Influence of silver nanoparticles and magnetic field on phytochemical, antioxidant activity compounds and physiological factors of Phaseolus vulgaris

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1 Technical Journal of Engineering and Applied Sciences Available online at TJEAS Journal / ISSN TJEAS Influence of silver nanoparticles and magnetic field on phytochemical, antioxidant activity compounds and physiological factors of Phaseolus vulgaris Saeedeh Najafi 1*, Reza Heidari 2, Rashid Jamei 2 1,2. Department of Biology, Faculty of Science, Urmia University, Iran Corresponding author : Saeedeh Najafi ABSTRACT: In order to study the effect of silver nanoparticles and magnetic field on Phaseolus vulgaris, an experiment was conducted in Experiment was carried out with 3 treatments in 10 days. Treatments were including (T1) control, (T2) magnetic field withb=1.8 mtfor 30 min per day in 10 days, (T3) silver nanoparticles (50 ppm).results showed that silver nanoparticles (T3) had the highest phytochemical and chemical composition while rate of yield and chemical composition in group of treated by magnetic field (T2) had the lowest. Experiment treatments had significantly effects on traits such as the plant height, lenght of root and shoot. It seems that silver nanoparticles (50 ppm) had the best effect on Phaseolus vulgaris growth, yield and may be suitable alternative for chemical composition in compare to other treatments. Key words: Antioxidant activity, Phytochemical compounds, Physiological factors, Magnetic field, Silver nano particles, Phaseolus vulgaris INTRODUCTION Research work on the effect of magnetic field and nano particles on plants development and yielding has been started in the last century. The various effects on growth, seed germination and biomass yields of plants depend in a complex way on magnetic flux densities, frequencies the pretreatment of the material, and treatment duration.nanotechnology is a method for potential of massive societal andfast-developing industry, posing substantial impacts on economy, society and environment. Nanoparticles (nano-scal particles= NSPs) are atomic or molecular aggregates that its size is about 100 nm down to about 1 nm (Ball,2002; Roco, 2003a), that can drastically modify their physics and chemical properties compared to the bulk material. Engineered nano materials have received a particular attention for their positive impact in improving many sectors of economy and trade, including consumer products, loom, pharmaceutics, consmetics, transportation, energy and agriculture etc., and are being increasingly produced for a wide range of applications within industry (Novack and Bucheli, 2007; Roco, 2003b).The organisms and especially those that interact strongly with their immediate environments, are expected to be affected as a result to their exposition to NSPs. In the review of NAVARRO et al. (Navarro et al., 2008) there topics are underlined, 1: sources, transformation and fate of nano particles and experience in contact with algae, fungi and and plants and then the enhance and fate of these organisms; 2: the mechanism of engineered nano particles toxicity and their effect on organism and how these toxic effects might be transferred through food chains, thus affecting communities and whole ecosystems. Engineered nano materials can be grouped into four type: 1. Carbon based materials, usually including fullerene, single walled carbon nanotube (SWCNT) and multiwalled carbon nanotubes (MWCNT); 2. Metal based materials such as quantum dots. Nanogold, nanozinc, nanoaluminum, nanoscales metal oxides like TiO 2, ZnO, Al 2 O 3 and nanosilver; 3.Dendrimers which are nano-sized polymers built from branched units. Capable of being tailored to perform specific chemical function; 4.Composites which combine nano particles with other nano particles or with larger bulk-type materials (Yu-Nam and Lead, 2008). Galland and Pazur reported the unsystematic manner in which the research on magnetorasponse in biology has been carried out in the past and explains presently accepted mechanisms ofmagnetoreception (Galland and Pazur, 2005).

2 MATERIAL AND METHODS Seed pretreatment with MF Seeds of Phaseolus vulgaris (cv. talash) were obtained from Esfahan in Research Center in 2012, Iran. Seeds of Phaseolus vulgaris have been used for investigating the influence of magnetic field on the development of plants. The induction of magnetic field has been B= 1.8 mt, measured with a digital tesla-meter (PHYWE, Germany). Magnetic-field-induction value has been chosen according to the opinion that weaker magnetic field has stronger effect on plant productivity. The healthy uniform dry seeds 8.6% of moisture content were selected and seeds kept at the geometric Centre of coil assemblies. Control seeds were kept under similar condition local geomagnetic field only but in the absence of magnetic field. Exposure was 10 days for 30 min per day and control seeds were kept under the similar condition in the absence of the MF. The experiments have been performed in 2012 under laboratory conditions. The natural light cycle was 16 h light/8h darkness with daily temperature 25 Cand night temperature 22 C. Preparation of silver nanoparticles silver nanoparticles were prepared by means of the biological reduction of metal salt precursor (silver nitrate, AgNO 3 ) in water with aqueous extract of manna of hedysarum plant in the presence of extract of soap-root plant as a stabilizer (Forough and Farhadi, 2010). Briefly, 10 ml of freshly prepared extract of soap-root plant as a stabilizer agent was added to 100 ml of 3 mm aqueous silver nitrate solution and incubated in a rotary shaker for 2 h in dark conditions at 25 C, and then 15 ml of the aqueous extract of manna of hedysarum plant as a reducing agent was added into the mixture at 86 C. The mixture obtained, was purified by repeated centrifugation at 12,000 rpm for 20 min to obtain the fresh biologically Ag nanoparticles solution. Seed germination and seedling development MF pretreated and control seeds were surface sterilized with 1% NaOCl (w/v) for 5 min, washed thoroughly 3 times with distilled water and then propagated in pots containing soil and sand mixture (1:2). The pots were maintained under natural photoperiod with 35% (w/w) soil moisture content. Seed germination observed at 7th day, and germination seedlings were uprooted and measured the length of 10 days for both control and treated seedlings. Pigment contents (chlorophyll a and chlorophyll b) The photosynthetic pigments e.g., Chl a, b and Car were extracted in 5 ml of chilled 80% acetone by grinding the leaves of salt treated seedlings in a chilled mortar and pestle. The homogenate was centrifuged at 3000 rpm for 10 min at 4 C. The absorbance of the resulting supernatant was taken at 480, 645 and 663 nm. Different pigments were estimated using the following formula by Arnon as given below: Total Chl (a and b) (mg/l) ¼ 20.2 (A645) 8.02 (A663) Chl a (mg/l) ¼ 12.7 (A663) e 2.69 (A645) Chl b (mg/l) ¼ 22.9 (A645) e 4.68 (A663) Car (mg/l) ¼ A480 x 200 The pigment concentration was calculated in µg/g FW of sample and expressed as percent change (Bonilla et al., 2003). Total phenol Total phenolic were determined spectrophotometrically using Folin Ciocalteu s reagent as described by Singleton and Rossi (1965) and Bonilla et al. (2003). Briefly, 4 g fresh berries (the seed discarded) were ground in liquid nitrogen. A sample was then extracted in 2% HCl in methanol for 24 h in the dark and at room temperature. After centrifugation at 12,000 rpm for 20 min at 4 C, the supernatant was diluted with the same extract solvent at a suitable concentration for assaying total phenolic. Two hundred microliters of diluted extraction was introduced into a 5.0 ml test tube. One milliliters of Folin Ciocalteu reagent and 0.8 ml carbonate (7.5%) were then added and the contents mixed and allowed to stand for 30 min. Absorption at 765 nm was measured in a Shimadzu UV Vis spectrophotometer (Shimadzu UV-1601).Total phenolic content was expressed as Gallic acid equivalents (GAE) in milligrams per gram of sample using a standard curve generated with 50, 100, 150, 200, 250, 300, 350, 400, and 500 mg/l of gallic acid (Arnon, 1949). Determination of flavonoid content The flavonoid contents of the extracts were determined by the colorimetric method described by Jia et al. (Jerman et al., 1989), with some modifications. The Phaseolus vulgaris extract (0/1 ml) was mixed with 1.25 ml of 2813

3 distilled water and 75 μl of a 5% NaNO 2 solution. After 5 min, 150 μl of a 10% AlCl 3 H 2 O solution was added. Aft er 6 min, 500 μl of 1 M NaOH and 275 μl of distilled water were added to the mixture. The solution was mixed well and the intensity of the pink color was measured at 510 nm. Catechin was used to calculate the standard curve ( mm; y = x; R2 = ) and the results were expressed as milligrams of catechin equivalents per gram of extract (mg CEs/g extract). Statistical analysis The data obtained from the experiments were analyzed and calculated. As the experimental design is completely randomized design and data for each experiment were analyzed by one-way ANOVA with factorial arrangement to determine the effects of magnetic treatment. Means were compared using Duncan s multiple-range test at a 5% level of significance by SPSS. Result and discussion: Harris and Bali in 2008 reported the limits of uptake and the distribution of silver nanoparticles in Brassica junceaandmedicago sativa (Harris and Bali, 2008). In contrast to Brassica juncea, Medicago sativa observed an increase in metal uptake with a corresponding increase in the substrate of metal concentration and exposure time. The silver nanoparticles were located in the nucleus and applying the definition of McGrath and Zhao in 2003, the outhor showed that both Medicago sativa and Brassica junceawerehypercumulators of silver (McGrath and Zhao, 2003). Figure 1. Influence of magnetic field on rate of seed germination of Phaseolus vulgaris. Bars represents means ± standard error. Means followed by the same letter are not significantly different (P<0.05) as determined by Duncan s multiple-range test. Racuciu and Creanga in 2007 analysed the influence of magnetic nanoparticles coated with tetramethylammoniumhydroxid on the growth of Zea mays plant in early ontogenetic stages (Racuciu and Creanga, 2007). Montgomery and Smith investigated a slight enhancement of barley growth rate under moderate-strength magnetic fields (Montgomery and Smith, 1963). Our observations suggest that in addition to a possible stimulating influence, in accord with previous observations, there is a definite inhibition effect of magnetic exposure and enhancement effect of silver nanoparticles on the seeds.petti et al.(piattiet al., 2002) have observed a negative effect on rate of seed germination. 2814

4 Figgure2.Influence of magnetic field on Phaseolusvulgarisseedling height, length of root and stem. Bars represent means ± standard error. Means followed by the same letter are significantly different (P<0.05) as determined by Duncan s multiple-range test. Belyavskaya in 2004 observed that weak magnetic field exhibited negative effects on plant growth, such as inhibition of primary root growth (Belyavskaya, 2004). In soils, the mobility of silver nanoparticles in pore water is an essential condition for interactions with plant roots. Yano et al. in 2004 showed that exposure to magnetic field inhibited early growth of radish seedlings with decrease in the weight and leaf area (Yano et al., 2004). Figure3.Influence of magnetic field on pigments content (Chlorophyll a and b) of Phaseolusvulgarris. Bars represent means ± standard error. Means followed by the same letter are significantly different (P<0.05) as determined by Duncan s multiple-range test. When light impinges on photosynthetic material a plant leaf, an alga or a photosynthetic bacterium it is absorbed by an array of light collecting pigments. Through resonant energy transfer the absorbed quantum of light is transported to a trap, the reaction centre. Within such a trap, a specialized (bacterio)chlorophyll complex is able to eject from its excited state an electron. This electron is captured by an adjacent acceptor, which in turn donates the electron to a second acceptor, and so on. Thus, light energy is converted into chemical energy which is ultimately used in the metabolic processes of the cell. An explanation for the higher or lower chlorophyll content is the increase or decrease in the ion accumulation by the nutrient solution, which in general change the metabolic activity of the plants. The central atom of chlorophyll is magnesium; the supply of this element is therefore essential for the synthesis of chlorophyll (Woodwardet al., 1960). Furthermore, several steps in photosynthetic pigment metabolism are dependent on iron (Briat et al., 2007). The results have shown that the total chlorophyll content decreased with magnetic field in the range about 1.8mT. 2815

5 Figure4.Influence of magnetic field on flavonoid and Phenol letter are significantly different (P<0.05) as determined by Duncan s multiple-range test. Flavonoid and Phenol content can be confine free radicals over extended periods that are long enough to allow an external magnetic field to affect spin evolution. The content of phenolic and flavonoid compounds reduced with magnetic field in the range about 1.8mT while these compoundswere increased bytreatment with silvernanoparticles. additionally a significant correlation was found between total phenol content and antioxidant activity. Therefore, it is possible that the phenolic compounds significantly contributed to the antioxidant activity in plants. However, other substances, e.g. glucosinolates, might contribute to the antioxidant activity in plants as well. The highest increase of antioxidant activity was found in the control group and was significantly increased in comparison to other groups. Also, the highest increase of flavonoid and phenol content were found in the in the grouptreatedwithsilver nanoparticles. Compared to this study(kaimoyo et al., 2008; Dannehl et al., 2011)have found that sub-lethal levels of electric current can be used to induce plant defence reactions and activity as an abiotic elicitor to enhance the secondary metabolite production in fenugreek, chickpea roots, and tomatoes. REFERENCES Arnon DI Copper enzymes in isolated chloroplasts.polyphenoloxidase in Beta vulgaris. Plant Physiol. 24: Ball P Natural strategies for the molecular engineer.nanotechnology. 13: Belyavskaya NA Biological effects due to weak magnetic field on plants. Adv. Space Res.34: Bonilla EP, Akoh CC, Sellappan S, Krewer G Phenolic content And antioxidant capacity of Muscadine grapes. Agric. Food Chem. J. 51: Briat JF, Curie C, Gaymard F Iron utilization and metabolism in plants. Current Opinion in Plant Biology. 10(3): Dannehl D, Huyskens-Keil S, Eichholz I, Ulrichs C, Schmidt U Effectsof direct-electric-current on secondary plant compounds and antioxidantactivity in harvested tomato fruits (SolanumlyxopersiconL.).Food Chemistry.126: Forough M, Farhadi K Biological and green synthesis of silver nanoparticles.turk J Eng. EnvironSci. 34: Galland P, Pazur A Magnetoraception in plants. Plant Res. J. 118(6); Harris AT, Bali R On the formation and extent of uptake of silver nanoparticles by live plants.journal of Nanoparticles Research. 10: Kaimoyo E, Farag MA, Sumner LW, Wasmann C, Cuello JL, VanEtten H Sub-lethal levels of electric current elicit the biosynthesis of plantsecondary metabolites. Biotechnology Progress. 24(2): McGrath SP, Zhao FJ Phytoextraction of metals and metalloids from contaminated soils.current Opinion in Biotechnology. 14: Montgomery DJ, Smith AE A search for biological effects of magnetic fields. Biomedical Science of Instrumentation, Vol. 1, Plenum Press, New York. Navarro E, Baun A, Behra R, Hartmann NB, Filser J, Miao A, Qigg A, Santschi PH, Sigg L Environmental behaviour and ecotoxicity of engineered nanoparticles to algae, plants and fungi. Ecotoxicology. 17: Novack B, Bucheli TD Occurrence, behavior and effect of nanoparticles in the environment.environmental Pollution. 150: Piatti E, Albertini MC, Baffone W, Fraternale D, Citterio B, Piacentini MP, Dacha M, Vetrano F, Accorsi A Antibacterial effect of a magnetic field on Serratiamarcescens and related virulence to Hordeumvulgare and Rubusfruticosus callus cells.comp.biochem. Physiol B BiochemMol. Biol. 132: Racuciu M, Creanga DE TMA-OH coated magnetic nanoparticles internalized in vegetal tissues.romanian Journal of Physics. 52: Roco MC. 2003a. Broader societal issue of nanotechnology.journal of Nanoparticle Research. 5: Roco MC. 2003b.Nanotechnology convergence with modern biology and medicine.current Opinion in Biotechnology. 14: Woodward RB, Ayer WA, Beaton JM, Bickelhaupt F, Bonnett R, Buchschacher P, et al The total synthesis of chlorophyll.journal of the American Chemical Society. 82(14): Yano A, Ohashi Y, Hirasaki T, Fujiwara K Effects of a 60 Hz magnetic field on photosynthetic CO2 uptake and early growth of radish seedlings.bioelectromagnetics. 25: Yu-Nam Y, Lead R manufactured nanoparticles: an overview of their chemistry interactions and potential environmental implications. Science of the Total Environment. 400: