Impaginato 93 Adv. Hort. Sci., 2020 34(1): 93­104 DOI: 10.13128/ahsc­8405 The toxicity potential of Ag nanoparti­ cles synthesized from Cordia myxa L. Z. Akbarnejad­Samani 1, M. Shamili 1 (*), F. Samari (2) 1 Horticulture Department, Faculty of Agriculture, University of Hormozgan, Iran. 2 Department of Chemistry, Faculty of Science, University of Hormozgan, Iran. Kew words: antioxidant capacity, chemical AgNPs, germination value, green AgNPs, lipid peroxidation, phenol, protein. Abstract: Plant­mediated nanoparticles synthesis is considered as one of the appealing options in bio­nanoparticles synthesis, however, there is contradicto­ ry information about the positive or negative impacts of nanoparticles on plants. Investigating the toxic effects of Ag NPs on model plants, such as onion, can reveal the probability of damage. Thus, the present study was conducted to compare the germination indices and biochemical parameters of edible onion seeds treated with different concentrations of two types of silver nanoparticles (green synthesized and chemical synthesized) to examine the oxidative stress. Based on our results, the interaction between leaf extract and silver salt result­ ed in a color change from pale yellow to dark brown, the first sign of the AgNPs formation. The green AgNPs treatment improved the onion germination indices. The green AgNPs­exposed seeds displayed a no­significant reduction in protein content and same protease activity as the control treatment, but chem­ ical AgNPs­treated displayed a significant rise of protease and reduction in pro­ tein content in concentration more than 0.06 gL­1. In chemical AgNPs­treated seeds both peroxidase and catalase displayed an ascending linear trend and the most activities belonged to chemical AgNPs at 0.05 g L­1 (315.62 and 51.45 μmol min­1g­1 FW, respectively). Both nanoparticle types made an increase in MDA, but green AgNPs did not significantly differ with control treatment. It can be concluded that the green synthesis of nanoparticles is a safe and suitable alter­ native for chemical­synthesized metal nanoparticles. 1. Introduction The production, manipulation, and use of nanoparticles (NPs), due to their distinctive and definite capabilities, has become one of the most attractive research topic in different area of science, including biology, chemistry, engineering, medicine, physics, agriculture and food. Nano­ dimension structures involved in many aspects of plant biology such as ionic transport and molecular transmissions. The diameter of plant cell wall pores are in the range of 5 to 20 nm. Furthermore, plasmodesmata, the intracellular channels to facilitate molecular transitions, are also nano­scale (50 to 60 nm in diameter) (Zambryski, 2004). (*) Corresponding author: shamili@ut.ac.ir Citation: AKBARNEJAD­SAMANI Z., SHAMILI M., SAMARI F., 2020 ­ The toxicity potential of Ag nanoparticles synthesized from Cordia myxa L.. ­ Adv. Hort. Sci., 34(1): 93­104. Copyright: © 2020 Akbarnejad­Samani Z., Shamili M., Samari F. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 15 October 2019 Accepted for publication 8 January 2020 AHS Advances in Horticultural Science http://https://www.google.com/search?q=plant+plasmodesmata&tbm=isch&source=hp&sa=X&ved=2ahUKEwiXnpql9cPiAhUGjqQKHVYZCDYQsAR6BAgIEAE http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2020 34(1): 93­104 94 The nanotechnologies based­products are expect­ ed to upturn (Maynard et al., 2006; Rejeski and Lekas, 2008). Nanotechnology applications in agricul­ tural sector involve in precision farming, smart feed­ ing, enrichment of food quality, products packaging, products labeling, nano­pesticide, nano­fertilizer, and nano­herbicide (Thiruvengadam et al., 2018). The dif­ ferent chemical and physical methods are applied to the synthesis of nanoparticles such as sol­gel, elec­ trochemical, hydrothermal, sono­chemical, and microwaves techniques. Chemical methods had low efficiency, need high temperature, high pressure and high energies during the reaction process. Besides they need the expensive reagent and complex equip­ ment. Furthermore, chemical methods require some non­degradable chemical components, as reducing or stabilizing agents, which finally caused environ­ mental pollution (Senapati et al., 2012). The potential of biological materials to synthesize metal nanoparticles has provided a low­cost and eco­ friendly route. Biological synthesis or green synthesis of nanoparticles, uses biological material as the reducing and capping agents (Veerasamy et al., 2011). The green synthesis uses microorganisms such as fungus, bacteria (Ahmad et al., 2005), yeast and actinomycetes (Kowshik et al., 2003) or macro­organ­ isms e.g., plants and algae, as intermediate agents (Niemeyer and Mirkin, 2004; Dubey et al., 2010; Prasad et al., 2012). Plant­mediated nanoparticles synthesis is consid­ ered as one of the appealing options in bio­nanopar­ ticles synthesis, due to high variety and abundance, no need for a complicated growth conditions, bulk­ biomass production, no need for special nutrient media, cost­effectiveness, richness of various effec­ tive metabolites, simple single­step synthesis process and suitable for the large­scale synthesis of nanopar­ ticles. Polyphenolic contents of the plant act as reducing agents and eventually act as coating and stabilizing agent of nanoparticles (Dubey et al., 2010). Cordia myxa (L.) or sapistan­tree, a member of Boraginaceae family, is native to tropical areas of Asia. It well documented that C. myxa contained flavonoids, glycosides, sterols, terpenoids, saponins, phenolic acids, alkaloids, tannins, coumarins, resins ; and mucilage (Inas et al., 2011; Rashed et al., 2014): a suitable candidate for green NPs synthesis. However, there is contradictory information about the positive or negative impacts of nanoparti­ cles on plants. Titanium dioxide nanoparticles increased the dry and fresh weight of spinach by increasing the light absorption, boosting rubisco enzyme activity (Lin and Xing, 2007) andrising the nitrogen metabolism (Yang et al., 2007). Zinc and copper nanoparticles showed significant differences in growth, and toxicity symptoms in treated plants (Lee at al., 2008; Monica and Cremonini, 2009; Musante and White, 2012; Prasad et al., 2012). Titanium nanodioxide (40 μg ml­1) improved seed germination, dry and fresh weight of onion seedling; however, the higher concentrations (50 μg ml­1 and more) had a reverse result (Raskar and Laware, 2013). Conversely, this nanoparticle did not affect the length and quantity of the onion root (Klancnik et al., 2011). The symptoms have depended on the form and coating type of nanoparticles (Barrena et al., 2009). Due to the growing rate of production and release of nanoparticles in nature (as Nano­fertilizers, Nano­ toxins, and Nano­carriers), there are increasing con­ cerns about the possibility of toxicity and oxidative damage (whether chemical or green synthesis) of these particles on the ecosystem. The WHO (World Health Organization) and FAO (The Food and Agriculture Organization) at the meeting on the application of nanotechnologies in the food and agri­ culture sectors, in Rome in 2010, recognized the potential concern of nanotechnology in agriculture and food sectors. The main concern was about the exponential developing global knowledge on nano­ technology and its applications, which may affect the balance of advantage to risk (FAO/WHO expert meet­ ing report, 2010). The Ag NPs is known to have antioxidant, antibac­ terial and antifungal properties and may be used in food, medicine and cosmetics products (El­Nour et al., 2010; Abdel­Aziz et al., 2014). So, investigating the toxic effects of these particles on model plants, such as onion, can reveal the probability of damage. Thus, the present study was conducted to compare the germination indices and biochemical parameters of edible onion seeds who treated with different con­ centrations of two types of silver nanoparticles (green synthesized and chemical synthesized) to examine the oxidative stress. 2. Materials and Methods Research site and plant material collection The current study was conducted at the Horticultural Laboratory of Agriculture and Natural Resources Faculty, Chemical Laboratory of Science Akbarnejad‐Samani et al. ‐ nanoparticles impact on Cordia myxia L. 95 Faculty, Central Laboratory (University of Hormozgan, Bandar Abbas, Iran) and Molecular Research Center (Hormozgan University of Medical Sciences, Iran), during 2018. Preparation of Cordia myxa leaf extract Leaves of Cordia myxa L. (a member of Boraginaceae family), were collected from Rooydar city, Hormozgan Province, Iran (57° 6΄ E, 60° 3΄ N, Elevation: 50 m, RH: 70%, Mean temperature: 28±1°C) during winter, 2018. The healthy leaves were choosing, then washing in tap water followed by rins­ ing in distilled water and air drying (6 days under the shade conditions at 24±1°C). The dried leaves were powdered using an electric grinder. The leaf extract was prepared by mixing 10 g of dried powder along with 150 mL of deionized water and then heating the mixture at 80°C for 30 min. The solution was pre­fil­ tered through Whatman No. 42 filter paper and re­fil­ tered through Whatman No. 1 filter paper. The obtained extract was collected and stored in the refrigerator (Samari et al., 2018). Synthesis and purification of silver nanoparticles (AgNPs) using C. myxa leaf extract For AgNPs synthesis, 2.0 ml of C. mixa leaf extract was added to 25 ml of 7.0 mM aqueous solution of AgNO3 (Merck, Germany) and the reaction pH was adjusted to 11.0 using NaOH. The mixture was con­ tinuously stirred at room temperature (24±1°C) for 3 h. The yellow­mixture turned to dark­brown (Samari et al., 2018). The green synthesized AgNPs were cen­ trifuged at 10000 rpm for 30 min and rinsed with deionized water. Then, the obtained sediments were re­dispersed in deionized water to get rid of any free phyto­molecules. This process (centrifugation and re­ dispersion) was repeated three times to ensure the purification of nanoparticles and separation of unbonded compounds. Characterization of silver nanoparticles Preliminary characterization of the green synthe­ sized AgNPs was carried out using a S­3100 UV­Vis spectrophotometer (Scinco, Korea, which is operated at a resolution of 2 nm and is equipped with a 10 mm quartz cuvette). An aliquot of the dried purified pellets, obtained from centrifugation, was used for X­ray diffraction (XRD). The XRD pattern was obtained using a powder X­ray diffractometer (Bruker D8 Advance powder dif­ fractometer) with Cu­Kα radiations (λ=1.5406 nm, in a 2θ range from 20° to 80°). Fine configuration of the green synthesized AgNPs was measured using a transmission electron microscopic examination (TEM). For this, the colloidal AgNPs were allowed for sonication and then a thin film was prepared on the carbon coated grid (Cu Mesh 300). TEM observations were made with (Zeiss­EM10C) operated at an accel­ erating voltage of 80 kV. Preparation of colloidal solution of AgNPs (green and chemical synthesized) The chemical AgNPs wasprovided from Iranian Nano­biotechnology company. Chemical and green nanoparticles were dispersed separately in distilled water and placed in an ultrasonic apparatus (SONI­ CA® Ultrasonic Cleaners) for 30 min for homogeniza­ tion. Then, colloidal solutions containing each nanoparticle were prepared at the concentrations of 0, 0.03, 0.06, 0.012, 0.025 and 0.05 g L­1. For germination assay, red onion seeds (Allium cepa L.), disinfected (0.04% of sodium hypochlorite solution, 3 min), rinsed in distilled water (3 times, each for 1 min), dipped in Nano­solutions for 30 min and finally rinsed in distilled water. Seeds were then cultured in sterilized glass Petri dishes (80 mm in diameter, covered with filter papers). Each Petri dish contained 100 seeds. Germination indices Germination percentage and germination value. Germination test was conducted under a laboratory condition (24±1°C). The seeds were placed on 80 mm petri dishes and were covered with filter papers. The seeds were irrigated daily with double distilled water. Counting was done by the emerge of the roots (the seed with root length equal or more than seed diam­ eter were assumed as germinated). The counting continued until 3 days fixed germination. The germi­ nation indices including germination percentage and germination value were calculated using the equa­ tions (1) and (2) (Okoro, 1976). Germination (%)= The total number of germinated seeds / total seed) x 100 (1) GV = MDG x PV (2) where GV is germination value and MDG is mean daily germination and calculated by the dividing of germination percentage by the number of days to the end of the test. PV or peak value, derived from all the cumulative germination percentages on any day divided by the number of days to reach these per­ centages. The length of radicle and plumule. Seedling root and stem length was measured using a ruler (express in mm). Adv. Hort. Sci., 2020 34(1): 93­104 96 Biochemical analysis Preparation of enzyme and protein extract. About 0.5 g of the root was homogenized in liquid nitrogen. Then, 1.0 ml of extraction buffer (in 100 ml: contain­ ing of 50 mM potassium phosphate buffer with pH: 7.0, 0.0372 g EDTA and 1.0 g PVP) was added and centrifuged (15 min, 12000 rpm, 4°C). At last, the supernatant was used to assay protein content, and the activity of catalase, peroxidase, and protease (Dhindsa et al., 1981). Protein content assay. The Bradford method (1976) was used to determine protein content. Briefly, 1.0 ml of Bradford reaction solution (contain­ ing 0.01 of Coomassie Brilliant Blue G250, 5.0 ml of 96% ethanol and 10.0 ml of 85% phosphoric acid) was added to 50 μl of protein extract. The optical absorption of the extracts was determined by a spec­ trophotometer (Cecil CE2501 model) at 595 nm with a plastic cuvette (Bradford, 1976). Peroxidase activity assay. After adding 33 μl of the enzyme extract to 1.0 ml of the peroxidase reaction solution (containing 13 mM guaiacol, 5 mM hydrogen peroxide and 50 mM phosphate potassium buffer at pH=7.0), the absorption of the extracts was recorded at 470 nm (Chance and Maehly, 1995). Catalase activity assay. For catalase assay 50 μl of the enzyme extract was mixed with 1.0 ml catalase reaction solution (containing 50 mM phosphate potassium buffer at pH=7.0 and 15 mM hydrogen peroxide). The absorption was recorded at 240 nm (Dhindsa et al., 1981). Protease activity assay. After adding 350 μl of 50 mM sodium phosphate buffer (pH=7.5) and 800 μl of 1% casein (W/V) to 50 μl of the enzyme extract, the mixture was incubated under laboratory condition (24±1°C) for 10 min. Then, 400 μl of 10% trichloroacetic acid (w/v) was added and the mixture re­incubated for 20 more min. Finally, after centrifu­ gation (10000 rpm, 5 min), the optical absorption of extracts was determined at 280 nm with a quartz cuvette (Kwmbhavi et al., 1993). Malondialdehyde assay (MDA). About 0.1 g of the root was homogenized with 5.0 ml of 1% trichloroacetic acid. The extract was then centrifuged (10000 rpm, 5 min) and the supernatant (250 μl) was placed in a water bath (95°C for 30 min), after adding one ml of MDA (containing 20% trichloroacetic acid and 5% thiobarbituric acid). The samples were placed on ice and then re­centrifuged (1000 rpm, 5 min). Finally, the optical absorption of the extracts was recorded at 532 and 600 nm (Alexieva et al., 2001). Total phenol assay. The total phenol was assayed using the Spanos and Wrolstad (1990) technique; about 0.1 g of root sample was homogenized with 15.0 ml of 80% methanol. The extract was then centrifuged (10000 rpm, 10 min). Then, 490 μl of distilled water and 500 μl of Folin reagent were added to 10 μl of the supernatant and were placed under dark condition (24±1°C, for 3 min). Subsequently, 500 μl of 1% sodium carbonate (1.0 g sodium carbonate ­ 100 ml distilled water) was added to each sample and re­placed under dark condition (24±1°C) for 30 more min. Finally, the absorbance of extracts was determined at 765 nm. Antioxidant capacity (DPPH assay). The DPPH assay was followed by Brand­Williams et al. (1995) procedure. Briefly, about 0.1 g of root sample was homogenized with 15.0 ml of 80% methanol and incubated under dark laboratory condition (24±1°C, for 24 h). Then, the extracts were centrifuged (10000 rpm, 10 min). After adding 40 μl of methanol, 350 μl of DPPH and 1550 μl of 80% methanol to 600 μl of the supernatant, the samples were kept under the dark condition at 4°C, for 20 min. Finally, the optical absorption was recorded at 517 nm and the antioxi­ dant capacity was calculated using the equation (3): Antioxidant capacity%= [(Acont ­ Asamp)/Acont)] x 100 (3) where Acont and Asamp are the standard and sample optical absorption. Data analysis The research was conducted as a factorial experi­ ment based on complete random design, with six replications (each replicon contains 100 seed). The statistical analysis was done using SAS Version 9.1.3 (SAS Institute Inc. Cary, NC, USA, 1990). The factors were AgNPs type (green AgNPs and chemical AgNPs) and AgNPs levels (0, 0.03, 0.06, 0.012, 0.025 and 0.05 g L­1). The Shapiro­Wilks test confirmed the data nor­ mality (procedure: Proc Univariate, SAS). The Multivariate Analysis of variance, considering the AgNPs type and AgNPs levels as independent vari­ ables were done (procedure: Proc Glm, SAS). Pillai’s trace test confirmed the variance homogeneity (pro­ cedure: Proc Glm, SAS). Tukey’s test was performed for mean comparison (procedure: Files, Sedit, Factor, Range, P<0.01, MSTATC). Excel 2013 was used to draw the figures. Akbarnejad‐Samani et al. ‐ nanoparticles impact on Cordia myxia L. 97 The green AgNPs treatment improved the onion ger­ mination. Most germination percentage belonged to green AgNPs (100% in 0.05 g L­1), and the least value 3. Results Synthesis and characterization of C. myxa synthesized AgNPs The interaction between leaf extract and silver salt resulted in a color change from pale yellow to dark brown, the first sign of the AgNPs formation. This process occurred under room temperature (24±1°C) and lasted 3 hours (Fig. 1). UV­Vis Spectroscopy is one of the most extensive techniques to confirm the production of nanoparti­ cles. The UV­Vis spectrum of synthesized AgNPs using C. myxa leaf extract (Fig. 2A) showed a significant peak with λmaxaround 410 nm due to SPR (Surface Plasmon Resonance) of AgNPs. TEM images were used to detect the surface mor­ phology and size distribution of synthesized AgNPs by C. myxa leaf extract. TEM micrographs of the AgNPs confirmed the spherical shape of particles, 3­10 nmsize, well­distributed and little aggregation in solu­ tion (Fig. 2B); however, the average size of particles was found to be 5.8 nm. The XRD patterns of synthesized AgNPs showed prominent Bragg reflections at 2θ values of 38.1, 44.25, 64.55 and 77.2 (Fig. 3A), which is related to the (111), (200), (220) and (311) Bragg reflections of face­centered cubic (FCC) AgNPs. SEM analysis con­ firmed the nano dimension of chemical AgNPs (Fig. 3B). Germination indices Germination percentage and germination value. The germination percentage was influenced by the type and concentration of nanoparticles (Table 1). Fig. 2 ­ The SPR spectrum of C. myxa synthesized with the AgNPs (A), and TEM image of AgNPs synthetized by C. myxa leaf extract (B). Fig. 1 ­ The mixture of C. myxa leaf extract and AgNO3 (Left), green­synthetized AgNPs colloidal solution (Right). Fig. 3 ­ The XRD patterns of the C. myxa synthesized AgNPs (A) and electron microscopic scanning image of chemical AgNPs (B). 98 Adv. Hort. Sci., 2020 34(1): 93­104 (70%) belonged to chemical AgNPs treatment at con­ centrations more than 0.06 g L­1 (Fig. 4A). The germination value also influenced by the interaction of treatments (Table 1). Green AgNPs treatment did not differ with control, while treat­ ment with the chemical AgNPs caused a significant reduction in the germination value (from 1.88 in con­ trolreached to 0.12 in the 0.05 g L­1). Most and least germination values (1.90 and 0.12) were observed in onion seeds treated with the high concentration of green AgNPs and chemical AgNPs, respectively (Fig. 4B). The length of radicle and plumule. The effect of type and concentration of AgNPs was significant on the length of plumule (Fig. 5). There was no signifi­ cant difference between the green AgNPs and the control. Even, the chemical AgNPs was not different from the control up to the concentration of 0.06 g L­1. The higher amount of chemical AgNPs, reduced the Fig. 4 ­ The influence of AgNPs type and concentration on germi­ nation percentage (A) and germination value (B) in Allium cepa. Means ± SD of six replicates. The same letter denotes lack of a statistically significant difference (Tukey, p<0.01). Fig. 5 ­ The influence of AgNPs type and concentration on plu­ mule length (A) and radicle length (B) in Allium cepa. Means ± SD of six replicates. The same letter denotes lack of a statistically significant difference (Tukey, p<0.01). Table 1 ­ The interaction of AgNPs type and concentration on Allium cepa biochemical parameters (z) The mean square values are given. * and **: state significant at 5 and 1% respectively AgNPs type ×AgNPs concentration: states the interaction of AgNPs type and AgNPs concentration SOV Germination percentage Germination value Radicle length Plumule length Protein Peroxidase Catalase Protease MDA Phenol IC50 NPs type 4408.33 ** (z) 89.89 ** 284.21 ** 926.64 ** 0.11 540853.35 ** 3600.65 ** 9151.33 ** 0.112 * 886.31 ** 14.57 NPs concentration 3.80 0.07 9.98 ** 5.11 ** 0.27 * 40965.25 ** 13476.80 ** 1.40 * 0.030 ** 17.84 34.52 * NPs type × NPs concentration 15.53* 0.31 ** 4.90 ** 1.70 0.15* 38719.77 ** 7566.10 ** 1.87 * 0.020 ** 17.89 ** 21.90 ** Error 13.10 0.26 1.17 1.92 0.16 5852.71 683.03 1.14 0.020 55.16 24.61 Akbarnejad‐Samani et al. ‐ nanoparticles impact on Cordia myxia L. 99 plumule length (from 7.40 mm in the control treat­ ment reached to 3.28 mm in the 0.05 g L­1 of chemi­ cal AgNPs) (P≤0.01) (Fig. 5A). The radicle length had a different pattern in response to the type and concen­ tration of the nanoparticles. Green AgNPs improved radicle length (from 4.53 mm in control treatment reached to 7.76 mm in high concentration) (P<0.01). The chemical AgNPs made a decreasing trend in this parameter and reached it to 2.22 mm in the concen­ tration of 0.05 g L­1 (Fig. 5B). Biochemical indices Protein content and protease activity. Despite the significant effect of two AgNPs types on protein con­ tent and protease activity (Table 1, P≤0.01), the inter­ action between AgNPs type and concentration had a diverse pattern (Fig. 6A and B). The green AgNPs­ exposed seeds displayed a no­significant reduction in protein content (from 1.1 to 1 mg g­1). The reduction of protein content in chemical AgNPs­treated seeds was dose­dependent (Fig. 6A). Green AgNPs­treated seeds had the same protease activity as the control treatment. Chemical AgNPs displayed a significant rise in concentration more than 0.06 gL­1 (from 1.77 μmol min­1 g­1 FW in control treatment to 6.21 μmol min­1 g­1 FW at the high concentration) (Fig. 6B). Antioxidant enzymes (peroxidase and catalase activities). By the results, the activity of both antioxi­ dant enzymes was influenced by the type and con­ centration of the AgNPs (Table 1). Green AgNPs­ treated seeds indicated no significant difference with control. In chemical AgNPs­treated seeds both enzymes displayed the linear trend, so most peroxi­ dase and catalase activities belonged to chemical AgNPs at 0.05 g L­1 (315.62 and 51.45 μmol min­1g­1 FW, respectively) (Fig. 7A and B). Malondialdehyde (MDA), phenol and DPPH (antioxidant capacity). The MDA influenced by AgNPs treatment (Table 1) and the less value was observed in control treatment (0.03 mg g­1 Fw). Both nanopar­ ticle types made an increase in MDA, but green AgNPs did not significantly differ with control treat­ ment. Also, the content of MDA had a linear relation Fig. 7 ­ The influence of AgNPs type and concentration on activi­ ties of peroxidase (A) and catalase (B) in Allium cepa. Means ± SD of six replicates. The same letter denotes lack of a statistically significant difference (Tukey, p<0.01). Fig. 6 ­ The influence of AgNPs type and concentration on pro­ tein content (A) and activity of protease (B) in Allium cepa. Means ± SD of six replicates. The same letter deno­ tes lack of a statistically significant difference (Tukey, p<0.01). Adv. Hort. Sci., 2020 34(1): 93­104 100 with chemical AgNPs concentration (from 0.03 mg g­1 Fw in the control reached to 0.25 mg g­1 FW at high concentration of chemical AgNPs) (Fig. 8A). The type and concentration of AgNPs influenced phenol content. The most value was assigned to chemical AgNPs (15.16 mg Gallic g­1 FW at 0.05 g L­1), and the least value to control treatment (4.10 mg Gallic g­1 FW) (Fig. 8B). The amount of antioxidant capacity was also affected by NPs treatments (Table 1). This parameter was expressed in mg g­1 fresh weight based on the­ half­maximal inhibitory concentration (IC50). The con­ trol value (10.67 mg g­1 FW) followed a descending trend in both AgNPs types. It reached to 8.97 mg g­1 FW in green AgNPs while to 1.01 in chemical AgNPs (Fig. 9). 4. Discussion and Conclusions Plants contain flavonoids, phenols, aroma, latex and alcohols, and some of these compounds are responsible for the reduction of metal ions and pro­ duction of metal NPs from the metal salts. It has been stated that the leaf extract of Cordia species possesses phenolic and flavonoids derived com­ pounds such as robinin, rutin, datiscoside, hes­ peridin, dihydrorobinetin, chlorogenic and caffeic acid (Al­Ati, 2011). The hydroxyl and ketonic groups of such compounds, construct chelate structures by binding to metal ions (Issaabadi et al., 2016). In pre­ sent work, the leaf extract of C. myxa was used as a reducing and stabilizing agent for phyto­synthesis of AgNPs. This eco­friendly procedure was free­hazard and non­toxic. The successful synthesis of green AgNPs from plant extracts has already reported in Chenopodium album, Camellia sinensis and Rhus cori‐ aria (Song and Kim, 2009; Dwivedi and Gopol, 2010). The NPs synthesis basis is oxidation of hydroxyl, carbonyl, and aldehyde and subsequently, reduction of metal ions during the neutralization of the electric charge (Sivaraman et al., 2009). In present research the C. myxa extract turned to dark brown, due to the surface plasmon resonance of AgNPs and confirmed the successful synthesis of the AgNPs (Kasthuri et al., 2009; Roopan et al., 2013). In this study, the synthe­ sis process lasted 3 hours at room temperature con­ dition. Other reports have also mentioned that this rapid NPs synthesis dose not require high tempera­ tures (Sivaraman et al., 2009). The FCC crystal struc­ ture of green synthesized AgNPs was found by com­ paring the obtained XRD data with JCPDS File No. 04­ 0783 (Sutradhar and Saha, 2016), clearly indicate the highly crystalline structure of green AgNPs. Nanoparticles play an important role in many areas of biology, chemistry and agriculture (Navarro et al., 2008), but their adverse potential on the envi­ ronment is being subjected to extreme discussions. NPs penetrate to various parts of the plants, some stored within the cell and some in extracellular space (Lee et al., 2008). High AgNPs concentrations pass through the cell by diffusion, cause mitochondrial Fig. 9 ­ The influence of AgNPs type and concentration on antioxidant capacity in Allium cepa. Means ± SD of six replicates. The same letter denotes lack of a statistically significant difference (Tukey, p<0.01). Fig. 8 ­ The influence of AgNPs type and concentration on MDA (A) and total phenol (B) in Allium cepa. Means ± SD of six replicates. The same letter denotes lack of a statistically significant difference (Tukey, p<0.01). Akbarnejad‐Samani et al. ‐ nanoparticles impact on Cordia myxia L. 101 mal­function, generation of ROS (He et al., 2012; Roh et al., 2012). On the other hand, easy penetration of nanoparticles into the seed shell develops water absorption and increases the activity of rubisco enzyme, which finally simplifies germination (Gao et al., 2006). In the present study, Cordia myxa AgNPs increased onion germination percentage and value. The improvement of seed germination has reported in nano­metal treated spinach (Gao et al., 2006), maize (Lin and Xing, 2007) and peanut (Prasad et al., 2012). The AgNPs have to penetrate plant cell walls, the natural sieves, and roots plasma membranes to enter the xylem tissues and then dislocated to stems and finally leaves (Dietz and Herth, 2011). In fact, the roots are the first target for lethal materials (Sresty and Rao, 1999). There are disagreeing reports regard­ ing the effects of nanoparticles on the plant root and system. The NPs treatment made a decrease in radish root length (Wang et al., 2015), but an increase in the shoot and root length of rice (Hao et al., 2016). Also, ZnO NPs caused root elongation in radish, lettuce, corn, and cucumber (Lin and Xing, 2007). ZnO NPs (500 mg L­1) increased soybean’s root length, while higher concentrations resulted in a sig­ nificant reduction (Lopez­Moreno et al., 2010). The root cap cells of AgNPs­exposed Lolium multiflorum were damaged and malformed, which lastly reduced root growth and dry matter (Yin et al., 2011). Plant root inter­connect with physical and chemi­ cal factors of the root zone. The elongation zone of onion root may act as a sensitive receiver for external signals. The length and morphology of onion roots is an important parameter that reflects the toxicity of the chemical compounds (Odeigah et al., 1997). It has reported that AgNPs lessened root length of onion whichrelatedto a reduction in water absorp­ tion and cell division (Kumari et al., 2009). In this study, an inhibition in radicle and plumule develop­ ment in chemical AgNPs­treated seeds indicate its toxicity potential. Moreover, the root length reduced more than the shoot length in AgNPs­treated sam­ ples. The toxicity of AgNPs in biological systems is closely related to its surface oxidation, releasing the Ag ions, and interaction with macromolecules (Reidy et al., 2013), especially with sulfur­containing mole­ cules e.g. proteins, due to silver­ sulfur strong ten­ dency (Liu et al., 2011). The nano­dimension plant pores, the surface charge of NPs (more negative AgNPs limit the cell­particle interactions and made lower toxicity) and coating type (chemical or biologi­ cal) influence the intensity of AgNPs’ toxicity (Choi and Hu, 2008; El Badawy et al., 2011). In the present study, protein content and pro­ tease activity of green AgNPs treated seeds did not significantly differ from the control. However, the high concentration of chemical AgNPs obviously decreased protein content and boosted protease activity. This alignment related to the role of pro­ tease in protein catabolism. High concentrations of AgNPs denature the membrane and release LPS (lipopolysaccharide) and purines. Then limit the pro­ ton mobility, react with thiol groups of some enzymes, deactivate enzymes, bind to protein groups, denature proteins, produce hydrogen perox­ ide, which finally causes oxidative stress (Hwang et al., 2008; Zhu et al., 2008). The regulated production of free radicals in organ­ isms maintains the oxidation and reduction home­ ostasis cycle; however, there is a group of antioxi­ dant enzymes who prevent and deactivate ROS. Also, extracellular antioxidant molecules, such as ascor­ bate, scavenge free radical molecules (Shams et al., 2011). Increasing the activity of antioxidant enzymes, catalase and peroxidase, inplant’s exposed to the chemical metal NPs treated plants (Krishnaraj et al., 2012; Singh et al., 2013; Wang et al., 2015; Cvjetko et al., 2017) confirms our results. Nanoparticles interact with the cell membrane (Khan et al., 2011), then depending on their nature and concentration, reduce the destructive effects of oxidative stress, cause programmed cell death (Lei et al., 2008). Silver nanoparticles could damage cell divi­ sion, cause chromatin bridge, disturbed metaphase, make multiple chromosomal breaks and final cell dis­ integration (Kumari et al., 2009). According to Burman et al. (2013), zinc oxide NPs linearly increased ROS and MDA content. The MDA content of tomato and tobacco plants who exposed to AgNPs was higher than control plants (Cvjetko et al., 2017). Also, an increase in phenol content has already been reported in metal NPs treated plants (Singh et al., 2013). IC50 is a measure of the potency of a substance in inhibiting the biological function and indicates how much of this substance is required to inhibit the bio­ logical process. In our research, the chemical AgNPshad IC50around 8 times more than green AgNPs, which confirms the more toxicity risk of chemical AgNPs. The same trend was observed in green AgNPs synthesized from Aegle marmelos extract (Patil et al., 2015). The results of the present work displayed that green AgNPs significantly enhanced antioxidant abilities by stimulating polyphenols and ascorbic acid in onion. http://https://en.wikipedia.org/wiki/Potency_(pharmacology) Adv. Hort. Sci., 2020 34(1): 93­104 102 Globally, incredible changes in agricultural pro­ duction patterns have taken place, through the appli­ cation of modern labor­saving technologies, mecha­ nization, and improved crop varieties. In sustainable agriculture, the application of nano­fertilizers is a tal­ ented option to provide the food needed for the growing population worldwide. Green synthesis of nanoparticle as an environmental­friendly technique, by minimizing and reducing hazardous material, gradually has introduced itself in the commercial pro­ duction of nanoparticles. The present research used an eco­friendly and low­cost methodology, without the use of any danger or lethal chemicals for AgNPs synthesis from leaf extract of C. myxa under room temperature conditions. The phenolic and flavonoid contents of leaf extract acted as reducing and stabi­ lizing agent in nanoparticles synthesis and AgNPs with a good quantity and stability were synthesized. Various bioassays are available to assess the rela­ tive toxicity of chemicals in different organisms. However, there is no single test that can detect the damage of classes of chemical compounds. A germi­ nation test is a sensitive tool used in physiological and cytogenetic studies. According to our findings, the green nanoparticle synthesis, not only had no oxidative effect on onion germination parameters, but also some stimulant effect was observed. The chemical nanoparticle motivated the plant’s defense responses, by inducing oxidative stress and limited germination in a dose­dependent manner. As a final conclusion, it can be noted that the low­cost and easy synthesis of nanoparticles from plant sources, is a safe and suitable alternative for chemical­synthe­ sized metal nanoparticles. Acknowledgements The authors thank the Head of Research and Technology Center (University of Hormozgan, Iran) and Dr. Ebrahim Eftekhar, the head of Molecular Research Center (Hormozgan University of Medical Sciences, Iran), for technical support. 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