CC BY
33ISSN 2072-5981
aänetic Resonance in Solids
Electronic Journal
Volume 18, Issue 1 Paper No 16104, 1-5 pages 2016
http: //mrsej. kpfu. ru http: //mrsej. ksu. ru
Established and published by Kazan University Sponsored by International Society of Magnetic
Resonance (ISMAR) Registered by Russian Federation Committee on Press,
August 2, 1996 First Issue was appeared at July 25, 1997
© Kazan Federal University (KFU)*
"Magnetic Resonance in Solids. Electronic Journal" (MRSey) is a
peer-reviewed, all electronic journal, publishing articles which meet the highest standards of scientific quality in the field of basic research of a magnetic resonance in solids and related phenomena. MRSey is free for the authors (no page charges) as well as for the readers (no subscription fee). The language of MRSey is English. All exchanges of information will take place via Internet. Articles are submitted in electronic form and the refereeing process uses electronic mail. All accepted articles are immediately published by being made publicly available by Internet (http://MRSe/. kpfu.ru).
Editors-in-Chief Jean Jeener (Universite Libre de Bruxelles, Brussels) Boris Kochelaev (KFU, Kazan) Raymond Orbach (University of California, Riverside)
Executive Editor Yurii Proshin (KFU, Kazan) mrsej@kpfu.ru editor@ksu.ru
Editors
Vadim Atsarkin (Institute of Radio Engineering and Electronics, Moscow) Yurij Bunkov (CNRS, Grenoble) Mikhail Eremin (KFU, Kazan) David Fushman (University of Maryland,
College Park)
Hugo Keller (University of Zürich, Zürich) Yoshio Kitaoka (Osaka University, Osaka) Boris Malkin (KFU, Kazan) Alexander Shengelaya (Tbilisi State University, Tbilisi) Jörg Sichelschmidt (Max Planck Institute for Chemical Physics of Solids, Dresden) Haruhiko Suzuki (Kanazawa University,
Kanazava) Murat Tagirov (KFU, Kazan) Dmitrii Tayurskii (KFU, Kazan) Valentin Zhikharev (KNRTU, Kazan)
In Kazan University the Electron Paramagnetic Resonance (EPR) was discovered by Zavoisky E.K. in 1944.
Paramagnetic properties and antioxidant activity of metal-containing bionanocomposites based on humic substances
N.I. Tikhonov 1, S.S. Khutsishvili 1*, M.V. Lesnichaya1, G. Dolmaa2, T.I. Vakul'skaya1, G.P. Aleksandrova1, B.G. Sukhov1
1 A.E. Favorsky Irkutsk Institute of Chemistry of the Siberian Branch of Russian Academy of Sciences, Favorskogo st. 1, Irkutsk 664033, Russia
2 Chemistry and Chemical Technology Institute of the Mongolian Academy of Sciences, Enkhtaivan av. 54b, Ulaanbaatar 13330, Mongolia
*E-mail: khutsishvili_sp@yahoo.com
(Received November 22, 2016; revised December 16, 2016; accepted December 20, 2016)
Paramagnetic properties of novel bioactive nanocomposite materials based on humic substances and the precious metals (Ag, Au) are investigated by electron paramagnetic resonance and compared to their antioxidant properties. The possible mechanisms of antioxidant activity in the light of the free radical activation or inhibition of oxidation processes are analyzed.
PACS: 61.46.+w, 76.30.-v, 81.65.Mq, 87.15.-v, 81.65.Mq.
Keywords: antioxidant activity, EPR, spin density, nanocomposite, humic substance
1. Introduction
In recent years, the search for new biogenic antioxidants that combine low toxicity and ability to effectively inhibit free radical processes in living organisms is gathering momentum [1-4]. Among such antioxidants, the compounds bearing the phenol moiety continue to attract a special research interest for many years [4].
Humic substances (HS) are considered to be very promising antioxidants. They represent complex polymeric organic compounds of aromatic and carbohydrate nature. Also, HS comprise various oxygen-containing groups, including phenolic hydroxyls. It is known that HS possess diverse biological activity [5, 6]. In particular, they are natural polymer antioxidants that are capable of neutralizing the free radicals formed in the inflammatory locations, and are inductors of destabilizing and breaking the cell membrane [7]. Previously, we have demonstrated the possibility of successful application of HS derived from biogenic sediments for the synthesis of water-soluble nanocomposites [8, 9]. Thus, the obtained silver-containing nanocomposites are expected to combine specific properties of HS (antioxidant activity and water solubility) with those of gold and silver nanoparticles, such as antimicrobial properties. A comparative study of the antioxidant activity of the resulting nanocomposites as well as the initial HS shows that all the samples in vitro inhibit the peroxidation model processes of the living cell membrane lipids [10].
Despite the advances in the studies of bioantioxidant properties of natural compounds, many issues related to the relationships between the structural organization and antioxidant properties of the substances still remain poorly understood. Here we report the results of comparative analysis of the paramagnetic characteristics and antioxidant properties of the obtained metal-containing nanocomposites with the original matrixes. Also, the general regularities of the change in their oxidising properties are elucidated.
2. Experimental
All the target HS-based nanocomposites were characterized by a complex of modern spectral and physicochemical methods [8, 9]. Silver (HS-Ag) and gold (HS-Au) nanocomposites were synthesized by the reaction of silver and gold ions with the corresponding humic substances, extracted from medical muds (HSm) of Mongolian Lake Gurvan Nuur, shales (HSs) of Shine Hudag and brown coals
Table 1. Metal content and spin concentration of HS and nanocomposites on its base.
Samples HS silver nanocomposites gold nanocomposites
Fe, % N, spin/g Ag, % Fe, % N, spin/g Au, % Fe, % N, spin/g
HSm 0.7 3.4x1018 9.8 1.0 3.6x1018 7.1 0.5 1.7x 1018
HSs - 4.4x1017 10.5 0.1 9.9x1017 6.8 1.1 8.4x1017
HSc - 1.6x1019 10.8 1.5 1.3x1019 8.6 0.6 1.4x 1019
(HSc) of Baga Nuur field, in the aqueous-alkaline medium for 20 min at 90°C according to previously published protocols [8, 9]. The formation of Ag0 and Au0 nanoparticles was proved by the appearance of intensive plasmon absorption in the visible spectral region (Ag 408-432 nm, Au 510-540 nm) due to collective excitation of conduction electrons of gold or silver.
The metal percentage of nanocomposites has been determined by atomic absorption analysis using HITACHI TM 3000, detector SDD XFlash 430-H. The metal mass percentages of the nanocomposites on the basis of HS matrix are presented in tab. 1.
The X-ray diffraction (XRD) analysis has been made on powder diffractometer D8 ADVANCE (Cu radiation). The estimated size of metal nanoparticles was determined by XRD analysis, their linear dimensions were calculated by the Scherrer formula [11]. XRD analysis of nanocomposites based on HSm, HSc and HSs showed that the average region size of the coherent scattering (L, nm) of silver nanoparticles in the obtained samples varied in the range of 6-14 nm. The average size of the coherent scattering of gold nanoparticles in the nanocomposites obtained was 10-17 nm (XRD data).
The antioxidant activity was determined by the measurement of the inhibitory effect of a potential antioxidant on the in vitro model of Fe2+ ascorbate induced oxidation of polyoxyethylene sorbitan monooleate (Tween-80) to malondialdehyde with air oxygen [10]. The measurement precision of antioxidant activity was ±1.0%.
Continuous wave electron paramagnetic resonance (EPR) spectra were recorded with FT X-band Bruker ELEXSYS E-580 spectrometer (X-wave range 9.7 GHz). The EPR-spectra were recorded under the following conditions: amplitude modulation 1.0 G, modulation frequency 100 kHz, time constant 0.02 s, conversion time 0.06 s, microwave power 0.6325 mW, field range 6000 G / centre field 3480 G and field range 100 G / centre field 3443 G, averaged scans 10, receiver gain 30 dB at room temperature. The concentrations of paramagnetic centers were calculated by the known method [12] with the use of diphenylpicrylhydrazyl as a standard (tab. 1).
3. Result and Discussion
The initial HS possess paramagnetism and give in the EPR spectra the main high intensity signal with g-factor of 2.005 and width of about 5 G (fig. 1). Narrow symmetrical lines of the samples correspond to the concentration of unpaired spins of 1017-1019 spin/g, see tab. 1. Paramagnetic properties of HS are caused by polyaromatic strongly condensed nature of the conjugated bonds system. The molecular structure of the investigated substances is characterized by the presence of aromatic and aliphatic components, especially the so-called "core" of the macromolecule. Furthermore, in the HS structure, the aromatic moieties predominate over the chained aliphatic ones. Ratios (degree of benzenoid) H/C for the selected HS products vary in the range of 0.067-0.083 and shows a high content of aromatic structures in their composition. The presence of a well-developed system of conjugated bonds in HS structure is consistent with high concentration of paramagnetic centers. The intensive signal is often present in such natural macromolecules, due to including free radicals in these compounds (co-called macroradicals) [13, 14]. The most intense signal is shown in the spectrum of substances extracted from brown coal, due to the natural complicated structure of these compounds (with a high degree of unpaired electrons delocalization) and high spin concentration [15].
70 n
60
50
a 40
30
20
10
3000 4000 Magnetic Field, G
Figure 1. A typical EPR spectrum of started HS (measured at room temperature).
llhl
HSm HSs HSc Succinic To no I acid
HSm
Figure 2. Antioxidant activity of the started HS in comparison with the activity of known antioxidants.
The study of the antioxidant activity of the starting HS has revealed that all samples in vitro have a higher inhibitory effect on oxidative processes within 42-52% (fig. 2), the amount of antioxidant activity being greater than that for the known antioxidant succinic acid. The diagram shows that the greatest inhibition of the peroxidation products accumulation (52%) is observed when HSs is used as an antioxidant, HSm possesses lesser antioxidant activity (43.6%) and HSc exerts the lowest (42.3%) the inhibitory effect. Different antioxidant ability of the substances is probably due to the difference between elemental and functional composition of the macromolecules. High characteristics of antioxidant activity and high spin concentrations allow to trace their change in the obtained metal-containing bionanocomposites.
In the EPR spectra of the silver- and gold-containing nanocomposites HS-Ag and HS-Au, intense main signal is also observed (fig. 3), as in the original matrixes. The narrow line for metal-containing samples broaden to 6.5 G, g-factor increases (in range of 2.0060-2.0065) and deviates from the values of a free electron ge that indicates the contribution of the spin-orbit interaction of the electrons with a heavy nucleus. It can be concluded that the narrow line is a superposition of signals, caused by free radicals of the original matrix and the zero-valent metal nanoparticles, which is consistent with the EPR data for the conductivity electrons of zero-valent metal particles in nanoscale systems [16-18]. It is assumed that the main contribution to the reduction of metal cations to the zero-valent state is made by the HS aldehyde groups (with different aliphatic and aromatic substituents) and the phenolic hydroxyls. In addition, the nanocomposites are characterized by a high degree of oxidation as compared to the initial HS samples, which also testifies to their oxidation in the process of metal reduction.
The diagram shows that the greatest inhibition of the oxidation products accumulation is observed in the case of HS-Ag nanocomposites (22-52%) and HS-Au nanocomposites (44-52%) (fig. 4). The antioxidant properties of silver composites either decrease or remain almost unchanged (HSs-Ag). The smallest inhibitory effect (22%) is found for silver-nanocomposites having a maximum content of silver in 10.8% for HSc-Ag. For gold composites, there is a slight increase (or a very weak decrease in the case HSs-Au), i.e. the value of antioxidant activity slightly differs from that of the original matrices. It can be assumed that the antioxidant activity of nanocomposites based on HS, like many other bioantioxidants, is caused by the presence of phenolic groups. The observed decrease in antioxidant activity of nanocomposites with maximum silver content can be attributed to a decrease in the concentration of the phenolic groups of the obtained nanocomposite due to their one-electron oxidation by silver cations in the synthesis process.
1000 2000 3000 4000 5000 6000 Magnetic Field, G
Figure 3. A typical EPR spectrum of gold-containing HS-based nanocomposites (measured at room temperature).
An Ag An
7.1% 10.5% An 8.6%
6.8%
A g
9.8% Ag
10,8%
HSm HSs HSc
Figure 4. Antioxidant activity of the metal-containing HS-based nanocomposites (metal contents specified in the figure).
Note the changes of the spin concentration also are not unequivocally (tab. 1). So free radicals together with the phenolic groups can also act as a reducing agent of silver and gold ions owing to interaction of metal ions with an electron-donor centers during the formation of nanosystems. This can be explained by the fact that the introduction of a larger amount of metal in the matrix leads to a greater decreasing of the spin concentration in the composite, which is consistent with the hypothesis on the interaction of metal ions with free radicals. Reduction process occur due to different types of free radicals in such complicated macromolecule structures (HSs and HSm are more oxidized unlike substances HSc of deeper decomposition), which explains the dissimilar expenditure of free radicals. Note that the reduction of Au3+ ions to zero-valent state requires the use of 3e that should cause substantially greater decrease of spin concentration in the HS-Au, as compared to HS-Ag compounds. However, not so high difference in concentrations could be explained by the different content of metals in the nanocomposites. Moreover, one should consider the contribution in a spin concentration from the zero-valent metal nanoparticles. It is important to note that the complexities of the changes in the antioxidant properties can be attributed to other processes, so the antioxidant properties of phenolic compounds are determined not only by their anti-radical action, they are also able to interact with transition metals and can thus contribute to the formation of free radicals. As a result, reduction of metal ions can activate the processes of hydroperoxides decompositions (Fenton reaction type) [4], and the phenolic group may act as prooxidants. Thus, by interacting with the transition metal ions, they can both inhibit and activate free radical oxidation processes. Moreover, iron, presented in the humic substances in amount 0.1-1.5%, can act as an additional initiator of free radical peroxidation process [10].
4. Summary
Bionanocomposites based on HS of Mongolian deposits preserve their paramagnetic properties, at least for one year, and represent biologically active substances containing a lot of stable nanoparticles of zero-valent silver and gold (6-14 nm). Attempts to correlate antioxidant activity and spin concentration of HS samples have shown its ambiguity, that makes impossible to approach the evaluation of an antioxidant activity based on EPR characteristics in such complicated substances. In addition, all obtained nanocomposites exhibit high antioxidant activity comparable with that of the known antioxidants, ionol and succinic acid. Thus, the HS-based bionanocomposites find new applications in the synthesis of novel pharmaceuticals with antioxidant properties, in the manufacture of composite materials for the healing with antimicrobial properties of HS-Ag and softening the effects of pollution on living organism.
Acknowledgments
This work was supported by the President Grant of Russian Federation (Grant MK-7149.2016.3). The authors are grateful to the Baikal Analytical Center for the special measurements.
References
1. Assodeh A., Homayouni-Tabrizi M., Shabestarian H., Emtenani S., Emtenani Sh. J. Food and Drug Analysis 24, 332-342 (2016)
2. Yoo D., Guk K., Kim H., Khang G., Wu D., Lee D. Int. J. Pharm. 450, 87-94 (2013)
3. Öchsner A., Shokuhfar A. New Frontiers of Nanoparticles and Nanocomposite Materials, Springer-Verlag, Berlin Heidelberg (2013)
4. Menshchikova E.B., Lankin V.Z., Kandalintseva N.V. Phenolic Antioxidants in Biology and Medicine, LAP LAMBERT, Saarbrücken (2012)
5. Steinbüchel A., Marchessault R.H. Medical Aspects and Applications of Humic Substances, in Biopolymers for Medical and Pharmaceutical Applications, R. Klöcking, B.A. Helbig eds. (Wiley-VCH, Weinheim, 2005) pp. 3-16
6. Xu J., Wu J., Y. He Y. Functions of Natural Organic Matter in Changing Enviroment, Springer Netherlands, Dordrecht (2013)
7. Conner E.M., Grisham M B. Nutrition 12, 274-277 (1996)
8. Lesnichaya M.V., Aleksandrova G.P., Dolmaa G., Sapozhnikov A.N., Sukhov B.G., Regdel D., Trofimov B.A. Dokl. Chem. 456, 72-75 (2014)
9. Lesnichaya M.V., Aleksandrova G.P., Sukhov B.G., Trofimov B.A., Dolmaa G., Nomintsetseg B., Regdel D., Sapozhnikov A.N. Dokl. Chem. 460, 13-16 (2015)
10. Dolmaa G., Lesnichaya M.V., Aleksandrova G.P., Sukhov B.G., Regdel D., Trofimov B.A. Dokl. Chem. 11, 453-454 (2013)
11. Barret C.A., Massalsky T.B. Structure of Metals: Crystallographic Methods, Principles, and Data, McGraw-Hill, New York (1966)
12. Poole C.P. Electron Spin Resonance: A Comprehensive Treatise on Experimental Techniques, 2nd ed., Dover Publications, Dover (1997)
13. Jezierski A., Czechowski F., Jerzykiewicz M., Chen Y., Drozd J. Spectrochim Acta A Mol Biomol Spectrosc. 56A, 379-385 (2000)
14. González-Pérez M., Martin-Neto L., Colnago L.A., Milori D.M.B.P., de Camargo O.A., Berton R., Bettiol W. Soil & Tillage Research 91, 95-100 (2006)
15. Piawa B., Wittkowski A.B. Res. Chem. Intermed. 33, 825-839 (2007)
16. Khutsishvili S.S., Vakul'skaya T.I., Kuznetsova N.P., Ermakova T.G., Pozdnyakov A.S., Prozorova G.F. J. Phys. Chem. C 118, 19338-19344 (2014)
17. Mitrikas G., Deligiannakis Y., Trapalis C.C., Boukos N., Kordas G. J. Sol-Gel Sci. Technol. 13, 503-508 (1998)
18. Eichelbaum M., Rademann K., Hoell A., Tatchev D.M., Weigel W., Stößer R., Pacchioni G.
Nanotechnology 19, 1-9 (2008)
