SYNTHESIS OF SILVER NANOPARTICLES USING CONDENSED AND HYDROLYSABLE TANNINS. Hana KŘÍŽOVÁ, Jakub WIENER

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1 SYNTHESIS OF SILVER NANOPARTICLES USING CONDENSED AND HYDROLYSABLE TANNINS Hana KŘÍŽOVÁ, Jakub WIENER Technical University of Liberec, Liberec, Czech Republic, EU, Abstract The aim of this work was the synthesis of silver nanoparticles using two types of tannins, hydrolysable and condensed, and comparing the properties of incurred colloids. Both tannins are powerful antioxidants (reductants) which differ in their chemical structure as well as certain properties, which influences the formation of silver particles as well as the composition and properties of the resulting mixture. There was compared the size and stability of reduced silver nanoparticles according to the type of tannin and the concentration of silver and further measured the residual antioxidant capacity of tannins in these solutions. Keywords: Silver nanoparticles, tannins, antioxidant 1. INTRODUCTION Silver nanoparticles are widely used in many fields, such as optics, electronics, chemical catalysis and in biological applications. Biogenic synthesis of silver nanoparticles is based on the reduction of Ag+ ions by enzymes of some microorganisms or by various natural substances and plant extracts. Preparation of silver nanoparticles in aqueous solution using reducing properties of tannins is one of many possibilities of their green synthesis. These preparation methods are very environmentally friendly because they are prepared without using hazardous chemicals. 1.1 Antibacterial properties of silver At a time when it is difficult to develop new antibiotics against increasingly resistant bacterial strains, silver nanoparticles come more and more to the forefront of medicine, mainly because of their high antibacterial properties. Bacteria are very sensitive to the action of silver ion at very low concentrations (35 ppb) [1], because they are very reactive and react with proteins and enzymes in the cell wall as well as cytoplasm, they also react with nitrogen bases of nucleic acids and thereby destroy particularly simple microorganisms structurally and functionally. But even here there is some risk of possible bacterial resistance, as many bacteria possess detoxifying mechanisms having evolved against heavy metal ions. The metal form of silver has unlike the ionic form relatively low cytotoxicity and high biocompatibility, which is very favorable from the viewpoint of potential environmental toxicity of silver nanoparticles. The mechanism of antibacterial activity of nanosilver metal is not entirely clear, but it is certain that the antibacterial activity of silver particles increases with a decrease in their size and it has been demonstrated that an important role has also the particle morphology. Most effective are silver nanoparticles of size about 25 nm; their antibacterial activity is very close to the antibacterial activity of silver ions. [2] 1.2 Tannins Tannins are water-soluble plant polyphenolic compounds located in various parts of plants, such as wood, bark, fruit peels, pods, leaves, roots and plant galls. They provide typical phenolic reactions such as proteins coagulation and chelation of metal ions, in which are predominantly applicable hydroxyls in the orthopositions. They are also powerful antioxidants (reductants) capable of eliminating reactive unpaired electrons in oxygen radicals by delivery of electrons which are in sufficient quantities in many hydroxyl groups.

2 Similarly, they are able to provide electrons for the reduction of Ag+ ions at the atomic silver. The principle of this mechanism is similar to so-called Tollens reagent (ammoniacal solution of silver ions), which is used for example for the identification of reducing substances such as aldehydes. In the case of tannins, phenols are oxidized to quinones or quinoid structures. Tannins are generally divided into hydrolyzable ones whose monomer units are gallic and ellagic acid. Their typical example is tannic acid which was first described in the extract from the Chinese sumac. This substance is part of pharmacopoeias since the middle of last century and has proven antibacterial effects, but it is also suspected of hepatotoxicity and cytotoxicity. [3] Condensed tannins are the second group of tannins, so called proanthocyanidins, whose monomer units of flavanols are combined by fixed covalent bonds. They occur typically in the peels and seeds of grapevine and arise by similar metabolic pathways such as anthocyanins contained in red wine. These tannins are due to covalent bonds more stable than the hydrolyzable ones and as polyphenols they have with them also other comparable properties astringent, antioxidant and complexation ones - without their cytotoxicity being reported. [4] The combination of vegetable tannins with silver nanoparticles extends their treatment options, especially extends a spectrum of their antibacterial effect. 2 EXPERIMENTAL PART 2.1 Material Silver nitrate, AgNO3, M=169,87 g/mol (Lach-Ner) Sodium carbonate anhydrous, Na2CO3 (Lach-Ner) Tannin grape - product with high content of condensed tannins for viticultural use (Erbslöh) Tannic acid, g/mol C76H52O46 (Sigma-Aldrich) Stable radical 1,1-diphenyl-2-picryl hydrazyl, DPPH (Sigma-Aldrich) Ethanol p.a. 96 % (Lach-Ner) 2.2 Devices VIS spectrophotometer Helios Epsilon (Thermo Scientific) Zetasizer Nano ZS (Malvern) Dyeing apparatus Ahiba Nuance ECO (Datacolor) 2.3 Methods Preparation of samples There were prepared solutions of AgNO3 at a concentration of 6.00, 0.60, 0.06 and mm/l, solutions of tannic acid (TA) and of Tannin Grape (mixture of condensed tannins, CT) at concentrations of 1.00 and 0.10 g/liter. In case of the tannic acid this corresponds to a concentration of 0.60 and 0.06 mm/l. Since the CT solution of unknown composition was prepared from the same weight as the amount of TA, there will be both tannins for simplification further mentioned at molar concentrations that correspond to TA. 1 g of crushed dried seedless grapes was for 1 hour boiled at 100 C in 100 ml of distilled water in a closed cartridge of dyeing apparatus with microwave heating. The acquired extract (E) was used as a source of condensed tannins for comparison with Tannin Grape product. Mixing the solutions of silver ions and tannins in ratio 1:1, incurred solutions at half concentrations (TA/Ag, CT/Ag and E/Ag). The resulting mixtures were adjusted by adding sodium carbonate solution to approximately ph 7. The presence of silver nanoparticles indicates a change in solution discoloration (various shades of yellow, brown to gray or black colour), which

3 is dependent primarily on the size of the resulting particles and their aggregation. The size and stability of the particles were measured after about 2 hours at room temperature Size of silver nanoparticles Size of silver nanoparticles was measured by Zetasizer Nano device using dynamic light scattering. The particles are illuminated by a laser and the analysis of fluctuations in scattered light intensity (large particles scatter more light than small particles) states Brownian motion of particles in relation to the size of the particles. The instrument measures particles in the range of 0.6 nm to 6 microns Stability of silver nanoparticles The stability of colloidal silver nanoparticles is another application of apparatus Zetasizer Nano, which measures the zeta potential by means of laser Doppler electrophoresis. The stability of nanoparticles depends on the ratio between attractive (Van der Waals forces) and repulsive interactions (electrostatic repulsive forces). In ionic silver state predominate the repulsive forces among particles that keep colloidal solution homogeneous and stable. The neutral atoms form gradually weakly cohesive agglomerates, or formation of tightly associated aggregates of colloidal particles. Metal colloids are generally less stable than colloids of conductive materials due to their easy polarizability. The size of electrostatic repulsive forces of colloidal nanoparticles (namely size of the electric charge on the surface) cannot be measured directly, so it is used an indirect method of measuring of so-called zeta potential. Zeta potential is related to electrokinetic phenomena (e.g. electroosmosis or electrophoresis) that occur in an interaction of charged colloidal particles with electric field. Zeta potential is measured at the interface of liquid and at the outside part of the electrical double layer of colloidal particles (so-called slipping plane). The size of the zeta potential indicates a possible stability of the colloidal systems. If all the particles in the suspension have a large negative or positive zeta potential, then they will tend to repel each other and the system is relatively stable. However, if the particles have low values of zeta potential, then outweigh attractive forces between the particles of electrorepulsive forces and particles tend to aggregate and flocculate. In general, the boundary between stable and unstable suspension is considered to be the value of zeta potential of ±30 mv. Values from 0 to ±10 mv indicate the rapid coagulation or flocculation Antioxidant activity of tannin/silver mixture Dissolving 10 mg of the stable radical DPPH in 150 ml of 96 % ethanol created dark purple solution with the absorbance about 1.7 AU at the absorption maximum 517 nm. A degression of DPPH is occurred by adding of antioxidant. This is reflected by differently rapid discoloration of the violet solution from decrease the intensity of purple until the yellow colour which is also associated with a decrease in absorbance at 517 nm. 100 µl of sample was added to 2 ml of DPPH in the cuvette, every 5 minutes the solution was mixed thoroughly and after 15 minutes was subtracted the decrease from the initial absorbance. Each measurement was performed 5 times and calculate the average value. 2.4 Results Results in Table 1-4 indicates that: Size of the silver nanoparticles reduced by tannic acid and by mixture of condensed tannins is by using of comparable concentrations fundamentally not different. With the decrease in the concentration of silver ions in the mixture with tannin increases the size of created silver nanoparticles. With increasing concentrations of tannin in the mixture increases the size of created silver nanoparticles. Higher concentration of tannin in the mixture leads to the formation of more stable colloid.

4 When using an aqueous extract of grape seeds there are forming colloidal particles at a size of hundreds of nanometers. This colloid has high stability which is probably due to the content of other ballast substances (such as carbohydrates), which stabilize the colloid. Antioxidant strength of tannin in a mixture with a molar ratio of 1:10 (tannin: AgNO3) decreased by reduction of silver ions to about half and this value further slightly decreased after 2 hours compared to colloid 30 minutes old. At the molar ratio of 1:100 is the antioxidant activity of tannin by reduction of silver completely spent. (Table 4) The optimum molar ratio for the synthesis of particle size (which was reported as the most effective for their antibacterial activity about 25 nm), lies between 1:1 to 1:10 (tannin: AgNO3). Table 1 Silver nanoparticles reduced by tannic acid Concentration of tannin (mm/l) Concentration of AgNO 3 (mm/l) Molar ratio (tannin:agno 3) Size (nm) Zeta potential (mv) : : : : : : ,6 Table 2 Silver nanoparticles reduced by condensed tannins (Tannin Grape) Concentration of Concentration of Molar ratio Size (nm) tannin (mm/l) AgNO 3 (mm/l) (tannin:agno 3) /; Zeta potential (mv) : : : : : : Table 3 Silver nanoparticles reduced by extract from grape seeds Conc. of the extract from seeds (g/l) Concentration of AgNO 3 (mm/l) Size (nm) Zeta potential (mv) Table 4 Changes in antioxidant activity of tannin/silver mixtures Molar ratio (tannin : AgNO 3) TA (30 min.) TA (120 min.) CT (30 min.) CT (120 min.) BLANK (TA/DV) (TA/DV) (CT/DV) (CT/DV) 10: : , : , :

5 ΔA 517 (30 min.), ΔA 517 (120 min.) decrease in absorbance of DPPH solution (initial value 1.7 absorption units), after adding 100 µl of colloid, which was 30 or 120 minutes old. BLANK. (mixture tannin/distilled water), ie. decrease in absorbance of DPPH solution by tannin before the reaction with silver. TA tannic acid, CT condensed tannins (Tannin Grape), DV distilled water. 3. CONCLUSION Experiments performing at ph 7 showed no major differences in the size of silver nanoparticles prepared by hydrolysable and condensed tannin. At the molar ratio of tannin and silver ions in a mixture of 1:1 to 1:10 were forming sufficiently small silver nanoparticles while maintaining roughly half of the antioxidant ability of tannin against the original value. This study was conducted as part of the development of textile materials for primary protection and the treatment of wounds where tannins are of very good use. Tannins may have a beneficial effect on wounds closure, are suitable anti-flammatory and destruct free oxygen radicals in the wound. Tannic acid itself has proven antibacterial effects which may the silver nanoparticles still enhance and extend the scope of their action. But the question is, what biological effect can be expected from quinoid structures logically arising in the mixture after the reduction of silver as well as by oxidation of phenolic groups of tannins. ACKNOWLEDGMENTS The paper has been elaborated with support of the grant project SGS (provided by Faculty of Textile of Technical University of Liberec) and grant project TA program Alpha (provided by Technology Agency of the Czech Republic). LITERATURE [1] CHAMBERS, C. W., PROCTOR, C. M., KABLER, P. W.: Bactericidal Effect of Low Concentrations of Silver. Journal of the American Water Works Association 54 (1962), p [2] KVÍTEK, L.: Studium přípravy a aplikačních možností nanočástic stříbra (habilitační práce). PřF Univerzity Palackého v Olomouci, [3] HUPKENS et al.: Tannic acid as a topical agent in burns: historical considerations and implications for new developments. Burns 21, (1995), p [4] SCHOFIELD, P., MBUGUA, D. M., PELL, A. N.: Analysis of condensed tannins: a review. Animal Feed Science and Technology 91 (2001), p