Elsevier

Journal of Chromatography B

Volume 960, 1 June 2014, Pages 126-132
Journal of Chromatography B

Functionalized magnetic nanoparticles coupled with mass spectrometry for screening and identification of cyclooxygenase-1 inhibitors from natural products

https://doi.org/10.1016/j.jchromb.2014.04.032Get rights and content

Highlights

  • New Fe3O4@SiO2–COX-1 nanoparticles were synthesized for ligand fishing.

  • Immobilized COX-1 displays more convenient separation and higher reusability.

  • Four curcuminoids in turmeric were fished out and identified.

  • Comprehensive mass fragmentation pathways of curcuminoids were established.

Abstract

Development of simple and effective methods for high-throughput, high-fidelity screening and identification of cyclooxygenase-1 (COX-1) inhibitors from natural products are important for drug discovery to treat inflammation and carcinogenesis. Here, we developed a new screening assay based on cyclooxygenase-1 (COX-1) functionalized magnetic nanoparticles (i.e. Fe3O4@SiO2–COX-1) for solid phase ligand fishing, and then mass spectrometry (MS) was applied for structural identification. Incubation conditions were optimized. High specificity for isolating COX-1 inhibitors was achieved by testing positive control, indomethacin, with active and inactive COX-1. Moreover, high stability of immobilized COX-1 (remained 95.3% after ten consecutive cycles) allows the analysis reproducible. When applied to turmeric extract, four curcuminoids (i.e. curcumin, demethoxycurcumin, bisdemethoxycurcumin, and 1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-(1E,6E)-1,6-heptadiene-3,5-dione), difficult to be distinguished from original MS spectrum of turmeric extract, were isolated as main COX-1 inhibitors. Their structures were characterized based on their accurate molecular weight and diagnostic fragment ions. The results indicated that the proposed method was a simple, robust and reproducible approach for the discovery of COX-1 inhibitors from complex matrixes.

Introduction

Enzymes are attractive drug targets, and enzyme inhibitors represent almost half the drugs used in clinical today [1]. Therefore, discovery of new enzyme inhibitors has been one of the major interests and challenges in drug discovery and development process [2]. Historically, majority of new drugs were generated from natural products or compounds derived from natural products [3]. From 1981 to 2010, 50% of all the marketed-new chemical entities were shown to be of natural origin [4]. Undoubtedly, natural products continue to play a highly significant role in the discovery of drug leads.

Natural products embody hundreds or even thousands of secondary metabolites, but a few of them are responsible for the pharmacological activity. The key step in natural products research is to develop high throughput, high fidelity methods for discovery of bioactive components. Moreover, the process of modernization and globalization of natural products necessitate the biochemical profiles analysis for quality control purpose. To address these requirements, biofingerprint chromatogram based on ligand fishing has been proposed to provide unique information on the multiple bioactive compounds direct interaction with drug targets (i.e. protein, enzyme and receptor). Ligand fishing assay has been considered as the most convenient and efficient technology with high-selectivity and high-throughput in early stage bioactive components discovery, which include, but are not limited to, centrifugation [6], ultrafiltration [7], [8], [9], equilibrium dialysis [10], microdialysis [11], magnetic solid phase fishing [7], [12], and surface plasmon resonance [13], [14]. Immobilized drug targets on solid surface are more robust and resistant to environmental changes than solution phase drug targets [7], [15]. At the same time, magnetic nanoparticles could be separated from solution conveniently. Therefore, ligand fishing based on functionalized magnetic nanoparticles has been proved to be an exciting method to screen ligands from natural products [7], [12].

COX-1 is known as a housekeeping enzyme constitutively expressed in almost all the mammalian tissues. The prostaglandins produced from arachidonic acid transformation through the participation of COX-1 play an important role in inflammation and carcinogenesis [16]. Importantly, platelet COX-1 is the target of one of the most efficacious antithrombotic agents used for prevention of vascular occlusive events (i.e. aspirin) [17], which thereby provides the rationale for the development of COX-1 inhibitors. Many reports have focused on the detection of COX-1 inhibitory activity of commercial isolated compounds from natural products or synthesized compounds [17], [18], [19], [20]. However, much less attention has been paid to develop facile screening assay to discover COX-1inhibitors from natural products.

Turmeric is dried powder from rhizomes of Curcuma longa L. (Zingiberaceae), which has been used as a traditional medicine for its various pharmacological activities such as anti-inflammatory, antiviral, antioxidant, anti-infectious activities, anti-parasitic infection, anti-mutagenic effect, and anticancer [21], [22]. The underlying mechanisms of these effects involve the recognition of various molecular targets, such as enzymes and protein kinases. Recent studies showed that curcuminoids, the major yellow pigment and active components of turmeric [23], had significantly higher inhibitory effects on the peroxidase activity of COX-1 than COX-2 [24]. However, no reports systematically analyzed COX-1 inhibitors in turmeric.

Herein, we report proof of principle for the first time of integration of COX-1 functionalized magnetic nanoparticles and direct infusion MS for facile, specific screening and identification of COX-1 inhibitors from complex natural products. Four curcuminoids (i.e. curcumin [1], demethoxycurcumin [2], bisdemethoxycurcumin [3], and 1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-(1E,6E)-1,6-heptadiene-3,5-dione [4], Fig. 1) with COX-1inhibitory activity were isolated from turmeric.

Section snippets

Chemicals and reagents

Ovine COX-1, arachidonic acid, prostaglandin E2 (PGE2), [d4]-PGE2, co-factors l-epinephrine and hematin, 3-aminopropyltrimethoxysilane (APTMS), tetraethyl orthosilicate, and glutaraldehyde (25%, w/v aqueous solution) were acquired from Sigma–Aldrich Chemicals (St. Louis, MO, USA). The HPLC grade acetonitrile and methanol were bought from Tedia Company, Inc. (Ohio, USA). Ultrapure water (18.2 MΩ) was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). Buffer solution

Characterization of Fe3O4@SiO2–COX-1 nanoparticles

Magnetic nanoparticles (e.g. Fe3O4) were widely used for enzyme immobilization and separation due to its higher surface area, lower mass transfer resistance and easier solid–liquid separation. Fe3O4 based ligand fishing has been successfully applied for identifying bioactive components from natural products, such as binders of serum albumin [12], [7], [26], tyrosinase [7], α-glucosidase [27], [28], maltase [29], and xanthine oxidase [30]. In addition, Fe3O4@SiO2 could promote enzyme stability

Conclusion

We have proposed a new assay based on Fe3O4@SiO2–COX-2 ligand fishing for facile and rapid screening COX-1 inhibitors from complex natural products, and then direct infusion MS was developed for structural elucidation. The proposed method was verified by COX-1 inhibitor, indomethacin, which indicated the method could successfully isolate COX-1 inhibitors in rid of nonspecific binding. Another particularly noteworthy advantage of the developed method was that immobilized COX-1 was stable, which

Acknowledgements

Partial support of this work by a grant from the National Natural Science Foundation of China (21275163), the Science and Technology Program of Hunan Province, China (2012FJ2006), the Shenghua Yuying project of Central South University, China, aid program for Science and Technology Innovative Research Team (Chemicals of Forestry Resources and Development of Forest Products) in Higher Educational Institutions of Hunan Province, China, and the Postdoctoral Science Foundation of Central South

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