Bioactivity screening, extraction, and separation of lactate dehydrogenase inhibitors from Polygala tenuifolia Willd. based on a hyphenated strategy.

Stroke is the second leading cause of death worldwide. Lactate dehydrogenase inhibitors are currently widely used in the treatment of ischemic stroke, and natural products are considered promising sources of lactate dehydrogenase inhibitors. In this study, ultrafiltration liquid chromatography coupled with mass spectrometry was used for the screening and identification of lactate dehydrogenase inhibitors from Polygala tenuifolia. Furthermore, five lactate dehydrogenase inhibitors, sibiricose A5, 3,6'-di-O-sinapoyl-sucrose, glomeratose A, tenuifoliside B, and tenuifoliside C, were selected as target lactate dehydrogenase inhibitors. In addition, the five target compounds with purities of 96.45, 97.65, 96.38, 94.34, and 93.29% were extracted and isolated using a new hyphenated strategy of microwave-assisted extraction coupled with countercurrent chromatography with a two-phase solvent system of n-hexane/n-butanol/ethanol/water (5.321:1.00:1.664:6.647). The bioactivities of the isolated compounds were analyzed using PC12 cells and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results also demonstrated that microwave-assisted extraction coupled with countercurrent chromatography is an efficient method of isolating chemical constituents from medicinal herbs. Moreover, the research route consisting of activity screening, extraction, separation, and activity verification of the compounds has the advantages of being efficient, orientated, and objective.

[1]  T. Abbruscato,et al.  In vitro and in vivo efficacy of a potent opioid receptor agonist, biphalin, compared to subtype-selective opioid receptor agonists for stroke treatment , 2015, Brain Research.

[2]  Bin Wu,et al.  A strategy for screening of high-quality enzyme inhibitors from herbal medicines based on ultrafiltration LC-MS and in silico molecular docking. , 2015, Chemical communications.

[3]  Jae-yong Cho,et al.  Protective effect of quercetin, a natural flavonoid against neuronal damage after transient global cerebral ischemia , 2006, Neuroscience Letters.

[4]  P. Haddad,et al.  Simple, quantitative method for low molecular weight dissolved organic matter extracted from natural waters based upon high performance counter-current chromatography. , 2016, Analytica chimica acta.

[5]  C. Teng,et al.  Synthesis and Antithrombotic Effect of Xanthone Derivatives , 1996, The Journal of pharmacy and pharmacology.

[6]  Sung-jin Kim,et al.  Inhibitory Effect of the root of Polygala tenuifolia on Bradykinin and COX 2-Mediated Pain and Inflammatory Activity , 2013 .

[7]  H. Kong,et al.  Neuroprotective effects of berberine on stroke models in vitro and in vivo , 2008, Neuroscience Letters.

[8]  M. Guardia,et al.  Microwave-assisted extraction of pyrethroid insecticides from soil , 2004 .

[9]  P. Tu,et al.  Xanthone O-glycosides from Polygala tenuifolia. , 2002, Phytochemistry.

[10]  Xinmin Liu,et al.  An LC-MS/MS method for simultaneous determination of three Polygala saponin hydrolysates in rat plasma and its application to a pharmacokinetic study. , 2015, Journal of ethnopharmacology.

[11]  Gaoxue Wang,et al.  In vitro immunocompetence of two compounds isolated from Polygala tenuifolia and development of resistance against grass carp reovirus (GCRV) and Dactylogyrus intermedius in respective host. , 2014, Fish & shellfish immunology.

[12]  L. Kaczmarek,et al.  MMP-9 Inhibition: a Therapeutic Strategy in Ischemic Stroke , 2013, Molecular Neurobiology.

[13]  P. C. Nagajyothi,et al.  Antioxidant and anti-inflammatory activities of zinc oxide nanoparticles synthesized using Polygala tenuifolia root extract. , 2015, Journal of photochemistry and photobiology. B, Biology.

[14]  Shu Liu,et al.  Ultrafiltration LC-PDA-ESI/MS combined with reverse phase-medium pressure liquid chromatography for screening and isolation potential α-glucosidase inhibitors from Scutellaria baicalensis Georgi , 2014 .

[15]  M. Iinuma,et al.  Antibacterial Activity of Xanthones from Guttiferaeous Plants against Methicillin‐resistant Staphylococcus aureus , 1996, The Journal of pharmacy and pharmacology.

[16]  L. Arab,et al.  Tea, flavonoids and stroke in man and mouse. , 2010, Archives of biochemistry and biophysics.

[17]  Yao Qin,et al.  Screening and isolation of potential lactate dehydrogenase inhibitors from five Chinese medicinal herbs: Soybean, Radix pueraria, Flos pueraria, Rhizoma belamcandae, and Radix astragali. , 2016, Journal of separation science.

[18]  J. Frank,et al.  Beneficial Effects of Ethanolic and Hexanic Rice Bran Extract on Mitochondrial Function in PC12 Cells and the Search for Bioactive Components , 2015, Molecules.

[19]  Xiaoqing Chen,et al.  Separation of α-amylase inhibitors from Abelmoschus esculentus (L).Moench by on-line two-dimensional high-speed counter-current chromatography target-guided by ultrafiltration-HPLC. , 2015, Journal of separation science.

[20]  Shu Liu,et al.  Screening and determination for potential α-glucosidase inhibitors from leaves of Acanthopanax senticosus harms by using UF-LC/MS and ESI-MS(n). , 2012, Phytochemical analysis : PCA.

[21]  Ming-cang Chen,et al.  Analysis and detection of the chemical constituents of Radix Polygalae and their metabolites in rats after oral administration by ultra high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. , 2013, Journal of pharmaceutical and biomedical analysis.

[22]  M. Pompili,et al.  Suicide in Stroke Survivors: Epidemiology and Prevention , 2014, Drugs & Aging.

[23]  Liangliang Liu,et al.  Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high-performance liquid chromatography and high-speed countercurrent chromatography. , 2014, Journal of separation science.

[24]  Y. Ito.  pH-zone-refining counter-current chromatography: origin, mechanism, procedure and applications. , 2013, Journal of chromatography. A.

[25]  Stella Brudin,et al.  Analytical methodology for sulfonated lignins. , 2010, Journal of separation science.

[26]  Mineyuki Mizuguchi,et al.  Structural Insight into the Interactions between Death-Associated Protein Kinase 1 and Natural Flavonoids. , 2015, Journal of medicinal chemistry.

[27]  W. Schulz-Schaeffer,et al.  Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions , 2015, Experimental Neurology.

[28]  Yuchi Zhang,et al.  Development of a method to screen and isolate potential α-glucosidase inhibitors from Panax japonicus C.A. Meyer by ultrafiltration, liquid chromatography, and counter-current chromatography. , 2015, Journal of separation science.

[29]  K. Robards.  Strategies for the determination of bioactive phenols in plants, fruit and vegetables. , 2003, Journal of chromatography. A.

[30]  S. Lim,et al.  Identification of tyrosinase specific inhibitors from Xanthium strumarium fruit extract using ultrafiltration-high performance liquid chromatography. , 2015, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[31]  M. Zikic,et al.  Current status of stroke epidemiology in Greece: a panorama. , 2014, Neurologia i neurochirurgia polska.

[32]  M. Mancuso,et al.  Genetics of ischaemic stroke in young adults , 2014, BBA clinical.

[33]  Yoichiro Ito,et al.  Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. , 2005, Journal of chromatography. A.

[34]  V. Orsat,et al.  Microwave-Assisted Extraction of Flavonoids: A Review , 2012, Food and Bioprocess Technology.