Antioxidant SiO2-HALP nanomaterials utilizing HumicAcid Like Polycondensates
E. BLETSA (a), P. STATHI (b), M. LOULOUDI (b), Y. DELIGIANNAKIS (a)*
(a) Department of Physics, University of Ioannina, 45110 Ioannina, Greece
(b) Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
* E-mail:
Keywords: nanoantioxidant, DPPH radical, HALP, covalent grafting
Abstract. Nanoantioxidant materials have been engineered by covalently grafting a Humic-Acid-Like Polycondensate (HALP) on SiO2 nanoparticles (NPs). The radical-scavenging capacity (RSC) of the SiO2-HALP NPs was quantified based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical method. RSC of the SiO2-HALP NPs was quantified in comparison with HALP in solution using electron paramagnetic resonance (EPR) and UV−vis spectroscopy. SiO2-HALP NPs are shown to perform success fully H-Atom Transfer (HAT) from the phenolic OH-groups of HALP.
Introduction
A well characterized synthetic Humic Acid [Humic-Acid-like polycondensate (HALP)] synthesized by oxidative polymerization of natural polyphenolic substances, consist of several major functional groups, predominantly phenolic (OH) and carboxyl (COOH) [1]. Polyphenol are widely used as antioxidants in nutrients, medicine and polymers [2].The antioxidant activity of polyphenols, and their derivatives, is determined by the number and positions of ring-OH groups [3]. Antioxidants, preventing harmful free-radical reactions in biological systems and materials attract intense scientific and economic interest in human health, food and polymer industries [2].
Silica nanoparticles [NPs] in the commercial form of fumed silica are nowadays among the largest industrial nanotechnology products, and find many applications (pharmaceuticals and among others). Immobilizing antioxidants on nanosized SiO2 particles [nanoantioxidants] offers a unique opportunity to exploit the potential of natural antioxidants [2]. Here, three different types of commercially available well characterized SiO2 with different particle sizes (40, 20 and 7nm) are used and functionalized with HALP on their surface. The successful attachment of HALP is verified TGA, EPR and FTIR spectroscopy. The radical-scavenging capacity (RSC) of the so synthesized SiO2-HALP NPs was quantified in comparison with pure HALP based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical method, using UV−vis spectroscopy [5].
Materials and methods
Synthesis of HALP. Synthesis HALP has been performed by oxidative copolymerization of gallic acid (GA) (3,4,5-trihydroxybenzoic acid) and protocatechuic acid (PA) (3,4-dihydroxybenzoic acid) with no use of a catalytic material, according to Giannakopoulos et al [1].
Preparation of SiO2-HALP NPs. The preparation protocol for the SiO2-HALP NPs consisted of two main steps. First, we prepare aminopropyl- SiO2 (APTES- SiO2) by reacting the SiO2 NPs with APTES. Covalent immobilization of HALP on the formed aminopropyl- SiO2 has been achieved by formation of amide bonds between the amine groups of aminopropyl- SiO2 and the carboxyl group of HALP activated by EDC coupler [2].
Results and Discussion
EPR Characterization
EPR spectroscopy was used: (i) To determine the maximum concentration of radical-forming phenolic moieties; (ii) To monitor the radical reactions, for example, between DPPH and SiO2-HALP NPs. Maximum radical concentration was achieved by oxidation of HALP by O2 in aqueous solution at alkaline pH [1].
Figure 1. EPR spectra of maximum concentration of radicals of various SiO2-HALP NPs. lower Specific Surface Area NPs (per same mass of material) give higher concentration of HALP -phenolic radicals.
As shown in Fig.1 SiO2-HALP NPs are able to form high concentrations of phenolic-type radicals (g-value=2.0040), and lineshape proves that these radicals are localized on the phenolic-ring oxygens [1]. This is of immediate relevance for the antioxidant activity of the phenolic oxygens which are the reactive sites on HALP that perform fast H-Atom Transfer to DPPH radical. Spin quantitation data of the phenolic radicals on SiO2-HALP NPs in comparison to reference HALP, normalized per same mass of HALP [according to the loading of HALP on NPs surface], are listed in Table 1.
Table 1. Spin quantitation data of the phenolic radicals
Material / μmol phenolic radicals per g of HALPHALP reference / 4.1
SiO2[50]-HALP / 2.2
SiO2[90]-HALP / 2.4
SiO2[300]-HALP / 2.3
The data show that the SiO2-HALP NPs retain almost 50% of phenolic radicals in comparison to the reference HALP.
DPPH-Radical Scavenging Capacity
Figure 2 displays kinetic traces recorded for different masses of SiO2[90]-HALP NPs.
Figure 2. Kinetics of decay of absorbance at 515nm for DPPH radicals [(DPPH) 0=30±0.1 μM] reacting with SiO2[90]-HALP NPs.
In Figure 2, we observe the two different types of kinetic reactions for SiO2-HALP NPs: (i) fast kinetic phase for reaction times below 2 min, and (ii) slow decay phases persisting at prolonged times. The initial fast decay of the DPPH radical (DPPH•) interacting with a phenolic antioxidant is due to rapid reactions 1 and 2:
ArOH+DPPH•→ArO•+DPPH−H (1)
ArO•+DPPH•→−ArO-quinone (2)
These are H-Atom Transfer reactions [HAT] from the phenolic OH groups to the DPPH-radical. The slow-decay phases resolved for [DPPH:SiO2-HALP] may involve radical−radical dimerization/polymerization reactions [2].
Figure 3. DPPH radicals scavenged by SiO2-HALP NPs for each type of NPs.
The total DPPH radicals scavenged by the SiO2-HALP NPs normalized per same SSA are presented in Fig. 3.
These data prove that: [i]SiO2-HALP NPs have significant antioxidant capacity, via the Ηydrogen Αtom Transfer mechanism; [ii] bigger NPs [SiO2[50]-HALP NPs] have better antioxidant capacity in comparison to smaller SiO2[300]-HALP NPs.
Figure 4 shows EPR spectra for SiO2-HALP NPs interacting with the DPPH radical.
Figure 4. EPR spectra of DPPH radical interacting with each SiO2-HALP NPs.
The EPR spectrum [black line] in Figure is typical for DPPH-radicals in methanol. The interaction of DPPH radical with the NPs resulted in decay at the EPR signal and provides solid evidence that the spectral changes observed in DPPH radical scavenging by the NPs.
References
1. Giannakopoulos E., M. Drosos, Y. Deligiannakis. Journal of Colloid and Interface Science 336 (2009) 59–66.
2. Deligiannakis, Y. G., A. Sotiriou, and S. E. Pratsinis. ACS Applied Materials and Interfaces4 (2012) 6609-6617.
3. Stathi P., M. Louloudi, Y. Deligiannakis. Chemical Physics Letters 472 (2009) 85–89.
4. Stathi P. and Y. Deligiannakis. Journal of Colloid and Interface Science 351 (2010) 239–247.
5. Brand-Williams, W.; Cuvelier, M. E.; Berset, C. LWT-Food Sci. Technol. 28(1995) 25−30.
Acknowledgment. This research has been co-financed by the European Union (European Social Fund–ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF)-Research Funding Program: THALIS. Investing in knowledge society through the European Social Fund.