Screening of natural inhibitors against peptidyl arginine deiminase 4 from herbal extracts by a high-performance liquid chromatography ultraviolet-visible based method.

Peptidyl arginine deiminase 4 (PAD4) is an important biocatalytic enzymes involved in the conversion of protein arginine to citrulline, its dysregulation has a great impact on many physiological processes. Recently, PAD4 has emerged as a potential therapeutic target for the treatment of various diseases including rheumatoid arthritis (RA). Traditional Chinese Medicines (TCMs), also known as herbal plants, have gained great attention by the scientific community due to their good therapeutic performance and far fewer side effects observed in the clinical treatment. However, limited researches have been reported to screen natural PAD4 inhibitors from herbal plants. The color developing reagent (COLDER) or fluorescence based methods have been widely used in PAD4 activity assay and inhibitor screening. However, both methods measure the overall absorbance or fluorescence in the reaction solution, which are easy to be affected by the background interference due to colorful extracts from herbal plants. In this study, a simple, and robust high-performance liquid chromatography ultraviolet-visible (HPLC-UV) based method was developed to determine PAD4 activity. The proposed strategy was established based on COLDER principle, while used hydrophilic l-arginine instead of hydrophobic N-benzoyl-l-arginine ethyl ester (BAEE) as a new substrate to determine PAD4 inhibition activity of herbal extracts. The herbal extracts and PAD4 generated hydrophobic l-citrulline were successfully separated by the HPLC, and the developed method was optimized and validated with a known PAD4 inhibitor (GSK484) in comparison with COLDER assay. The IC50 value of GSK484 measured by HPLC-UV method was 153 nM, and the detection limit of the citrulline was 0.5 nmol, respectively, with a linear range of 0.5 nmol to 20 nmol. The IC50 value of the HPLC-UV method was improved by nearly three times compared with COLDER assay (527 nM), and the results indicated the reliability of PAD4 inhibition via HPLC-UV method. The inhibitory effect against PAD4 were fast and accurately screened for the twenty-four extracts from eight herbs. Among them, Ephedra Herba extracts showed significant inhibitory activity against the PAD4 with the IC50 values of three extracts (ethanol, ethyl acetate and water) ranging from 29.11 µg/mL to 41.36 µg/mL, which may help researchers to discover novel natural compounds holding high PAD4 inhibition activity.

Effect of preparation conditions on protein secondary structure and biofilm formation of kafirin.

Various extraction and drying conditions for the isolation of kafirin from dry-milled, whole grain sorghum have been investigated, with a view to optimizing extraction of the protein for commercial food coatings and packaging films. The addition of sodium hydroxide to an aqueous ethanol extractant increased the yield and solubility of kafirin. Subsequent heat drying at 40 degrees C was shown to cause the kafirin to aggregate as indicated by an increase in intermolecular beta-sheets. Extraction of the flour using ethanol (70%, w/w) with 0.5% (w/w) sodium metabisulfite and 0.35% (w/w) sodium hydroxide at 70 degrees C followed by freeze-drying of the protein was found to produce a yield of 54% kafirin with good film-forming properties. The kafirin films were assessed for their sensory properties, tensile strength, strain, and water vapor permeability. Fourier transform infrared spectroscopy was used to study the secondary structure of the extracted kafirins. The best films were made with kafirin containing a large proportion of nativelike alpha-helical structures with little intermolecular beta-sheet content as indicated by the Fourier transform infrared reflectance peak intensity ratios associated with these secondary structures. The principal factor affecting the secondary structure of the protein appeared to be the temperature at which the protein was dried. Heat drying resulted in a greater proportion of intermolecular beta-sheets. Any industrial-scale extraction must therefore minimize protein aggregation and maximize native alpha-helical structures to achieve optimal film quality.