The application of pharmaceutical quality by design concepts to evaluate the antioxidant and antimicrobial properties of a preservative system including desferrioxamine

Abstract

Background: The purpose of the present study was to investigate the antioxidant and antimicrobial activities of a conventional preservative system containing desferrioxamine mesylate (DFO) and optimize the composition of the system through mathematical models.

Methods: Different combinations of ethylenediaminetetraacetic acid (EDTA), sodium metabisulfite (SM), DFO and methylparaben (MP) were prepared using factorial design of experiments. The systems were added to ascorbic acid (AA) solution and the AA content over time, at room temperature and at 40 °C was determined by volumetric assay. The systems were also evaluated for antioxidant activity by a fluorescence-based assay. Antimicrobial activity was assessed by microdilution technique and photometric detection against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus brasiliensis. A multi-criteria decision approach was adopted to optimize all responses by desirability functions.

Results: DFO did not extend the stability of AA over time, but displayed a better ability than EDTA to block the pro-oxidant activity of iron. DFO had a positive interaction with MP in microbial growth inhibition. The mathematical models showed adequate capacity to predict the responses. Statistical optimization aiming to meet the quality specifications of the ascorbic acid solution indicated that the presence of DFO in the composition allows to decrease the concentrations of EDTA, SM and MP.

Conclusion: DFO was much more effective than EDTA in preventing iron-catalyzed oxidation. In addition, DFO improved the inhibitory response of most microorganisms tested. The Quality by Design concepts aided in predicting an optimized preservative system with reduced levels of conventional antioxidants and preservatives, suggesting DFO as a candidate for multifunctional excipient.

Keywords: Antioxidant; Desferrioxamine; Design space; Experimental design; Preservative.

Fig. 1

Response surface 3D plots showing the effect of the percentage (w/v) of ethylenediaminetetraacetic acid (X1), sodium metabisulfite (X2) and desferrioxamine mesylate (X3) on the ascorbic acid concentration (mg/mL) over time at room temperature (Y1) and at 40 °C (Y2). When not showed in the plot, the level of the factors X1 and X3 was fixed at zero

Fig. 2

Response surface 3D plots showing the effect of the percentage (w/v) of ethylenediaminetetraacetic acid (X1), sodium metabisulfite (X2) and desferrioxamine mesylate (X3) on the antioxidant activity expressed as fluorescence units/min (Y3). When not showed in the plot, the level of the factor X1 was fixed at zero, X2 at 5.3 μM (0.1% w/v) and X3 at 2.7 μM (0.1% w/v)

Fig. 3

Response surface 3D plots showing the effect of the percentage (w/v) of sodium metabisulfite (X2), desferrioxamine (X3) and methylparaben (X4) on antimicrobial activity against Staphylococcus aureus (Y4) expressed as percentage of microbial growth inhibition. When not showed in the plot, the level of the factors X1, X2, X3 and X4 was fixed at 0.1% (w/v)

Fig. 4

Response surface 3D plots showing the effect of the percentage (w/v) of ethylenediaminetetraacetic acid (X1), sodium metabisulfite (X2), desferrioxamine (X3) and methylparaben (X4) on antimicrobial activity against Escherichia coli (Y5) expressed as percentage of microbial growth inhibition. When not showed in the plot, the level of the factors X1, X2, X3 and X4 was fixed at 0.1% (w/v)

Fig. 5

Response surface 3D plots showing the effect of the percentage (w/v) of ethylenediaminetetraacetic acid (X1), sodium metabisulfite (X2), desferrioxamine (X3) and methylparaben (X4) on antimicrobial activity against Pseudomonas aeruginosa (Y6) expressed as percentage of microbial growth inhibition. When not showed in the plot, the level of the factors X1, X2, X3 and X4 was fixed at 0.1% (w/v)

Fig. 6

Response surface 3D plots showing the effect of the percentage (w/v) of ethylenediaminetetraacetic acid (X1), sodium metabisulfite (X2), desferrioxamine (X3) and methylparaben (X4) on antimicrobial activity against Candida albicans (Y7) expressed as percentage of microbial growth inhibition. When not showed in the plot, the level of the factors X1, X2, X3 and X4 was fixed at 0.1% (w/v)

Fig. 7

Response surface 3D plot showing the effect of the percentage (w/v) of desferrioxamine (X3) and methylparaben (X4) on antimicrobial activity against Aspergillus brasiliensis (Y8) expressed as percentage of microbial growth inhibition. When not showed in the plot, the level of the factors X1 and X2 was fixed at 0.1% (w/v)

Fig. 8

Overlapped contour plots and Design Space regions for the determination of ascorbic acid concentration (mg/mL) over time at room temperature (Y1) and at 40 °C (Y2), for the antioxidant activity expressed as fluorescence units/min (Y3), and for the antimicrobial activity expressed as percentage of microbial growth inhibition against Staphylococcus aureus (Y4), Escherichia coli (Y5), Pseudomonas aeruginosa (Y6), Candida albicans (Y7) and Aspergillus brasiliensis (Y8), as a function of the percentage (w/v) of ethylenediaminetetraacetic acid (X1), desferrioxamine mesylate (X3) and methylparaben (X4). When not showed in the plot, the level of the factor X1 was fixed at 0.025% (w/v), X2 at zero, X3 at 0.1% (w/v) and X4 at 0.025% (w/v)