ABSTRACT
Bioassay-guided fractionation of the ethanol extract of whole herbs of Achillea alpina led to the isolation of isochlorogenic acids A and B as transient receptor potential vanilloid 3 (TRPV3) channel antagonists by using a calcium fluorescent assay. The structures were identified by spectroscopic analysis and the inhibitory activities of isochlorogenic acids A and B were confirmed by whole-cell patch clamp recordings of human embryonic kidney 293 (HEK293) cells expressing human TRPV3. Molecular docking results revealed that these two compounds reside in the same active pocket of human TRPV3 channel protein with lower binding energy than the agonist 2-aminoethoxydiphenyl borate (2-APB). High-speed counter-current chromatography (HSCCC) coupled with a liquid-liquid extraction approach was successfully established for the separation of isochlorogenic acids A and B from the whole herbs of A. alpina. Ethyl acetate and n-hexane-ethyl acetate-water (3:3:4 and 1:5:4, v/v/v) were selected as liquid-liquid extraction solvent systems to remove high- and low-polarity impurities in the mixture. Sixty g of ethanol extract was refined by solvent partition to yield 1.7 g of the enriched fraction, of which 480 mg in turn obtained 52.5 mg of isochlorogenic acid B (purity 98.3%) and 37.6 mg isochlorogenic acid A (purity 96.2%) after HSCCC with n-hexane-ethyl acetate-water containing 1% acetic acid (1:4:8, v/v/v).
Subject(s)
Achillea/metabolism , Chlorogenic Acid/analogs & derivatives , Countercurrent Distribution/methods , Liquid-Liquid Extraction/methods , Plant Extracts/chemistry , TRPV Cation Channels/antagonists & inhibitors , Acetates/chemistry , Boron Compounds/chemistry , Boron Compounds/pharmacology , Catalytic Domain , Chlorogenic Acid/chemistry , Chlorogenic Acid/isolation & purification , Chromatography, High Pressure Liquid/instrumentation , Chromatography, High Pressure Liquid/methods , HEK293 Cells , Hexanes/chemistry , Humans , Molecular Docking Simulation , Solvents/chemistry , Spectrum Analysis , TRPV Cation Channels/agonists , TRPV Cation Channels/chemistry , Water/chemistryABSTRACT
Xanthine oxidase, an enzyme present in significant levels in the intestine and liver, metabolizes hypoxanthine to xanthine and xanthine to uric acid in the purine catabolic pathway. An inhibitory compound acting against xanthine oxidase was isolated from sweet white clover (Melilotus albus) by bioassay and high-performance liquid chromatography guided separation. It was identified as tricin by spectroscopic analysis. Tricin possessed a potent xanthine oxidase inhibitory activity with an IC50 value of 4.13 µM. Further inhibition kinetics data indicated it to be a mixed-type inhibitor and Ki and KI values were determined to be 0.47 µM and 4.41 µM. To find a rich source of tricin, the distribution of tricin in seven different tissues from four Gramineae species was investigated by high-performance liquid chromatography analysis. The highest amount (1925.05 mg/kg dry materials) was found in the straw of wheat, which is considered as a potentially valuable source of natural tricin.
Subject(s)
Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Melilotus/chemistry , Plant Extracts/pharmacology , Xanthine Oxidase/antagonists & inhibitors , Chromatography, High Pressure Liquid , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Flavonoids/chemistry , Flavonoids/isolation & purification , Models, Molecular , Molecular Conformation , Plant Extracts/chemistry , Protein Binding , Solubility , Structure-Activity RelationshipABSTRACT
WRKY transcription factors are novel transcriptional regulatory factors, which play an important role in regulating plant development, metabolism and other physiological processes. In this study, a new Dendrobium officinale WRKY transcription factor, designated as DoWRKY1 was cloned by using RT-PCR and RACE (GenBank Accession No. KF953910). Bioinformatic analysis demonstrated that, the full-length cDNA of DoWRKY1 was 1,704 bp. And DoWRKY1 contained a 1,629 bp open reading frame (ORF) that encoding a peptide of 542 amino acid residues. The putative DoWRKY1 protein contained two conserved WRKY domains and it belonged to the group I WRKY family protein. Yeast one-hybrid experiment showed that DoWRKY1 had transcriptional activation ability in yeast, and it could activate the expression of downstream report genes (His3 and Ade2). Semi-quantitative RT-PCR experiment showed that DoWRKY1 expressed in roots, stems, leaves and protocorm-like bodies. Real-time qRT-PCR proved that DoWRKY1 could be induced by methyl jasmonate (MeJA) and chitosan (Chitosan), and the expression level of this gene can reach the expression peak at 2 h and 1 h, respectively. These results are useful for further determination of the regulation function of this gene in secondary metabolism of D. officinale.