Separation Of Bioactive Molecules Research Articles

In silico and Biochemical Approach for Isolated, Purified, and Characterized Protease Inhibitor from Limonia acidissima

Proteases produced by various bacterial and fungal pathogens are associated with pathogenicity and cause septic hazards. Thereby inhibition of such proteases by protease inhibitors from natural sources is effective strategy to combat infectious diseases. Protease inhibitors originating from plant source are abundant in nature and play an important role in defense mechanism against virulent microbes. Many protease inhibitors can be isolated and purified from plant sources and formulated as therapeutic drugs to combat human diseases. In spite of their applications in biomedicines, it is necessary to explore the novel biochemical characteristics of protease inhibitors with improved efficacy. The objective of the current study was extraction, purification and biochemical characterization of protease inhibitor from L. acidissima. The protease inhibitor was isolated from L. acidissima by buffer extraction method and confirmed for anti-protease activity using standard trypsin assay. The inhibitor was found to be active between pH ranging from 5-9 with maximal activity at pH 7. The inhibitor showed thermal stability at a temperature of 50-60°C and retained 70% activity at 50°C for 2 hr. Inhibitor activity was enhanced by thermal stabilizers glycine, urea, Calcium chloride (CaCl2), glycerol, and sucrose at 50°C; metal ions Cu2+ and Al3+ and detergents like Sodium dodecyl sulfate (SDS), Triton-X 100, Tween-80. Protease inhibitor was also stable in the presence of oxidizing and reducing agents Dimethyl sulfoxide (DMSO), Hydrogen peroxide (H2O2), β-mercaptoethanol, and Sodium Thioglycolate. Thus, optimization and characterization studies indicate that the isolated inhibitor is active under natural conditions. Protease inhibitor showed positive results for anticancer, anti-diabetic and anti-inflammatory activities indicating the therapeutic potential of inhibitor molecule. Further identification and separation of bioactive molecules from the buffer extract of L. acidissima was carried out by Flash chromatography followed by Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis that revealed the presence of Diisobutylphthalate as bioactive phytoconstituent. Docking studies performed revealed the anti-diabetic, anti-inflammatory, and anti-cancer potential of Diisobutylphthalate. Thus, in silico studies support the activity of our isolated protease inhibitor to ensure a promising future in the development of therapeutics.

Magnetic Technologies and Green Solvents in Extraction and Separation of Bioactive Molecules Together with Biochemical Objects: Current Opportunities and Challenges

Currently, magnetic technology and green solvents are widely used in chemical engineering, environmental engineering and other fields as they are environmentally friendly, easy to operate and highly efficient. Moreover, a magnetic field has positive effect on many physicochemical processes. However, related new methods, materials, strategies and applications in separation science still need to be developed. In this review, a series of meaningful explorations of magnetic technologies for the separation of natural products and biologic objects, including magnetic ionic liquids and other magnetic solvents and fluids, magnetic nanoparticles and magnetic fields, and the development of magnetic separators were reviewed. Furthermore, the difficulties in the application and development of magnetic separation technology were discussed on the basis of comparison and data analysis, especially for the selection of magnetic materials and magnetic field sources. Finally, the progress in the development of magnetic separators was also elaborated for researchers, mainly including that of the new high-efficiency magnetic separator through multi-technology integration and the optimization of traditional magnetic separators, which help current techniques break through their bottleneck as a powerful driving force.

Solvation Free Energy for Selection of an Aqueous Two-Phase System: Case in Paeonol Extraction from Cortex Moutan.

Aqueous two-phase system(s) (ATPS) has/have been widely employed in the extraction and separation of bioactive molecules from herbs due to its various advantages such as high efficiency and good selectivity. For selecting the type and amount of organics and salts in ATPS, onerous experimental trials are required to ensure the reliability. We intended to develop a theoretical method to select ATPS in the case of paeonol extraction from cortex moutan. The solvation free energies (Esolv) of paeonol in the top phase of 54 ATPS (ATPS-acetone, ATPS-acetone-EA, ATPS-THF, ATPS-THF-EA, ATPS-EtOH, and ATPS-EtOH-EA) were calculated with Gaussian 09, and the extraction yields with 54 ATPS were determined. By comparison of Esolv and yield, the Esolv rank was effective to select the optimal organic type and organic solvent fraction and aqueous salt concentration. With each series of 18 ATPS (ATPS-acetone plus ATPS-acetone-EA; ATPS-THF plus ATPS-THF-EA; or ATPS-EtOH plus ATPS-EtOH-EA), the paeonol yield was correlated with Esolv, suggesting that the optimal organic type and fraction and the aqueous NaH2PO4 concentration could be selected by using theoretical Esolv, or at least, the theoretical Esolv rank could offer effective guidance for experimental design, and thus, tedious and onerous experimental work for optimization in ATPS extraction can be significantly reduced.

Construction by Molecular Dynamics Modeling and Simulations of the Porous Structures Formed by Dextran Polymer Chains Attached on the Surface of the Pores of a Base Matrix: Characterization of Porous Structures

Significant increases in the separation of bioactive molecules by using ion-exchange chromatography are realized by utilizing porous adsorbent particles in which the affinity group/ligand is linked to the base matrix of the porous particle via a polymeric extender. To study and understand the behavior of such systems, the M3B model is modified and used in molecular dynamics (MD) simulation studies to construct porous dextran layers on the surface of a base matrix, where the dextran polymer chains and the surface are covered by water. Two different porous polymer layers having 25 and 40 monomers per main polymer chain of dextran, respectively, are constructed, and their three-dimensional (3D) porous structures are characterized with respect to porosity, pore size distribution, and number of conducting pathways along the direction of net transport. It is found that the more desirable practical implications with respect to structural properties exhibited by the porous polymer layer having 40 monomers per main polymer chain, are mainly due to the higher flexibility of the polymer chains of this system, especially in the upper region of the porous structure. The characterization and analysis of the porous structures have suggested a useful definition for the physical meaning and implications of the pore connectivity of a real porous medium that is significantly different than the artificial physical meaning associated with the pore connectivity parameter employed in pore network models and whose physical limitations are discussed; furthermore, the methodology developed for the characterization of the three-dimensional structures of real porous media could be used to analyze the experimental data obtained from high-resolution noninvasive three-dimensional methods like high-resolution optical microscopy. The MD modeling and simulations methodology presented here could be used, considering that the type and size of affinity group/ligand as well as the size of the biomolecule to be adsorbed onto the affinity group/ligand are known, to construct different porous dextran layers by varying the length of the polymeric chain of dextran, the number of attachment points to the base matrix, the degree of side branching, and the number of main polymeric chains immobilized per unit surface area of base matrix. After the characterization of the porous structures of the different porous dextran layers is performed, then only a few promising structures would be selected for studying the immobilization of adsorption sites on the pore surfaces and the subsequent adsorption of the bioactive molecules onto the immobilized affinity groups/ligands.