IMR Press / FBL / Volume 27 / Issue 8 / DOI: 10.31083/j.fbl2708229
Open Access Original Research
Lactate Dehydrogenase-Inhibitors Isolated from Ethyl Acetate Extract of Selaginella doederleinii by Using a Rapid Screening Method with Enzyme-Immobilized Magnetic Nanoparticles
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1 School of Pharmacy, Zunyi Medical University, 563000 Zunyi, Guizhou, China
2 Department of Pharmacy, Zunyi Medical and Pharmaceutical College, 563000 Zunyi, Guizhou, China
3 Department of Pharmacy, The Third Affiliated Hospital of Zunyi Medical University, 563000 Zunyi, Guizhou, China
*Correspondence: leshenge@163.com (Shenge Li); wg8855350@163.com (Gang Wang)
These authors contributed equally.
Academic Editor: Luigi De Masi
Front. Biosci. (Landmark Ed) 2022, 27(8), 229; https://doi.org/10.31083/j.fbl2708229
Submitted: 20 May 2022 | Revised: 17 June 2022 | Accepted: 13 July 2022 | Published: 26 July 2022
(This article belongs to the Special Issue Plant Bioactive Molecules)
Copyright: © 2022 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.
Abstract

Background: Lactate dehydrogenase (LDH) is one of the important enzyme systems for glycolysis and gluconeogenesis. It can catalyze the reduction and oxidation reaction between propionic acid and L-lactic acid, which is usually overexpressed in cancer cells. Therefore, inhibiting the activity of LDH is a promising way for the treatment of cancer. In this study, an effective method based on ligand fishing and ultra performance liquid chromatography-mass spectrum (UPLC-MS) was established to screen and identify active ingredients from Selaginella doederleinii with potential inhibitory activity for LDH. Methods: Firstly, LDH was immobilized on the magnetic nanoparticles (MNPs), three immobilization parameters including LDH concentration, immobilization time and pH were optimized by single factor and response surface methodology for maximum (max) immobilization yield. Then, a mixed model of galloflavin and chlorogenic acid (inhibitors and non-inhibitors of LDH) was used to verify the specificity of immobilized LDH ligand fishing, and the conditions of ligand fishing were further optimized. Finally, combined with UPLC-MS, immobilized LDH was used to simultaneously screen and identify potential LDH inhibitors from the ethyl acetate extract of Selaginella doederleinii. Results: The prepared fishing material was comprehensively characterized by scanning electron microscopy (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD) and fourier transform infrared spectrometer (FT-IR). The optimal immobilization conditions were obtained as LDH concentration of 0.7 mg/mL, pH value of 4.5, and immobilization time of 3.5 h. Under these conditions, the max immobilization yield was (3.79 ± 0.08) × 103 U/g. The specificity analysis showed that immobilized LDH could recognize and capture ligands, and the optimal ligand fishing conditions included that the incubation time was 30 min, the elution time was 20 min, and the concentration of methanol as eluent was 80%. Finally, two LDH inhibitors, amentoflavone and robustaflavone, were screened by immobilized LDH from the ethyl acetate extract of Selaginella doederleinii. Conclusions: The study provided a meaningful evidence for discovering the bioactive constituents in ethyl acetate extract of Selaginella doederleinii related to cancer treatment, and this ligand fishing method was feasible for screening enzyme inhibitors from similar complex mixtures.

Keywords
lactate dehydrogenase (LDH) inhibitor
functionalized magnetic nanoparticles
Selaginella doederleini
biflavonoids
ligand fishing
Figures
Fig. 1.
Funding
81860697/2018 National Science Foundation of China
No.10(E-361)/Zunyi Talent Team Project (2020)
[2020]4Y086/Guizhou Province Science and Technology Plan Project
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