A green approach to phytomediated synthesis of silver nanoparticles using Sambucus nigra L. fruits extract and their antioxidant activity

    A green approach to phytomediated synthesis of silver nanoparticles using Sambucus nigra L. fruits extract and their antioxidant activity Bianca Moldovan, Luminit¸a David, Marcela Achim, Simona Clichici, Gabriela Adriana Filip PII: DOI: Reference:

S0167-7322(15)30701-7 doi: 10.1016/j.molliq.2016.06.003 MOLLIQ 5905

To appear in:

Journal of Molecular Liquids

Received date: Accepted date:

30 September 2015 2 June 2016

Please cite this article as: Bianca Moldovan, Luminit¸a David, Marcela Achim, Simona Clichici, Gabriela Adriana Filip, A green approach to phytomediated synthesis of silver nanoparticles using Sambucus nigra L. fruits extract and their antioxidant activity, Journal of Molecular Liquids (2016), doi: 10.1016/j.molliq.2016.06.003

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ACCEPTED MANUSCRIPT A green approach to phytomediated synthesis of silver nanoparticles using

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Sambucus nigra L. fruits extract and their antioxidant activity

Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University, Cluj-Napoca,

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Filip3

Romania

Department of Pharmaceutical Technology and Biopharmaceutics, “Iuliu Hatieganu” University

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Bianca Moldovan1, Luminiţa David1*, Marcela Achim2, Simona Clichici3, Gabriela Adriana

of Medicine and Pharmacy, Cluj Napoca, Romania

Physiology Department, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj Napoca,

Romania

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Corresponding author:

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L. David, “Babeş-Bolyai” University, 11 Arany Janos Street, 400028, Cluj-Napoca, Romania. E-mail: [email protected]

Abstract

A simple and eco-friendly method for the phytomediated synthesis of silver nanoparticles (AgNPs) using European black elderberry (Sambucus nigra) fruit extracts was developed. The obtained AgNPs were characterized. The UV-Vis absorbtion spectrum of the biologically reduced silver showed a surface plasmon peak at 407 nm, characteristic for silver colloidal solutions. The average size of the biosynthesized, spherical in shape, nanoparticles was 26 nm, as revealed by transmission electron microscopy (TEM) analysis. The crystalline structure of

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ACCEPTED MANUSCRIPT metallic silver was confirmed by X-ray diffraction (XRD) analysis. The antioxidant properties of the obtained biomaterial were assessed in vitro, using the 2,2’-azinobis-3-ethyl-benzthiazino-6-

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sulphonic acid (ABTS) radical cation decolorization test. The in vivo antioxidant activity of the

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AgNPs was investigated against carrageenan-induced oxidative stress in Wistar rats. The results suggest that Black elderberry fruit extract mediated synthesized AgNPs present promising

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antioxidant activity and hence have a great potential in the development of therapeutic agents

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against oxidative tissue injuries.

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Key words: Sambucus nigra, green synthesis, silver nanoparticles, antioxidant effect

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1. Introduction

Nanotechnology is one of the most active research field in modern materials science,

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nanoparticles of noble metals gaining lately raising popularity, especially due to their biocompatibility. They present various applications in medicine, pharmacology, food industry and water purification [1-3].

Chemical and physical routes have been widely used to prepare metal nanoparticles. The physical approach involves several methods such as evaporation-condensation or laser ablation while the most employed method is the chemical reduction of the metal ions, followed by subsequent formation of metal clusters. Radiation-chemical methods such as ultraviolet and microwave radiation, photochemical and sonoelectrochemical methods were also used [4]. Traditional synthesis methods of nanomaterials require considerable amount of energy and generate of large amounts of toxic chemicals or hazardous substances. That’s why there is an

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ACCEPTED MANUSCRIPT increasing world-wide concern in obtaining nanoparticles using clean, non-toxic, environmental friendly procedures which follow the “green chemistry” principles.

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The green synthesis of noble metal nanoparticles which involves nondeleterious solvents

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and naturally occurring reducing agents such as plant extracts or biological microorganisms such as bacteria and fungus, which convert metal salts into elemental metal, have rapidly gained

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importance. The biosynthesis of nanomaterials represents a connection between nanotechnology

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and biotechnology, searching the appropriate biomaterials for the synthesis of nanoparticles being intensively developed. A consistent number of researches report the extracellular

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biosynthesis of AgNPs mediated by phytocompounds which act both as reducing and stabilizing agents [5-8].

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Among the metal nanoparticles, colloidal silver is one of the most remarkable nanomaterial especially due to its biological properties. Numerous researches demonstrated the

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potent inhibitory effect against microorganisms, the free radical scavenging and antiinflammatory properties, the wound healing properties, antitumor, antiviral, antibacterial and anti-angiogenetic effects of AgNPs [9-13]. Furthermore, some recent researches demonstrated that colloidal silver exerts cytotoxic, pro-inflammatory and pro-apoptotic effects mediated via reactive oxygen species (ROS) generated in normal and tumor cell lines [14-16]. Overproduction of ROS can be involved in various diseases like Alzheimer, artherosclerosis, arthritis, diabetes, neurodegenerative diseases, cancer and aging process [17]. Recent studies have demonstrated that several types of innorganic nanoparticles can act as efficient antioxidants or free radical scavengers [18, 19] Therefore, functionalization of AgNPs with natural antioxidants could reduce ROS production and protect the cell proteins and lipids and thus counteract the adverse reactions.

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ACCEPTED MANUSCRIPT Black or common elder (Sambucus nigra) it’s a wide spread deciduous shrub or small tree that grows in most parts of Europe, North America, Asia and North Africa. Elderberry

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cultivars are planted for ornamental purposes but the flowers are used in infusions, giving a

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commonly consumed refreshing drink in Northern and South-Eastern Europe. The dark-purple berries have been seldom used for fresh consumption, mostly being processed to juices, jams and

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jellies or as source of dietary supplements [20]. Traditional medicine mentioned the use of this

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plant by many native people and herbalists. Bark, flowers and fruits extracts of Sambucus nigra are reported to be efficient in the treatment of colds, bronchitis, asthma, Influenza,

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gastrointestinal disorders, viral infections and fever [21-23]. The Black elderberry fruit extract is particularly rich in primary metabolites such as organic acids and sugars but also in secondary

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metabolites among which anthocyanins are present in high amounts. Consumption of anthocyanins has become a matter of great interest, anthocyanin rich fruits and juices such as

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grapes, blueberries or blackberries, gaining increasing popularity. Less consumed berries such as those from Sambucus and Viburnum species have received lately great attention due to their high anthocyanin content and potent antioxidant activity [24-26]. Several studies also revealed the presence of high amounts of polyphenolic compounds in various Sambucus nigra L. cultivars [27]. These compounds are well known free radical scavengers reducing damages generated by oxidative stress to the human body [28]. The potential health benefits of polyphenols are the focus of numerous studies. In the last years, various important biological activities, such as antioxidant, anti-inflammatory, antiobesity, anticarcinogenic and antimutagenic, immune-stimulating, antiallergic and antibacterial properties were reported [29, 30]. The antioxidant and anti-inflammatory effects are closely related because

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ACCEPTED MANUSCRIPT cytokines and pro-inflammatory enzymes released in inflammatory conditions may increase ROS generation in non-phagocytic cells which in turn amplify the inflammation.

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The high content of anthocyanins, polyphenols and other antioxidant compounds

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encouraged us to choose Sambucus nigra fruits extract as reducing and capping agent for the green synthesis of metallic nanoparticles [3, 31]. Herein, we elaborated an environmental

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friendly method for the phytosynthesis of AgNPs based on the reduction of Ag+ ions by using

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biologically active compounds from Elderberry fruits extract [31]. The aim of the present study was to develop a new synthesis method of the silver colloid in order to obtain smaller size

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nanoparticles with improved biological properties. To this end, we modified the already published synthesis method by changing the reduction reaction conditions such as temperature,

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pH and extract: silver nitrate ratio. We also investigated, for the first time, the in vitro and in vivo antioxidant capacity of synthesized AgNPs in order to establish their possible biological

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applications.

2. Materials & Methods

2.1. Vegetal material and chemicals Samples of Sambucus nigra L. fruits were harvested in September 2014 from ClujNapoca, Romania. Fruits were packed in polyethylene bags and kept frozen at -18°C before being subjected to the extraction procedure. Silver nitrate, NaOH and EDTA-Na2 were obtained from Merck (Germany). All other chemicals and reagents were purchased from Sigma-Aldrich (Germany) and were of high grade purity. 2. 2. Preparation of the fruit extract Three grams of frozen fruits were crushed in a mortar and quantitatively transferred to an

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ACCEPTED MANUSCRIPT Erlenmeyer flask. 100 ml of double distilled water were added. The mixture was stirred for 1 h at room temperature and then filtered through Whatman no. 1 paper under vacuum. The extract was

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stored at 4°C for further use.

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2.3. Synthesis of AgNPs

The synthesis of AgNPs was achieved by reducing AgNO3 with Sambucus nigra L. fruits

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extract. 30 ml aqueous fruit extract were mixed with 70 ml of an aqueous solution of 1mM silver

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nitrate (AgNO3). The pH of the reaction mixture was adjusted to 8 by adding 0.1 N NaOH solution. Silver ion reduction occurred rapidly, as indicated by a brown color which appeared

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after 10 min. The nanoparticles were purified by centrifugation at 10.000 rpm for 20 minutes, washed with double distilled water and stored at 4°C until used.

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2. 4. Characterization of the AgNPs

The phytoreduction of silver ions was monitored by recording the UV–Vis spectrum with

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a Perkin Elmer Lambda 25 UV–Vis spectrophotometer. Measurements were performed at room temperature, in quartz cells, using silver nitrate (1 mM) as a blank, between 200 and 700 nm with a resolution of 1 nm. The morphological and topographical analysis of nanoparticles were performed by transmission electron microscopy (TEM) using an H-7650 120 kV Automatic TEM Hitachi, Japan. The infrared spectra of the fruits extract and of the green synthesized AgNPs were recorded on a Bruker Vector 22 FT-IR spectrometer from 4000 to 600 cm-1. Crystalline metallic silver was examined by X-ray diffraction (XRD). The XRD pattern was recorded using a D8 Advance diffractometer with CuKα radiation monochromatic filter. The diffracted intensities were recorded from 30 to 80° 2Ɵ angles. The X-ray photoelectron spectroscopy (XPS) spectra were recorded using a XPS spectrometer SPECS operated at 200 W Al Kα radiation. The XPS survey spectra were recorded at 30 eV and 0.5 eV/step. The high-

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ACCEPTED MANUSCRIPT resolution spectra for the individual elements (Fe, C, O, N, S, Si,Zn, Ba) were recorded at 30 eV and 0.1 eV/step. CasaXPS software with a Gaussian-Lorentzian product function was used to

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analyse and deconvolute the XPS peaks. The hydrodynamic diameters and Zeta potential of the

spectrometer (Malvern Instruments Ltd.; Malvern, UK).

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biosynthesized AgNPs were measured using a Zetasizer Nanoseries compact scattering

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2.5. In vitro evaluation of antioxidant activity using the ABTS assay: Trolox equivalent

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antioxidant capacity (TEAC)

The free radical scavenging ability was determined using the ABTS method of Arnao

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with some modifications [26]. 360 mg of ABTS were dissolved in 100 mL distilled water. The ABTS radical cation was prepared by mixing equal quantities of ABTS solution and 2.45 mM of

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potassium persulfate solution. The resulting solution was allowed to react in the dark for 24 h. A

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portion of the ABTS·+ solution (20 mL) was diluted with distilled water to an absorbance

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between 0.6 – 0.8, which was measured at 734 nm using a spectrophotometer. Fresh ABTS·+ solution was prepared for each assay. To 6 mL of diluted ABTS·+ solution, 0.1 mL fruit extract or colloidal silver solution were added and allowed to react in the dark for 15 minutes. The decrease of the absorbance was monitored at 734 nm. Trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid), a water soluble analog of Vitamin E was used as standard. The antioxidant activity was expressed in M Trolox equivalents using a calibration curve of the standard (0-400 M Trolox). 2.6. In vivo assessment of the antioxidant activity 2.6.1. Model of carrageenan-induced oxidative stress in Wistar rats Thirty two 110-130 g male Wistar rats obtained from the Animal Facility of "Iuliu Hatieganu" University of Medicine and Pharmacy, Cluj-Napoca, Romania were randomly

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ACCEPTED MANUSCRIPT divided into 4 groups of 8 animals each. Inflammation was induced by the injection of 100 μl carrageenan 1%, in the right hind food pad while the left hind foot pad was injected with the

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same volume of saline solution [32]. Prior to carrageenan injection, rats from group 1 were pre-

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treated daily with 15 mg/b.w. fruit extract, group 2 received orally 0.3 mg/b.w AgNPs suspension, for 4 days. A negative control, group 3, was treated with 0.5 ml 0.9% NaCl solution

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and a positive control group 4 was treated with Indomethacin (5 mg/b.w. in 0.5 ml

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carboxymethylcelulose 1.5%). At 2, 24 and 48 h after carrageenan injection, venous blood samples were collected and used to evaluate the oxidative/anti-oxidative status. Thereafter,

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animals were sacrificed and soft paw tissues were collected and used for oxidative stress quantification. All procedures for in vivo experiments were approved by the Ethics Committee

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on Animal Welfare of the "Iuliu Hatieganu" University, in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific

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Purposes, Council of Europe No 123, Strasbourg 1985. 2.6.2. Preparation of the samples

The soft tissues were washed with cold saline and homogenized with a Polytron homogenizer (Brinkman Kinematica, Switzerland) in 50 mM TRIS–10 mM EDTA buffer (pH 7.4). The homogenates were centrifuged (4000 rotation/min for 5 min) and the supernatants were collected. The protein content in homogenates was determined using the Bradford method [33]. Blood was centrifuged at 1000 g for 5 min to separate the plasma. Plasma and soft tissue homogenates from each group of animals were stored in aliquots at –80°C until used. 2.6.3. Oxidant/antioxidant status assessment

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ACCEPTED MANUSCRIPT 2.6.3.1. Malondialdehyde (MDA) was determined in plasma and soft tissue homogenate by measuring of thiobarbituric reactive species using the method described by Conti slightly

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modified [34]. MDA values were expressed as nmoles/mg protein.

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2.6.3.2. Reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured fluorimetrically using o-phtalaldehyde. The concentrations for GSH and GSSG were determined

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using standard curves [35, 36].

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2.6.3.3. Glutathione peroxidase activity (GPx) was determined by Flohe and Gunzler method, slightly modified [37]. GPx activity was expressed as µmoles of NADP

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produced/min/mg protein.

2.6.3.4. Catalase activity (CAT) in homogenates was measured using hydrogen peroxide

activity/mg protein [38].

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2.7. Statistical analysis

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in 50 mM potassium phosphate buffer, pH 7.4. The results were expressed as units of catalase

Data are reported as mean values of at least three experiments. Results were analyzed using one-way variance analysis (ANOVA). Analysis of variance was performed using XLSTAT Release 10 (Addinsoft, Paris, France). For comparison of multiple groups’ in the in vivo studies one-way ANOVA with Tukey post test was used. Differences at p<0.05 were considered statistically significant. 3. Results and discussion The Sambucus nigra fruits extract is a rich source of polyphenols and anthocyanins, efficient antioxidant compounds. The phytochemical composition of these fruits suggested that they can be exploited as reducing agents for the biosynthesis of silver nanoparticles and as efficient stabilizers of silver colloids.

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ACCEPTED MANUSCRIPT The present study investigated the antioxidant properties of the phytosynthesized AgNPs using in vitro and in vivo assays.

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3.1. Synthesis and characterization of AgNPs

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The bioreduction of silver ions from aqueous silver nitrate solution was achieved by adding Elderberry fruits extract. The pink-purple color of the water extract changed immediately

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to brown color indicating the formation of AgNPs (Fig. 1a).

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UV-Vis spectroscopy is one of the most applied analytical techniques used to determine the formation and stability of AgNPs. The measured UV-Vis spectrum of the biosynthesized

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AgNPs is presented in Fig. 1b. It was observed that the silver surface plasmon resonance (SPR)

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band appeared at 407 nm, confirming the reduction of silver ions to colloidal silver.

Figure 1. Color change of AgNO3 solution (A) by addition of Sambucus nigra fruits extract (B) by the synthesis of AgNPs (C) (a); UV-Vis spectrum of AgNPs (b) The morphology and particle size was determined by transmission electron microscopy. The TEM image (Fig. 2) revealed the obtaining of polydisperse silver nanoparticles with spherical shape. The particles were found to have different sizes ranging from 8 to 33 nm in diameter. The average particle size as obtained from TEM analysis was found to be 26 nm. The TEM images clearly revealed the presence of a faint thin layer on the surface of AgNPs prepared using the fruits extract of Sambucus nigra, which may be due to the organic molecules from the

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extract who also act as capping and stabilizing agents of the silver nanoparticles.

Figure 2 TEM image of AgNPs

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Black elderberry fruits contain high amounts of anthocyanins, especially cyanidin-3sambubioside and cyanidin-3-glucoside, and are particularly rich in flavonoids especially

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quercetin-3-rutinoside [20]. These polyphenolic derivatives can act as potential reducing agents

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by donating free electrons. The FT-IR spectrum of the water extract of Sambucus nigra fruits

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showed absorptions at 3388 (OH), 2930 (CH), 1726 (C=O), 1625 (C=C) cm-1 (Fig. 3).

Figure 3 FTIR spectrum of Sambucus nigra fruits extract and AgNPs The stretching vibration for OH of phenol groups appears at 3388 cm-1 [31]. The peak at 1726 cm-1 may be attributed to the vibrations of the carbonyl group [39] while the band at 1652 cm-1

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ACCEPTED MANUSCRIPT corresponds to the C=C vibrations [40]. The C-O stretching vibration of the phenolic groups appears as a peak at 1251 cm-1 [41]. All these data clearly indicate the presence of flavonoid

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derivatives in the European black elderberry fruit extract. The hydroxyl and carbonyl groups of

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the mentioned bioactive molecules can reduce the silver ions to metallic silver. The FT-IR spectrum of the phytosynthesized AgNPs showed absorption peaks at 3430, 2916, 1702, 1652,

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1407 and 1283 cm-1. By comparing the FTIR spectra of the extract and AgNPs, a shift in the

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absorption peak of the hydroxyl and carbonyl groups was observed, proving that the major biomolecules from the extract were capped on the AgNPs surface, showing their characteristic

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peaks in the IR spectrum of the colloidal silver solution.

The crystal structure of the biosynthesized AgNPs was determined by using X-ray

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diffraction. Fig. 4 shows the XRD pattern of the silver nanoparticles synthesized by Sambucus

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nigra fruits extract. Four major diffraction peaks at 2Ɵ values of 38.1°, 44.3°, 64.5° and 77.3°

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corresponding to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of face centered cubic silver were observed. The XRD profile also revealed the presence of some insignificant additional peaks which appeared at 2Ɵ values of 32.2°, 55.6° and 57.5° which might correspond to the bioorganic compounds from the Sambucus nigra fruit extract fact that was also observed by other authors [39].

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ACCEPTED MANUSCRIPT Figure 4 X-ray diffraction patterns of synthesized AgNPs The average crystallite size was calculated from the XRD pattern according to Scherrer

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equation:

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in which D is the average diameter of the crystallites, K is a shape factor,

is the radiation

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wavelength, θ is the Bragg angle and β1/2 is the width of the XRD peak at half height. The calculated average size of the silver nanoparticles was found to be 25 nm in fairly good

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consistency with the particle size observed in the TEM analysis. The XPS analysis of the green synthesized silver nanoparticles is depicted in Fig. 5. The

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high-resolution XPS spectrum (Fig. 5a) shows the binding energies of Ag 3d5/2 and Ag 3d3/2 at

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368.5 eV and 374.5 eV, respectively, with a difference of 6 eV characteristic to metallic silver

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[42]. The full XPS survey spectrum (Fig. 5b) was recorded to investigate the composition and the oxidation state of the obtained AgNPs. It indicates the presence of Ag, C and O in the sample. The Ag 3d peak observed at a binding energy of 371 eV clearly indicates the presence of metallic silver (Ag0). The C 1s and O 1s peaks stem from the adsorbtion of the biomolecules from the fruit extract on the surface of silver nanoparticles.

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Figure 5 XPS analysis of green synthesized AgNPs: Ag 3d (a); survey scan (b) The stability of the colloidal silver nanoparticles solution is dependent of the coating

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agent, being controlled by sterical and electrostatic repulsive forces [43]. The Zeta potential

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analysis of the phytosynthesized AgNPs showed a sharp peak at a negative value of -20.9 mV.

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The negatively charged surface of the nanoparticles indicates that anionic capping agents such as polyphenols from the Sambucus nigra fruits extract are coordinated to the surface of silver nanoparticles metallic shells. The determined Zeta potential value is commonly associated to sufficient mutual repulsion to ensure the stability of the colloidal silver dispersion [44]. 3.2. Antioxidant activity of silver nanoparticles The evaluation of the antioxidant behavior of phytosynthesized silver nanoparticles is useful to establish their potential applications in the therapy of many diseases caused by inflammation and oxidative stress. The antioxidant activity of the greenly synthesized silver nanoparticles was evaluated both by in vivo and in vitro methods. The in vitro method used to determine the antioxidant activity was the TEAC assay, which measures the ability of an antioxidant to reduce the ABTS·+ to ABTS, relating this ability to an antioxidant standard solution such as Trolox. The determined value of the antioxidant

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ACCEPTED MANUSCRIPT capacity of the silver nanoparticles was 177.09 µM Trolox, higher compared to the antioxidant activity of the aqueous Sambucus nigra fruits extract used for the synthesis of the AgNPs which

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was found to be 118.44 µM Trolox. These results are in agreement with previously reported data

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concerning the antioxidant activities of silver nanoparticles [19, 45].

Free radicals are highly reactive species which play an effective role in the pathogenesis

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of acute inflammation. Generally, in the inflammatory response numerous cytokines and pro-

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inflammatory enzymes such as cyclooxygenase - COX-2 and inducible nitric oxide synthase iNOS are released, inducing cellular metabolic stress and even tissue necrosis [46]. The pro-

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inflammatory cytokines may also increase ROS generation in non-phagocytic cells and produce reactive nitrogen species (RNS) by iNOS activation [47]. In order to investigate the in vivo

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protective role of the AgNPs on the oxidative stress generated by carrageenan-induced inflammation in Wistar rats, the oxidative stress parameters: MDA as a marker of lipid

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peroxidation and GSH/GSSG, GPx and CAT activities as markers of antioxidant defense, were assessed and compared to the reference non-steroidal anti-inflammatory drug (NSAID) Indomethacin. It has been reported that carrageenan produces inflammation as a consequence of free radicals generation and this effect was suppressed by administration of polyphenols [48]. The biological impact of AgNPs administration upon oxidative stress was evaluated in dynamic, comparatively in the paw tissue and in blood. The preadministration of Sambucus nigra fruits extract diminished the MDA levels (68% inhibition; p<0.05) at comparable levels with Indomethacin (69%; p<0.05) (Fig. 6a). The antioxidant status, evaluated by GSH/GSSG ratio in tissue, showed (Fig. 6b) an increase of GSH/ GSSG ratio after treatment with aqueous Sambucus nigra fruits extract only at 48h after carrageenan injection (3.79 fold compared to the control group; p<0.01). GPx activity in soft plantar tissue at 2h after carrageenan injection did

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ACCEPTED MANUSCRIPT not show significant variation (Fig. 6c). A notable variation at 24h in the enzymatic activity of GPx in rats treated with fruits extract (64% inhibition; p<0.05) and AgNPs (65% inhibition;

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p<0.05) was observed, compared to the control group. In contrast, the treatment with AgNPs

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resulted in a significant increase of the GPx and CAT activities after 48h (2.26 respectively 2.21 fold compared to control group; p<0.01) proving that the antioxidant effects of AgNPs were

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maintained up to 48h after induction of oxidative stress. CAT activity was also increased by

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extract at 48h (1.36 fold; p<0.05) (Fig. 6d).

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Figure 6 MDA (a), GSH/GSSG levels (b) and antioxidant enzymes (c and d) activities in soft tissue of paw at 2h, 24h and 48h after carrageenan injection in animals pretreated with AgNPs and natural extracts. Values are means±SD (n=8). Statistical analysis was done by a one-way ANOVA, with Tukey’s multiple comparisons posttest (*p<0.05; **p<0.001).

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ACCEPTED MANUSCRIPT A different pattern regarding these parameters was observed in serum (Fig. 7). The phytosynthesized AgNPs presented a protective effect on serum oxidative lipid peroxidation

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(36% inhibition; p<0.05) without influencing the antioxidant defense.

Figure 7 MDA (a) and GSH/GSSG (b) levels in serum at 2h, 24h and 48h after carrageenan injection in animals pretreated with AgNPs and natural extracts. Values are means±SD (n=8). Statistical analysis was done by a one-way ANOVA, with Tukey’s multiple comparisons posttest (*p<0.05; **p<0.001). The higher and more persistent antioxidant activities of AgNPs compared to the fruits extract might be due to enhanced permeability and retention effect of the nanoparticles in the edema region and also to AgNPs uptake by the lymphatic system.

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ACCEPTED MANUSCRIPT AgNPs presented promising in vivo effects, decreasing the MDA level at 48h after induction of oxidative stress, in parallel increasing the catalase and glutathione peroxidase

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activities.

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The results of the present study are consistent with previously reported data [49] which demonstrated that epicatechin or tea extract functionalized silver nanoparticles increase the

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mitochondrial function and up-regulate the superoxide dismutase, an antioxidant enzyme which

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converts superoxide anion into hydrogen peroxide. Suriyakalaa and co-workers [50] showed that Andrographis paniculata mediated synthesized AgNPs possess stronger in vitro radical

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scavenging properties compared to the leaf extract.

The efficient free radical scavenging properties of the synthesized silver nanoparticles

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could be due to the combined effect of both AgNPs as well as of the bioactive compounds present in the Sambucus nigra fruits extract. The results of this investigation suggest that the

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green synthesized AgNPs using Sambucus nigra fruits extract could be considered as alternative antioxidant compounds useful to generate new pharmaceutical products. 4. Conclusions

A novel eco-friendly, rapid and low cost method for the extracellular synthesis of silver nanoparticles using the polyphenols rich Sambucus nigra fruits extract was developed. The bioactive molecules from the extract acted both as reducing and capping agents, avoiding the use of any toxic chemical. The synthesized AgNPs were characterized by UV-Vis spectroscopy, FTIR, XRD, XPS techniques and TEM. The obtained nanoparticles presented almost spherical shape, their sizes ranging between 8 and 33 nm, were well separated from each other and no aggregation occurred.

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ACCEPTED MANUSCRIPT The antioxidant activity of AgNPs was investigated both in vitro (TEAC method) and in vivo (by assessing the oxidative stress parameters). The obtained data demonstrate that

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administration of silver nanoparticles suppress the free radical generation which occurs during

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the carrageenan induced inflammation in Wistar rats.

Based on these findings the current method can be suitable for the industrial scale

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production of biologically active silver nanoparticles. Due to their promising antioxidant activity,

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the obtained AgNPs have a great potential in the preparation of pharmaceutical products which

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can be used against various degenerative diseases caused by free radicals.

Acknowledgments

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This work was supported by the Ministry of Education and Scientific Research, Romania

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ACCEPTED MANUSCRIPT 36. D. Olteanu, A. Nagy, M. Dudea, A. Filip, A. Muresan, C. Catoi, P.A. Mircea, S. Clichici, Hepatic and systemic effects of rosuvastatin on an experimental model of bile duct ligation in

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ACCEPTED MANUSCRIPT 43. L. Xu, G. Han, J. Hu, Y. He, J. Pan, Y. Li, J. Xiang, Hydrophobic coating- and surface active solvent-mediated self-assembly of charged gold and silver nanoparticles at water-air and water-

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ACCEPTED MANUSCRIPT 50. U. Suriyakalaa, J.J. Antony, S. Suganya, D. Siva, R. Sukirtha, S. Kamalakkannan, P.B.T. Pichiah, S. Achiraman, Hepatocurative activity of biosynthesized silver nanoparticles fabricated

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using Andrographis paniculata, Colloids Surf. B. 102 (2013) 189-194.

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Graphical abstract

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Highlights:  Silver nanoparticles were synthesized using Sambucus nigra fruit extract.  The nanoparticles were characterized by UV-VIS, FTIR, TEM, XPS and XRD analysis.  Spherical silver nanoparticles with average size of about 26 nm were obtained.  The obtained AgNPs exhibited promising in vitro and in vivo antioxidant activity.

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