Optimization of Stable IgG Formulation Containing Amino Acids and Trehalose During Freeze-Drying and After Storage: a Central Composite Design.

The physical and structural stability of freeze-dried immunoglobulin G (IgG) were examined by applying trehalose and amino acids (glycine, phenylalanine, and serine). The efficacy of amino acids was statistically compared considering their side-chain characteristics. The amount of amino acids (X1) and trehalose (X2) was considered as independent variables. Size exclusion chromatography (SEC-HPLC) was utilized to calculate the soluble aggregates, as dependent variables. The amounts of excipients were optimized through the central composite design (CCD). The beta-sheet conformation of IgG was quantified by Fourier transform infrared spectroscopy (FTIR). Thermal behavior and molecular integrity of IgG were evaluated by differential scanning calorimetry (DSC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimized formulations of powders were as follows: 24.5 mg serine-139.5 mg trehalose, 14 mg glycine-118 mg Trehalose, and 25 mg phenylalanine-139.5 mg trehalose. The amounts of soluble aggregates after processing were 0, 4.50, and 2.20%, respectively. The corresponding induced aggregates following storage conditions were 1.02, 7.0, and 3.70%. In all preparations, there were no detectable fragments. The native conformation of IgG was well preserved in the presence of amino acids. Excluding the glycine-based sample with minor endotherm at about 45°C, serine and phenylalanine incorporating powders were fully amorphous at examination temperatures. Trehalose was more potent than the amino acids in the stabilization of IgG. Serine was the most effective amino acid; phenylalanine and glycine were the next ones, respectively. Glycine crystallization was assumed to have accounted for low stabilization capability. The statistically synergistic phenomenon was only observed in the co-application of trehalose and phenylalanine. Graphical abstract.

Potential antiviral effects of some native Iranian medicinal plants extracts and fractions against influenza A virus.

BACKGROUND: Influenza A virus (IAV) infection is a continual threat to the health of animals and humans globally. Consumption of the conventional drugs has shown several side effects and drug resistance. This study was aimed to screen some Iranian medicinal plants extracts and their fractions against influenza A virus. METHODS: Glycyrrhiza glabra (rhizome), Myrtus commonis (leaves), Melissa officinalis (leaves), Hypericum perforatum (aerial parts), Tilia platyphyllos (flower), Salix alba (bark), and Camellia sinensis (green and fermented leaves) were extracted with 80% methanol and fractionated with chloroform and methanol, respectively. The cytotoxicity of the compounds were determined by MTT colorimetric assay on MDCK cells. The effective concentrations (EC50) of the compounds were calculated from the MTT results compared to the negative control with no significant effects on cell viability. The effects of EC50 of the compounds on viral surface glycoproteins and viral titer were tested by HI and HA virological assays, respectively and compared with oseltamivir and amantadine. Preliminary phytochemical analysis were done for promising anti-IAV extracts and fractions. RESULTS: The most effective samples against IAV titer (P ≤ 0.05) were crude extracts of G. glabra, M. officinalis and S. alba; methanol fractions of M. communis and M. officinalis; and chloroform fractions of M. communis and C. sinensis (fermented) mostly in co- and pre-penetration combined treatments. The potential extracts and fractions were rich in flavonoids, tannins, steroids and triterpenoids. CONCLUSION: The outcomes confirmed a scientific basis for anti-influenza A virus capacity of the extracts and fractions from the selected plants for the first time, and correlated their effects with their phytochemical constituents. It is worth focusing on elucidating pure compounds and identifying their mechanism(s) of action.