% of the catalytic activity was maintained compared with free lipase. The specificity and reproducibility of the proposed approach was validated by screening a known lipase inhibitor (i.e., orlistat) from a mixture that contains active and inactive compounds. Moreover, we applied this approach with
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high performance liquid chromatography-mass spectrometry technique to screen
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lipase inhibitors from the Magnoliae Cortex extract, a medicinal plant used for
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treating obesity. Two novel biphenyl-type natural lipase inhibitors magnotriol A and
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magnaldehyde B were identified, and their IC50 values were determined as 213.03 and 2
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96.96 μM, respectively. The ligand–enzyme interactions of magnaldehyde B were
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further investigated by molecular docking. Our findings proved that enzyme-adsorbed
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nanotube could be used as a feasible and selective affinity medium for the rapid
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screening of enzyme inhibitors from complex mixtures.
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KEYWORDS. Affinity selection mass spectrometry; enzyme inhibitor; rapid
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screening; microextraction; traditional Chinese medicine
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1. INTRODUCTION
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Triglyceride hydrolysis is catalyzed by lipase in vivo, and this process can lead to the
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accumulation of free fatty acids and monoacylglycerol in the body. The re-absorption
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of these constituents by the intestinal system may induce obesity and other relevant
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diseases [1,2]. Lipase inhibitors, such as orlistat, which forms covalent binding to the
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enzyme (lipase) and hence inhibits the triglyceride hydrolysis [3,4], have become an
53
efficient way to control or treat obesity [5,6]. Natural products are promising
54
resources of lipase inhibitors. Various naturally derived lipase inhibitors have been
55
reported in recent years and have been shown to have potentials for drug development
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[7-9]. However, the screening and identification of lipase inhibitors from natural
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products are time-consuming and labor-intensive works that often involve repeated
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bioassay guided isolation and identification [8,10]. Thus, the rapid screening of lipase
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inhibitor from complex mixture of natural products is of great demand to discover novel therapeutics for diseases related to abnormal fat accumulation. During the last decade, various nanomaterials, such as carbon and TiO2 nanotubes have been used as microextraction mediums for the selective enrichment of specific
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compounds [11,12]. Furthermore, a number of affinity mass spectrometry- based
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approaches, such as ultrafiltration [13-15], enzyme-immobilized magnetic particles,
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enzyme reactors [16-18], and cell membrane chromatography [19], have been
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developed to explore potential ligands from complex mixtures. Recently, we have 4
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demonstrated that enzyme-immobilized magnetic beads can be used for the screening
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of active compounds from herbal extracts [20]. Compared with commonly used
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immobilization techniques based on covalent attachment or cross-linking, the
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adsorption of enzymes onto the carriers offers advantages, including (1) the retention
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of active conformation of free enzyme and (2) avoidance of complicated pretreatment
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and chemical modification processes. Meanwhile, the physical/chemical property of
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the carriers (e.g. surface area) is critical to the efficacy of ligand screening and to the
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extent of non-specific binding, which may confound the interpretation of results.
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Halloysite, a naturally occurring aluminosilicate nanotube, has attracted increasing
76
interest because of its outstanding physical characteristics. The inner diameter,
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external diameter and length of halloysite nanotubes (HNTs) are 20–30 nm, 30–50 nm,
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and 1–2 µm, respectively [21]. HNTs provide an ideal nanometer-scale entrapment
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system for the storage of drugs, enzymes, and biocides [22,23]. More importantly, the
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external (mainly composed of O-Si-O groups) and inner surfaces (consist of Al2O3) of halloysite nanotubes provide more selective enzyme binding and hence reduce the non-specific adsorption of ligands onto HNTs. Therefore, HNTs may be used as a novel medium for ligands screening.
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The aim of this study is to develop a rapid and effective method to screen and
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characterize lipase inhibitors from Magnoliae cortex by lipase-adsorbed nanotube
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combined with HPLC-MS analysis. Magnoliae cortex, the bark of Magnoliae
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officinalis L. (Magnoliaceae), is a medicinal herb commonly used for digestive
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diseases. Many other biological activities have been associated with the extracts and 5
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ingredients of Magnoliae cortex, such as anti-inflammatory[24-28], anti-oxidant
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[29,30], anti-microbial [31], anti-clastogenic activities [32], and modulating the
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proliferation and migration of vascular smooth muscle cells [33]. To date, limited
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studies have been reported to identify the active compounds responsible for its
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efficacy in treating digestive diseases. In this study, four neolignan compounds were
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found to be potential ligands of lipase. Two of them were identified, and their
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structures were confirmed by nuclear magnetic resonance (NMR). Furthermore, their
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lipase inhibitory activities were verified by a functional assay. This new type of lipase
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inhibitors from Magnoliae cortex was reported for the first time.
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2. Experimental
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2.1. Materials and reagents
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The halloysite nanotubes were obtained from Wenzhou XinchengShenfei Aluminum
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Alloy Co., Ltd., China. Cortex Magnolia officinalis was obtained from the Hangzhou
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traditional Chinese herbal medicine factory (Hangzhou, China). Lipase (from porcine pancreas), 4-methylumbelliferyl oleate, orlistat and 4-nitrophenyl palmitate (pNPP), were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, Missouri, USA). Schisandrin was purchased from Ronghe Pharmaceutical Technology Development
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Co. Ltd. (Shanghai, China). HPLC-grade methanol and acetonitrile were purchased
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from Thermo Fisher Scientific (New Jersey, USA). Formic acid (HPLC-grade) was
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purchased from Roe Scientific Inc. (Newark, DE, USA).
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2.2. Characterization of lipase immobilized halloysite nanotubes 6
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The morphological characteristics of the halloysite nanotubes and lipase-immobilized
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HNTs were obtained by transmission electron microscopy (TEM). Samples were
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prepared by drying drops of a diluted aqueous solution of empty HNTs or
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enzyme-immobilized HNTs onto a carbon-coated copper grid. TEM analysis was
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performed on a JEM-3010 TEM at 80 kV. The Fourier-transform infrared (FT-IR)
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spectra of these samples were recorded by a Thermo Nicolet Avatar 370 FT-IR
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spectrometer in transmission mode between 400 and 4000 cm−1 at a resolution of 4
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cm−1.s
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2.3. Optimization of operative parameters for lipase immobilization
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To improve the binding efficiency of lipase on HNTs, parameters including lipase
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concentration, HNTs-to-enzyme ratio, pH of Tris buffer, incubation time, and
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incubation temperature were optimized. Different amounts of HNTs (5, 10, 20, 40, 60,
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and 80 mg) were mixed with lipase (1mL, 2 mg mL−1) at various time intervals (30,
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60, 90, 120, 180, and 240 minutes). After incubation at a specific time, the mixture was washed three times with 1mL of Tris buffer, centrifuged at 13,000 rpm for 5 min to collect the supernatant, and stored at 4 °C until use. The amount of immobilized lipase on HNTs was calculated by obtaining the difference in the amount of protein contents in the supernatant before and after adsorption. The catalytic activity of
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immobilized lipase was also determined and compared with free lipase by measuring
129
the
130
spectrophotometrical method [34]. Moreover, different concentrations of lipase (1mL,
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0.5, 1, 1.5, 2, 3, and 4 mg mL−1) were incubated with a constant amount of HNTs (40
hydrolysis
of
p-nitrophenol
palmitate
by
using
previously
reported
7
Page 7 of 30
mg) to optimize the enzyme concentrations during the adsorption process. In addition,
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different pH values (5.6, 6.2, 6.8, 7.4, and 8.0) and temperatures (4, 25, and 37 °C) of
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the incubation solution were used to investigate the extent of enzyme adsorption. The
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stability of lipase-immobilized nanotube reactor was evaluated by comparing its
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catalytic capability with free lipase after 2 weeks’ storage.
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2.4. Optimization of operative parameters for lipase immobilized-HNTs based ligand
138
screening
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To test the specificity and to select the best conditions for ligand screening of the
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lipase-immobilized HNTs, a self-made test mixture containing orlistat (a lipase
141
inhibitor with IC50 of 0.423 μM) and schisandrin (a phytochemical without lipase
142
inhibitory activity) was used. Briefly, lipase-immobilized HNTs were incubated with
143
the mixture (1mL, containing 2mg mL−1 orlistat and 0.2 mg mL−1 schisandrin) for 2 h,
144
followed by three times of washing with 1mL of Tris buffer (ionic strength varied
146 147 148 149
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from 150mM to 1000mM; pH varied from 5.6 to 8.0). Degeneration solvent (10% to 100% acetonitrile) was added to release the potential ligands from the enzyme-loaded HNTs, whereas empty HNTs were operated in the same manner to exclude non-specific adsorbed compounds. The specificity of ligands screening was determined by the binding degree which was calculated by the following formula:
150
binding degree = (A–A0)/Atotal, where A and A0 are the peak areas of the compound in
151
the degeneration solution of lipase-coated HNTs and the empty HNTs, respectively.
152
Atotal represents the peak area of the compound in the original test mixture. The
153
detailed experimental conditions on the analysis of the mixture are found in 8
Page 8 of 30
Supporting Information S1.
155
2.5. Screening potential ligands from Magnoliae cortex using lipase immobilized
156
HNTs
157
The screening of potential lipase ligands from the extract of Magnoliae cortex was
158
performed as follows. A 1mL extract solution (5 mg mL−1) was incubated with
159
lipase-immobilized HNTs (40 mg, dissolved in 1mL pH 6.8 Tris buffer) for 2 h at
160
room temperature. After washing three times with 1mL of Tris buffer (to remove
161
unbound compounds), the potential ligands were then released by 1mL of 50%
162
acetonitrile and followed by centrifuging for 5 min. The supernatants were injected
163
into the HPLC-MS. Two groups were designed for this experiment, namely, the
164
sample group using the lipase-immobilized HNTs and the control group using the
165
blank HNTs, to distinguish the non-specific binding compounds.
167 168 169 170
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The HPLC-MS analysis of degenerated samples were performed on an Agilent
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1100 series HPLC system connected to the LCQ DecaXPplus mass spectrometer via an ESI source. Agilent Zorbax SB-C18 column (4.6 mm, 250 mm, 5μm) was used for separation. The flow rate was 0.8 mL min−1, and the column temperature was set at 30 °C. The mobile phase was 0.05% formic acid–water (A) and acetonitrile (B). The
171
linear gradient program was as follows: 0/20, 45/95, 50/95 (min/ B%). The MS
172
signals were acquired in both the positive and negative ionization modes. The
173
instrument settings were as follows: 60 arb sheath gas (N2), 10 arb auxiliary gas (N2),
174
capillary temperature 350 °C, ESI spray voltages –3.5 kV. 9
Page 9 of 30
2.6. Identification and characterization of active compounds
176
By using the preparative HPLC (Shimadzu LC-8A system with a photodiode array
177
detector) and Agilent Zorbax SB-C18 column (250 × 21.2 mm, 7 μm), two identified
178
compounds, namely, magnotriol A and magnaldehyde B, were isolated, and their 1H
179
NMR (500 MHz) and
180
AVANCE DMX 500 NMR spectrometer. The dose-dependent inhibitory activities of
181
two compounds were determined by the conventional lipase inhibitory assay reported
182
in our previous study[35]. The IC50 values of the compounds were calculated by the
183
GraphPad Prism.
184
2.7. Molecular docking of lipase and identified active compounds
185
To investigate how the identified active compounds interact with lipase, we employed
186
a molecular docking approach in the study. The high resolution crystal structure of
187
lipase was retrieved from the Protein Data Bank (PDB id = 1LPB). Three-dimensional
189 190 191
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C NMR (125 MHz) spectra were obtained by a Bruker
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13
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structures of the compounds were built, and energy was minimized using Molecular Operating Environment (MOE) (Montreal, CA). These structures were docked into the putative binding site of lipase by the MOE docking module. Each compound was docked 50 times, and the conformation with the highest docking score was kept for
192
further evaluation.
193
3. Results
194
3.1. Characterization of natural halloysite nanotubes and lipase-immobilized
195
halloysite nanotubes 10
Page 10 of 30
As shown in Figure 1A, HNTs form clear hollow tubular structure with 50 nm i.d., 80
197
nm o.d., and ~1.5 μm in length and open spaces on the surfaces. The nanotubes were
198
also open at the ends with an inner diameter of around 50 nm. After
199
incubation/fabrication, the enzyme was adsorbed on the surfaces of the HNTs (the
200
empty spaces were occupied, Figure 1B). The immobilization of lipase in HNTs is
201
also evidenced by the presence of characteristic bands at 1659 cm−1 (representing the
202
–C=O (amide I band) stretching vibration) and 1538 cm−1 (–NH (amide II band)
203
bending vibration from the peptide group), as observed in the adsorbed lipase (Figure
204
1C). The results suggest that the proposed incubation approach is feasible for
205
incorporating lipase into HNTs.
206
3.2. Optimization of operative conditions for lipase immobilization
207
To select the best parameters for lipase immobilization, the effects of lipase
208
concentration, the HNT-to-enzyme ratio, pH of buffer, incubation time, and
210 211 212 213
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incubation temperature were investigated. As shown in Figure 2A and 2B, the amount of adsorbed lipase and the relative enzyme activity increased with the increase in added lipase concentration, but then reached a plateau when the added lipase concentration was approximately 2 mg mL−1. The amount of adsorbed lipase and the relative enzyme activity also exhibited an increasing trend as the HNT-to-enzyme
214
ratio increased from 20 to 40 mg/mg and remained unchanged at higher ratio (Figure
215
2C and 2D). To minimize the oversaturated HNTs that may cause non-favorable
216
non-specific adsorption of chemicals during screening, a lower HNTs-to-enzyme ratio
217
should be chosen. Furthermore, no significant difference (p >0.05) was observed 11
Page 11 of 30
between the percentage of adsorbed lipase (82.78% to 84.02%, Figure 2E) and the
219
relative enzyme activity (70.9% to 72.7%, Figure 2F) at various buffer pH values
220
(from pH 5.6 to 8.0), though the maximum activity was observed at the physiological
221
pH (i.e., pH 6.8).
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218
The amount of loaded lipase and relative enzyme activity increased as the
223
incubation time increased from 30 min to 90 min, and then reached an equilibrium
224
(Figure 3A and 3B). Moreover, the highest amount of adsorbed lipase and the optimal
225
enzyme activity were achieved when the incubation temperature was set to 37°C
226
(Figure 3C and 3D). After incubation, the sample mixture was rinsed three times with
227
Tris buffer, and negligible protein was detected in the washes, suggesting that the
228
enzyme was strongly adsorbed onto HNTs. Furthermore, the hydrolytic activity of
229
immobilized lipase (after the washes) remained at a relatively high level (about 80%
230
of the activity of free lipase), which was in consistent with the previous finding that
231
the catalytic activity of adsorbed lipase can be retained/maintained on hydrophilic
235
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222
236
that the immobilized lipase could maintain more than 80% of the original activity for
237
14 days, whereas the stored free lipase rapidly loses its efficacy after 2 weeks’ storage
238
(Figure 3E). The reusability of the lipase-immobilized nanotube reactor was also
239
tested by the repeated use of the same sample for ligand screening. Figure 3F shows
232 233 234
carriers [36].
To test the stability of immobilized lipase at long term storage, the immobilized
lipase samples were stored at 4 °C for 2 weeks storage, and the catalytic activity was compared with those free lipase to examine the storage stability. The results showed
12
Page 12 of 30
240
that the immobilized lipase maintained over 50% of the original activity after seven
241
cycles. The immobilization of lipase on various types of carriers has been vastly
243
investigated in the past decades [34,37]. Compared with previous reports, the loading
244
amount (12.5 mg g−1) and stability of lipase on HNTs were satisfied because of the
245
quick and simple procedure of adsorption-based immobilization.
246
3.3. Optimization of operative conditions for lipase-immobilized HNTs based
247
screening
248
A mixture of orlistat (lipase inhibitor) and schisandrin (non-lipase inhibitor, used as
249
negative control) was used to evaluate the screening selectivity/feasibility of
250
lipase-immobilized HNTs. As shown in Figure 4A, an increase in the amount (peak
251
area) of orlistat in the dissociation solution after incubated with lipase-adsorbed HNTs
252
(when compared with those using unloaded HNTs) was observed, indicating that the
254 255 256
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adsorption of lipase was highly selectively. No change in the amount of schisandrin, was observed in both the lipase-adsorbed and unloaded HNTs. The washing steps after incubation play important roles in ligand fishing because
unbound or nonspecific compounds may lead to false positive results. Investigations
257
on different washing times (one to three times) were carried out. The results indicated
258
that all nonspecific binding orlistat were completely removed after three times of
259
rising (Figure 4B). The fourth wash was performed by different proportions of
260
acetonitrile–water solutions to release the bound ligands from the HNTs. We found 13
Page 13 of 30
that 50% acetonitrile–water (v/v) was the optimal degeneration solvent (Figure 4C).
262
As shown in Figure 4D, no apparent difference in the binding efficiency was observed
263
when the pH of the buffer varied from 6.8 to 8.0. Thus, buffer with pH 6.8 was
264
adopted in the screening steps. Figure 4E shows that an increase in the ionic strength
265
of the buffer medium reduced the amount of orlistat. This result may be explained by
266
the fact that the surface charge of lipase was neutralized by the excess ions in the
267
buffer solution and hence led to a significant decrease in the electrostatic interaction
268
between the orlistat and the lipase [38]. In summary, Tris buffer (150mM, pH6.8) for
269
washing three times and 50% acetonitrile-water(v/v) were found to be the optimal
270
operation conditions. After optimization, the screening procedure was repeated, and
271
the results were found to be reproducible (peak area of orlistat within 3.1% RSD,
272
n=6). The binding capacity of orlistat was calculated as 16.5% from the linearity
273
curve (Figure 4F). Those results indicated that the proposed lipase-adsorbed nanotube
274
reactor was stable, selective, reproducible, and convenient for ligand screening.
278
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279
remain unclear. To further investigate on this issue, a screening test was conducted for
280
this crude herbal extract. After the incubation of the crude extract with
281
lipase-adsorbed HNTs and subsequent sample washings and LC/MS analysis, four
282
abundant signals (peaks) were identified (Figure 5). The chromatographic portions of
275 276 277
3.4. Screening and identification of lipase inhibitor from extract of Magnoliae cortex
Our preliminary study demonstrated that the crude extract of Magnoliae cortex exhibits anti-lipase activity (IC50=14.46 μg mL−1). However, its active compounds
14
Page 14 of 30
peaks 1 and 4 were further isolated and identified (based on their NMR data,
284
Supporting Information Tables S-1 and S-2) as magnotriol A and magnaldehyde B,
285
respectively. Peaks 2 and 3 were tentatively identified as magnaldehyde A and
286
magnaldehyde D according to multistage tandem MS analysis. The binding degree of
287
magnotriol A (1), magnaldehyde A (2), magnaldehyde D (3), and magnaldehyde B (4)
288
were calculated as 4.5%, 5.0%, 7.0%, and 7.4%, respectively. Validation assay
289
showed that the IC50 values of magnotriol A (1) and magnaldehyde B (4) were 213
290
and 96.96 μM, respectively. It is obvious that magnaldehyde B with a higher binding
291
degree exerted better anti-lipase activity than magnotriol A, suggesting that the extent
292
of binding determined by this method was consistent with the lipase inhibitory
293
activity.
294
3.5. Molecular docking of lipase and magnaldehyde B
295
Figure 6A illustrates the complex of magnaldehyde B–lipase predicted by molecular
296
docking. As shown in Figure 6B, docking simulations revealed significant binding
300
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301
may inhibit lipase activity by occupying the catalytic site, therefore blocking the
302
lipolytic activity of the enzyme (Figure 6).
303
4. Discussion
297 298 299
interactions between magnaldehyde B and key amino acids in the catalytic pocket of the pancreatic lipase, such as the hydrogen bonding of the hydroxyl group with His263. In addition to the polar interactions with the enzyme, several hydrophobic contacts were observed for this compound. Our results suggested that this compound
15
Page 15 of 30
Nanomaterials, in particular inorganic nanomaterials with excellent physical
305
properties, including high surface area, excellent chemical and thermal stability, high
306
mechanical strength, and ultra-light weight, are important advanced materials
307
commonly used in biomedical research (e.g., biosensors, bioanalysis and biocatalysis)
308
[39,40]. For example, Hua et al. immobilized the enzyme on multi-walled carbon
309
nanotubes for glucose detection[41]. Miyahata and co-workers developed a graphene
310
oxide-based DNA sensing platform by using fluorescence resonance energy transfer
311
for DNA analysis[42]. However, application of nanomaterials in ligand screening is
312
restricted by the relatively high proportion of non-specific adsorption from
313
non-covalent hydrophobic and π-stacking interactions between the carriers and the
314
small molecules. Thus, the development of nanomaterials with limited non-specific
315
adsorption is of great importance to maximize their potential as carrier system for
316
ligand screening.
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In this study, we described a novel approach in identifying potential active
321
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304
322
HNTs exhibited high selectivity for the solid-phase extraction of trace Pd(II) [47]. To
323
the best of our knowledge, HNTs have never been explored as a carrier for drug
324
screening. This technique was applied to the crude extract of Magnoliae cortex and
325
two novel biphenyl-type neolignans with lipase inhibitory effects were identified.
318 319 320
ligands from complex mixtures by using enzyme-immobilized nanomaterials. Loading or immobilization of the enzymes in halloysite nanotubes has been reported in several literatures[43,44]. However most of them used HNTs as carriers for drug delivery [45] and catalytic reaction[46]. Li et.al. also reported that functionalized
16
Page 16 of 30
Moreover, our docking results indicated that magnaldehyde B can be bound to several
327
key amino acids in the catalytic pocket of the pancreatic lipase, suggesting that this
328
adsorption-immobilized enzyme reactor is a useful tool for screening inhibitors from
329
complex mixture.
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Our results showed that enzyme-immobilized HNTs can be utilized as an
331
alternative affinity based screening technique for ligand fishing from natural products.
332
Compared with magnetic materials based ligand fishing assays[48,49], which need
333
multi-step reaction to immobilize enzymes on carriers through covalent binding or
334
crosslinking, the proposed adsorption-based immobilization strategy is much quicker
335
and simpler. Meanwhile, in comparison with our previous proposed hollow
336
fiber-based immobilization, the loading amount and efficiency of enzyme adsorbed
337
onto the nanotube are much higher because of the large area-to-volume ratio of HNTs.
338
Furthermore, since HNTs can be packed in column for on-line solid phase extraction,
339
it can be applied as an automated immobilization devices and can be coupled with
341
342
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LC-MS for on-line screening and identification of ligands, which would dramatically reduce the screening time and hence improve productivity.
5. Conclusion
343
In this work, we demonstrated an adsorption immobilization of lipase on
344
nanotubes and its application in ligand fishing from natural products. It is a rapid,
345
convenient, and efficient technique for screening enzyme inhibitors when combined
346
with LC-MS. Two neolignans with lipase inhibitory effects were identified. Future 17
Page 17 of 30
work will focus on improving the adsorption capacity of the nanomaterials and
348
simplifying the operating procedure by incorporating these nanotubes with an on-line
349
analytical apparatus.
350
ACKNOWLEDGEMENTS
351
This study was supported by the National Key Scientific and Technological Project of
352
China (No. 2012ZX09304007).
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Figure Captions
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Figure 1. Characterization of HNTs and lipase immobilized HNTs. (A) TEM images
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of blank HNTs. (B) TEM images of lipase loaded HNTs. (C) FT-IR spectra of lipase
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(green), HNTs (blue), and lipase loaded HNTs (red).
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Figure 2. Optimization of operative parameters for lipase immobilization. Effects of
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lipase concentration on (A) binding efficacy and (B) relative activity, effects of ratio
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of HNTs to lipase on (C) binding efficacy and (D) relative activity, and effects of
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buffer pH on (E) binding efficacy and (F) relative activity.
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Figure 3. Effects of incubation time on (A) binding efficacy and (B) relative activity,
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and effects of incubation temperature on (C) binding efficacy and (D) relative activity.
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(E) Storage stability of free and immobilized lipase and (F) reusability of immobilized
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lipase.
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Figure 4. (A) Representative chromatograms of mixture incubated with
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lipase-adsorbed HNTs and blank HNTs in negative ion mode.
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Figure 6. Magnaldehyde B–lipase complex predicted by molecular docking (A) 3D
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view of docking results. Magnaldehyde B was colored by atom type (Carbon: grey,
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Oxygen: red) and the lipase is shown in cartoon representation. Interaction surface
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lipase-adsorbed HNTs and blank HNTs. Effects of (B) wash times, (C) acetonitrile proportion, (D) pH, and (E) ionic strength on the binding efficiency of orlistat. (F) Linearity of gradient concentrations of orlistat incubation with immobilized lipase. Figure 5. HPLC-MS chromatograms of Magnoliae cortex extract incubated with
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between ligand and protein is displayed and colored according to atom polarity (polar:
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magenta, hydrophobic: green, mild polar: blue). (B) Two–dimensional view of
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binding interaction of magnaldehyde B with key amino acids.
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