Abstract
Human health is increasingly affected by chronic inflammation, which can subsequently develop into chronic diseases such as cancer, arthritis, diabetes, and diseases of the cardiovascular and nervous systems. Traditional non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit cyclooxygenase-1 (COX-1) produce side effects, such as irritation of the gastrointestinal tract and renal toxicity. While commercially available inhibitors of the cyclooxygenase-2 (COX-2) enzyme, such as meloxicam and celecoxib, treat inflammation more-effectively than traditional NSAIDs, they associate with liver-toxicity and cardiovascular-disease risks. Modern pharmacological studies have shown that Panax ginseng, Panax notoginseng, and other traditional Chinese medicines have anti-inflammatory properties; these medicines mainly contain flavonoids, alkaloids, saponins, polysaccharides, and other anti-inflammatory components, are highly efficient, and exhibit few side effects, which is advantageous. Therefore, screening potential COX-2 inhibitors from traditional Chinese medicine is greatly significant for the development of safe and effective novel anti-inflammatory drugs. However, screening these traditional medicines in an easy, quick, accurate, and efficient manner presents problem that require urgent solutions because traditional screening methods are cumbersome and inefficient. In this study, we used the ligand fishing method instead of poorly efficient traditional screening methods, with magnetic nanoparticles used as the carrier owing to their advantageous fast separation speeds. In addition, we also maintained the spatial structure and activity of the fixed enzyme by combining immobilized metal affinity technology, and selected polydopamine (PDA) as a low-toxicity metal-chelating agent owing to its strongly adhesive nature and good chelating ability for various metal ions. The application of PDA solves the problems of metal-ion leakage and limited protein load associated with traditional chelating agents. Firstly, magnetic Fe(3)O(4) nanoparticles were hydrothermally synthesized, after which their surfaces were coated with PDA using the magnetic-stirring method, with Ni(2+) then chelated on the PDA surface and COX-2 fixed through metal-ion affinity, to afford a convenient, fast, green, and specific fishing tool. The structure and properties of the magnetic nanomaterial were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis, vibrating sample magnetometry (VSM), and other methods. The magnetic nanomaterials have core-shell structures with particles that are 250-300 nm in size; they also have a large specific surface area, are strongly magnetic, highly stable, and perform excellently. Celecoxib, a selective COX-2 inhibitor, was used as a fishing target during optimization of the fixed-COX-2 and fishing conditions, including the adsorption and elution conditions. The optimal settings were then successfully used to screen potential COX-2 inhibitors in Panax notoginseng leaves. Methodological experiments revealed that the instrument and method are highly precise, accurate, and repeatable, with a limit of detection (LOD) of 0.02 mg/mL and a limit of quantification (LOQ) of 0.07 mg/mL. Only celecoxib was detected in a fishing solution composed of glipizide, indomethacin, and celecoxib, which implies that immobilized COX-2 exhibits good fishing specificity, and that only components that strongly interact with COX-2 are caught. Thirteen components that interact with COX-2 were identified by liquid chromatography-mass spectrometry for a fishing solution of Panax notoginseng leaves, including notoginsenoside Fa and ginsenoside Rb(1), which are expected to be COX-2 inhibitors. Compared with traditional screening methods, the method developed in this study is simple, rapid, and highly specific and enzyme active, leading to the capture and enrichment of the specific target. This study provides guidance for the synthesis and development of new safe and effective anti-inflammatory drugs, and provides a reference for the efficient discovery of such drugs or lead compounds from complex traditional Chinese medicines; it also provides a new concept for the rational utilization of Panax notoginseng leaves.