Innate catalytic and free radical scavenging activities of silver nanoparticles synthesized using Dillenia indica bark extract

Accepted Manuscript Innate catalytic and free radical scavenging activities of silver nanoparticles synthesized using Dillenia indica bark extract Alfa S. Mohanty, Bhabani S. Jena PII: DOI: Reference:

S0021-9797(17)30217-5 http://dx.doi.org/10.1016/j.jcis.2017.02.045 YJCIS 22072

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

22 December 2016 27 January 2017 19 February 2017

Please cite this article as: A.S. Mohanty, B.S. Jena, Innate catalytic and free radical scavenging activities of silver nanoparticles synthesized using Dillenia indica bark extract, Journal of Colloid and Interface Science (2017), doi: http://dx.doi.org/10.1016/j.jcis.2017.02.045

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Innate catalytic and free radical scavenging activities of silver nanoparticles synthesized using Dillenia indica bark extract Alfa S. Mohanty a,b and a,b Bhabani S. Jena* a

CSIR- Institute of Minerals and Materials Technology, Bhubaneswar- 751 013, Odisha, India,

b

Academy of Scientific and Innovative Research, New Delhi

Abstract A green approach was envisaged for the rapid synthesis of stable silver nanoparticles in an aqueous medium using phenolic rich ethanolic bark extract from D. indica with marked free radical scavenging and reducing ability. Biosynthesis of silver nanoparticles (AgNPs) was confirmed and characterized by using UV-Visible spectroscopy, particle size analyzer, X-Ray diffractometry (XRD), Transmission Electron Microscopy (TEM) and Fourier Transform Infrared Spectroscopy (FT-IR). Bio-reduction of Ag+ was confirmed with the appearance of golden yellow coloration within 5-10 minutes at 45°C with maximum absorbance at 421 nm. XRD analysis of AgNPs indicated the crystalline nature of metallic Ag. As analyzed by TEM, AgNPs were found to be spherical in shape, well dispersed and size varied from 15-35 nm and dynamic light scaterring (DLS) studies showed the average particle size of 29 nm with polydispersity index (PDI) of 0.280. Synthesized AgNPs were showing surface functionalization as revealed through FTIR studies. These AgNPs were observed to be highly stable at room temperature (28±2°C) for more than 3 months, thereby indicating the ethanolic extract of D. indica was a reducing as well as a capping agent for stabilization of AgNPs. Moreover, these green synthesized AgNPs showed enhanced free radical scavenging and excellent catalytic activities when used in the reduction of 4nitrophenol and methylene blue dye, at room temperature.

Keywords: Dillenia indica; silver nanoparticles; free radical scavenging activity; catalytic activity; 4nitrophenol; methylene blue.

*Corresponding author Bhabani S. Jena R&D Planning and Environment Sustainability Department CSIR-Institute of Minerals and Materials Technology, Bhubaneswar- 751 013, Odisha, India, Email address: [email protected], [email protected] Page | 1

1. Introduction Silver nanoparticles have established a phenomenal impact in the field of nano science due to their applications in a diverse range of emerging fields such as catalysis [1], electronics [2], photonics [3], optics [4], metallic inks [5], plasmonics [6], medicine [7], electrochemical sensor [8] and surface enhanced raman scattering (SERS) substrate [9]. Silver nanostructures have been also exploited for their potential therapeutic applications such as anti-microbial [10], anti-angiogenic, anti-infective, antitumorogenesis and radiotherapy [11].The applicability of silver nanoparticles has been diversified due to their shape variation, size distribution, and morphology. The conventional way of synthesis includes chemical based reduction and stabilization of silver ions [12]. But, nanoparticles synthesized through biological means attracted a huge fame due to their minimal toxicity with reduced side effects in comparison to conventional nanoparticles. In addition to this, usage of a variety of plant extracts for green synthesis of silver nanoparticles has drawn the attention of researchers over chemical, physical and microbial based protocols due to a simple, wellorganised, environmentally favorable and cost effective process. Plants such as Aloe vera, Tinospora cordifolia, Mangifera indica, Lantana camara, Withania somnifera, Cocus nucifera, Boerhaavia diffusa, Azadirachta indica, Nelumbo nucifera, Piper betel, Vitis vinifera have been already reported for their significant reducing and stabilizing capability [13]. AgNP synthesis using plant extracts is the mostaccepted and eco-friendly method [14]. Dillenia indica L. (Dilleniaceae) is found in the moist and evergreen forests of the subHimalayan tract, from Kumaon and Garhwal eastwards to Assam, Bengal, and Orissa. It is an evergreen tree, and common names like Chulta (Bengali, Hindi), Ou (Oriya), Bhavya (Sanskrit) and Elephant apple (English) [15]. It is a spreading tree and has large white fragrant flowers with five petals, toothed margin and pointed leaves, and large globose fruits with small brown seeds. The tree flowers during May-August and fruits ripen during September-February and the fruits are edible. Various extracts from different plant parts of D. indica have been reported to have antidiabetic and hypolipidemic [16], antioxidant [17, 18], anti-leukemic [19], anti-inflammatory [20] and anti-diarrhoeal activities [21]. The crude methanolic extract from the stem of D. indica have shown antimicrobial, antioxidant and cytotoxic activities [22]. A variety of secondary metabolites has been reported in D. Indica [22, 23, and 24]. A cursory survey of the literature, showed that aqueous fruit extract of D. indica have been used for the green synthesis of silver nanoparticles [25]. The catalytic activities of silver nanoparticles are quite fascinating now a day’s [26, 27]. Nitro aromatic compounds like 2-nitrophenol, 4-nitrophenol and 2, 4-dinitrophenols are the priority pollutants. In Page | 2

particular, 4-nitrophenol/p-nitrophenol exposure causes many health problems in humans like drowsiness, headaches, cyanosis and nausea through acute inhalation or ingestion due to its cyto and embryonic toxicity, carcinogenic and mutagenic activities [28]. An important method to achieve this is the photoreduction of nitrophenols to aminophenols. Aminophenols are the major constituents in some biologically active compounds, medicines, and dyes. Among the various methods, reduction reactions using NaBH4 have been known to accelerate within the presence of appropriate catalysts [29]. On the other hand methylene blue is a highly toxic dye causes skin irritation, vomiting, nausea, gastrointestinal tract irritation, diarrhea in humans. This dye is mostly used by textile industries to colorize the products [30]. Techniques applied for dye removal and reduction has several major drawbacks such as the high energy consumptions and the high cost and insufficient degradation. In this context, there is a requirement of an easy, cost effective and environment- friendly method to be adopted for reduction and/or degradation of 4-nitrophenol and methylene blue. The catalytic activities of green synthesized nanoparticles towards degradation of nitrophenol and azo dyes have been reported earlier [28, 31, and 32]. In the present study, we have reported the synthesis of stable silver nanoparticles using an ethanolic extract from the bark of D. indica as reducing and as well as a stabilizing agent. Silver nanoparticles were characterized by UV-Visible spectroscopy,zeta sizer, X-Ray diffractometry (XRD), Transmission Electron Microscopy (TEM), and Fourier Transform Infrared Spectroscopy (FT-IR). Free radical scavenging activity synthesized silver nanoparticles was assayed through 2, 2-diphenyl-1picrylhydrazyl (DPPH) method. The catalytic activities of biosynthesized silver nanoparticles were studied for the reduction of 4-nitrophenol and methylene blue dye.

2. Materials and Methods

2.1. Plant material and chemicals The barks of Dillenia indica were obtained from Nalagaja, Mayurbhanj, Odisha, India. Gallic acid and butylated hydroxyanisole (BHA) were obtained from Sigma-Aldrich, USA. Sodium carbonate, ferric chloride, silver nitrate, potassium hydroxide, sodium borohydride, 4-nitrophenol, methylene blue, potassium ferricyanide & α,α-diphenyl-β-picrylhydrazyl (DPPH) were procured from Himedia, Mumbai; ascorbic acid and Folin’s Reagent from Merck, Mumbai and trichloroacetic acid from Merck, Germany . All other solvents and chemicals used were obtained from Merck, Mumbai, India.

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2.2. Preparation of the D. indica bark extract

Barks of D. indica were dried under sunlight and powdered (about 60 mesh size) in a mixer grinder. Initially, 250g of bark powder was soaked with 2 liters of hexane for 24 hrs and filtered. The residual bark powder was dried under shade and immersed in 1.25 liters of 90% ethyl alcohol for 72 hrs at room temperature (28±2 °C) in a conical flask. To obtain particle free extract, the content was filtered through Whatman No. 41 filter paper. The extraction process was repeated twice with the residue. Filtrates were pooled and concentrated by using flash evaporator and dried under vacuum at 45°C and the dried extract was used for the present study.

2.3. Determination of total phenolics

Determination of total phenolic content of the D. indica bark extract was done by the method of Singleton and Rossi [33]. The extracts were dissolved in a mixture of methanol and water (6:4 v/v). 0.1 ml of samples (1mg/ml) were made up to 10ml in test tubes with distilled water and subsequently mixed with 0.5 ml of dilute Folin-Ciocalteu reagents (1:1 with distilled water) and 1ml of 7.5% sodium carbonate solution was added to each tube and mixed well with a vortex mixture. After 30 minutes kept at room temperature, the optical density was measured at 765 nm against reagent blank in a UV-Visible spectrophotometer (Eppendorf, Hamburg, Germany). From the standard curve of gallic acid, total phenolic content of the extract was calculated as gallic acid equivalents. The determination of phenolic content in the extract was carried out in ten replicates and the results were averaged.

2.4. Free radical scavenging activity

The free radical scavenging activity of the ethanolic bark extract was determined by 2, 2diphenyl-1-picrylhydrazyl (DPPH) method essentially as described by Blois [34]. Different concentrations of D. indica bark extract and BHA (20, 40, 60, 80 and 100 ppm) were taken in separate test tubes. The volume of the content of each tube was adjusted to 0.1ml with methanol. To these tubes, 5.0 ml of 0.1 mM methanolic solution of DPPH was added and mixed well. Tubes were allowed to stand at room temperature (28±2 °C) for 20 min. A control tube was prepared as above without extract simultaneously. The absorbances of the samples were measured at 517 nm against methanol blank. The following formula was used to calculate the free radical scavenging activity as the percent inhibition: % Free radical scavenging activity = [(Control OD – sample OD)/ Control OD]× 100 Page | 4

Tests were performed as ten replicates and averaged. 2.5. Determination of reducing power

The reducing power of extract from the barks of D. indica was determined by a method as described by Oyaizu [35]. The extract was dissolved in methanol:water (6:4 v/v) and different concentrations(25,50,100 µg) of samples were taken. The volume was adjusted to 5.0 ml by adding phosphate buffer (0.2 M, pH 6.6) and followed by 2.5 ml of 1% potassium ferricyanide and incubated at 50oC for 20 min. At the end of the incubation, 2.5 ml of 10% trichloroacetic acid was added to the mixtures and allowed to centrifuge for 10 min at 5000 rpm. 2.5 ml of the upper layer was mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% ferric chloride, and the absorbance was measured at 700 nm in a spectrophotometer. Tests were performed as ten replicates and averaged.

2.6. Silver nanoparticles synthesis

200µl of aqueous bark extract (2 mg/ml) was mixed with 50µl of silver nitrate solution (2.0 M) and the final volume was made up to 10 ml with distilled water. By adding 0.01M KOH, the mixture was made slightly alkaline. This mixture was stirred by a magnetic stirrer over a hot plate at 45o C with 150 rpm. The formation of golden yellow coloration within 5-10 minutes indicated the formation of colloidal silver nanoparticles as shown in Fig. S.1. (support information).

2.7. Characterization of synthesized nanoparticles

Synthesized silver nanoparticles solution was centrifuged at 15,000 rpm for 10 minutes and the supernatant was discarded and the pellet was dispersed in distilled water. This process was repeated for three times for removal of impurities and used for further studies. The UV-Visible spectral scanning of the synthesized silver nanoparticle colloidal solution was analyzed using quartz cells between wavelengths of 200 to 700 nm. Blanks of each sample set were prepared with deionised water. To find out the stability of synthesized nanoparticles with their size distribution, the zeta potential and

dynamic light scattering (DLS) measurements were performed by using Zetasizer

analyzer (Malvern Zetasizer, Nano-ZS90,UK) and particle size analyzer (Microtrac Inc., USA)

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respectively. 50µl of synthesized nanoparticles solution was dispersed with 2ml of distilled water and was used for both zeta potential measurement and analysis of size distribution. The synthesized AgNPs solution was mounted on a glass slide in a drop wise manner and allowed for evaporation of the solution over a hot plate. This was subjected to using X'pert PRO (Pan Analytica, Netherland) X-ray diffraction (XRD) unit using Ni -filtered Cu Ka (l¼1.54˚ A) radiation in scanning range between 30º to 90º. Surface morphology and chemical properties of the synthesized silver nanoparticles were examined

through

TEM/EDX

(transmission

electron

microscope/

Energy-dispersive

X-ray

spectroscopy). Synthesized silver nanoparticles were subjected to sonication (Model 3000, ultrasonic homogenizer, Biologics, Virginia, USA) for proper dispersion (for 15 min at 50 Hz) and a drop of purified synthesized silver nanoparticles solution was placed on the carbon-coated copper grids and kept under a lamp for drying. TEM measurements were performed on an FEB Tecnai G2 20 (Netherlands) instrument operated at an accelerating voltage of 200 kV. Surface functional groups of the nanoparticle were examined by Fourier transmission infra-red (FTIR) analysis. The dried silver nanoparticles were pelleted with KBr and kept in the vacuum oven in order to avoid moisture and were analyzed in an FTIR system (Perkin Elmer SPECTRUM GX, USA) using the spectral range of 600-4000 cm-1 with the resolution of 4 cm-1.

2.8. Free radical scavenging activity of synthesized AgNPs

The free radical scavenging activity of synthesized AgNPs, D. indica bark extract and AgNO3 solution (concentration used for the synthesis of AgNPs) was determined by DPPH method as described elsewhere. Briefly, 100µL of each of D. indica bark extract, AgNO3, and Ag NPs solutions were added to 5.0 mL methanolic solution of DPPH separately. Resulting mixtures were kept at room temperature for 20 min followed by optical density measurement at 517 nm against methanol blank. % Free Radical Scavenging Activity = [(control absorbance − sample absorbance)/control absorbance] × 100. All the estimations were performed in triplicates and averaged. 2.9. Catalytic degradation of 4-nitrophenol and methylene blue

Experiments for the reduction of 4-NP were carried out at room temperature (28±2 °C) in a standard quartz cell with a path length of 1 cm. To investigate the catalytic activity, 0.5 mL of 4Page | 6

nitrophenol aqueous solution (0.2 M) was mixed with 0.5 ml of freshly prepared NaBH4 solution (25 mM ) and 0.5 ml of AgNPs directly from the freshly synthesized solution was added. The reaction was carried out at room temperature with constant stirring. Control experiments for extract, AgNO3, NaBH4 and AgNPs were carried out in different test tubes. Immediately after that, the UV–vis absorption spectra were recorded with a time interval at a scanning range of 200–600 nm at room temperature. The catalytic activity of synthesized silver nanoparticles was also examined using methylene blue dye, in which 0.5 ml of AgNPs solution was mixed with 0.5 ml of dye. Control experiments were carried out for extract and AgNO3 containing the same amount of extract and AgNO3 present in AgNPs solution. Degradation of dye was monitored by scanning the absorbance with UV-vis spectrometer in the range of 500-800 nm up to 30 minutes. In all the reaction mixtures total volume was made up to 1 ml. 3. Results and Discussion

3.1. Total phenolics

In our previous work we have reported that aqueous acetone extract from the bark of D. indica contained a substantial amount of phenolics possessing potent antioxidant activity as assayed through various in vitro methods [18]. However, in the present study, the yield of ethanolic extract from the bark of D. indica was found to be 10.1% (w/w). Plant phenolics are normally soluble in polar organic solvents and alcohols are proven to be effective solvents for extraction of phenolics [36, 37]. For the present study, ethanol was preferred over other because of its lower toxicity [38].Total phenolic content of ethanolic extract from the bark of D. indica was found to be 40% as gallic acid equivalents.

3.2. Free radical scavenging activity

DPPH method was employed for the estimation of free radical scavenging activity of the ethanolic extract from the bark of D. indica and the results are presented in the Fig. S.2. DPPH is a free radical with deep violet in color and upon accepting a proton from any hydrogen donor; it loses its chromophore leading to yellow color. Thus, the degree of discoloration of DPPH indicated the efficacy of free radical scavenging property of sample antioxidant [39].Here free radical scavenging activity of the extract and BHA was found to increase with the increase in the concentration and free radical scavenging activity of the extract was very much similar to standard antioxidant BHA. At 20 ppm Page | 7

concentration, the free radical scavenging activity of BHA and extract was 89.92% and 89.42%, respectively.

3.3. Reducing power

Reducing power of D. indica bark extract was evaluated by ferric chloride reduction method. The essence of this assay is that ferric ions are reduced to ferrous ions and changes color from yellow to bluish green which is measured spectrophotometrically at 700 nm and increase of absorbance indicates an increase of reducing power of sample analyte. In the present study, the efficacy of reducing ability of D. indica bark extract was compared with standard reducing agent ascorbic acid. The absorbance of the reaction mixture was increased with increasing concentration of extract and ascorbic acid. At 100 ppm concentration of extract and ascorbic acid absorbance was 0.379 and 0.526 respectively (Fig.S.3). Reducing power can be employed as a notable reflection of the antioxidant activity. It appears that antioxidant activity of D. indica bark extract is attributed to its hydrogen donating ability [40]. Thus, the ethanolic extract from the barks of D. indica found to be a potent reducing agent and was used for the synthesis of silver nanoparticles in the present study.

3.4. Characterization of synthesized AgNPs

Synthesis of silver nanoparticles obtained by reduction of metal ions exposing D. indica bark extract as a reducing agent was monitored by a UV-visible spectrophotometer. Fig.S.4 illustrates the absorbance spectra of the reaction mixture containing the aqueous solution of silver nitrate of different concentration (0.5 M, 1.0 M, and 2.0 M) and 1mg/ml of D. indica bark extract. The combination of all reaction mixture in this study showed an absorbance peak at around 421 nm, which is the characteristic of silver nanoparticles, due to its surface plasmon resonance absorption band. It was observed that the peak intensity increased with an increase in the concentration of silver nitrate solution from 0.5 M to 1.0 M keeping extract concentration constant at 1mg/ml. However, the peak intensity was reduced in the combination of 2.0 M AgNO3 and 1mg/ml of extract thereby indicating an insufficient reduction of Ag+ which may be due to inadequate reducing agent in the mixture. However, the synthesis of silver nanoparticle was found to be optimum with the combination of 2.0 M silver nitrate solution and 2mg/ml of D. indica bark extract as revealed with higher optical density and area which corroborates with the results obtained by Cheng et al. [41] in the case of aminocellulose as reducing agent. Phytochemical analysis of D. indica have shown the presence of the triperpene, lupeol, betulinaldehyde, betulinic acid Page | 8

and botulin, flavonol, flavonoid along with other phenolic compounds, stigmasterol, and glycosides and sulphates of flavonoid [22-24, 42 and 43]. In this study, the synthesis of AgNPs indicated that active constituents from bark extracts were responsible for reduction of Ag+ ions. Further. In our earlier (18) and present study, it was found that the extract from the bark of D. indica possessed marked reducing ability.

This synthesized colloidal solution of silver nanoparticles was found to be stable at room temperature (28±2º C) for more than 3 months and exhibited a stable UV-vis peak at 430 nm (Fig. 1).

Fig. 1. UV-vis spectra after 1 day, 5 days, 10 days, 50 days and 100 days, of synthesized silver nanoparticle solution stored at room temperature. Zeta potential value of AgNPs showed that all the synthesized AgNPs possessed –ve zeta potential value and the corresponding average zeta potential value was -31.9 mV indicating the highly stable AgNPs (Fig.2.a). As the bark extract from D. indica was rich in phenolics, the –ve zeta potential may due to the capping of phenolic compounds which was in accordance with the results reported by Priti et al., 2016 [44] and Naveen et al, 2015 [45]. Further, the polydispersity index (PDI) of the synthesized nanoparticles was found to be 0.280 which indicated that synthesized AgNPs were well dispersed in water . DLS particle size distribution indicated an average particle size of 29 nm. (Fig.2.b)

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Fig.2. a) Zeta potential analysis and b) particle size distribution of AgNPs.

The structural properties of synthesized silver nanoparticles were confirmed with XRD study. XRD pattern of silver nanoparticles corresponded to the diffraction planes at (111), (200), (220) and (311) of face-centred cubic Ag (JCPDS 04-0783) (Fig. 3).

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Fig. 3. X-ray diffraction pattern of the synthesized silver nanoparticles.

The elemental profile of the synthesized silver nanostructures was analyzed using TEM with an EDX setup. TEM analysis at high resolution revealed the formation of prominently spherical AgNPs, with a size variation between 15 nm and 35 nm as shown in Fig. 4.a and b. Moreover, the synthesized silver nanoparticles were well dispersed. Selected area electron diffraction (SAED) pattern obtained from synthesized AgNPs showed the diffraction rings from inner to outer associated with the [111, 200, 220, 311] atomic planes of Ag also indicating (Figure 4.c )the formation of crystalline silver nanoparticles [46].

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Fig. 4. TEM images at a) 20 nm, b) 100 nm, c) SAED of synthesized silver nanoparticles and d) EDX spectrum of synthesized silver nanoparticles.

The EDX results in Fig. 4.d showed Ag, C, O and Cu peaks, which suggested the presence of silver nanoparticles. The Cu and C, O peaks corresponded to the copper grid used during TEM analysis and plant extract respectively. It appears that D. indica bark extract reduced the silver ions to silver nanoparticles.

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A strong peak observed at 1615 cm−1 in the D. indica bark extract might be due to the C = C stretching of the aromatic ring, which was shifted to 1607 cm−1 in case of AgNPs. The stretching at 1098 cm−1 and 1078 cm−1 for extract and AgNPs respectively indicated the presence of amine group. FTIR analysis in Fig. 5 revealed the presence of bands common to the bark extract of D. indica and AgNPs thereby suggesting the presence of phytochemicals on the surface of the AgNPs.

Fig. 5. FTIR spectra of D.indica bark extract and synthesized AgNPs. In the region of 3200 to 3600 cm -1, a broad band with small shoulders was observed in both cases which may be due to the intermolecular hydrogen-bonding of the OH group of D.indica bark extract on the nanoparticle surface [47, 48]. In our earlier (18) and present study it was observed that bark extract from D. indica contained substantial amount of phenolics which are rich with OH groups in their structures. The phytochemical constituents found in D. indica have carboxylic, alkanes, aromatic rings, hydroxyl and carbonyl groups in their structures [22-24, 42 and 43]. It appears that ethanolic extract from D. indica bark was responsible for the reduction of silver ion to AgNPs and also as the capping agent to prevent agglomeration for stability which may be due to the presence of hydroxyl, carbonyl and carboxylic groups in the phytochemicals present in the extract [49]. 3.5. Free radical scavenging activity of D. indica bark extract and synthesized AgNPs

Free radical scavenging activity of AgNO3, D. indica bark extract, AgNO3 and D. indica, bark extract (reaction mixture at zero time) and synthesized AgNPs was determined by DPPH method and was found in the order of synthesized AgNPs (37.58± 0.19 %) > AgNO3 and D. indica, bark extract (reaction mixture at zero time) (30.94 ± 0.18 %) > D. indica bark extract (28.76± 0. 30 %) > AgNO3 Page | 13

(8.47± 0.23 %).(Fig.6) Thus, the enhanced free radical scavenging activity of synthesized silver nanoparticles could be due to the synergistic effect of both AgNPs and the bioactive compounds of the D. indica bark extract present in the surface of nanoparticles as the capping agent. The elevation of free radical scavenging activity was reported for silver nanoparticles synthesized by using the extracts from Elephantopus scaber [46] and Coleus aromaticum [50].

Fig. 6. Free radical scavenging activity of AgNO3, extract, the reaction mixture (0 min) and the reaction mixture after synthesis of AgNPs. Values are mean ± SD of triplicates.

3.6. Evaluation of catalytic activities of synthesized AgNPs

3.6.1. Catalytic reduction of 4-nitrophenol

The catalytic activity of the Dillenia indica bark mediated Ag NPs in the reduction of 4-NP to 4AP in the presence of NaBH4 in water was evaluated. In the absence of the AgNPs, the reduction process was not observed. The conversion of 4-NP to 4-AP in aqueous medium was monitored by UVVis spectroscopy at room temperature and the results are shown in Fig. 7.a. A shift of the peak of 4-NP from 317 to 400 nm was observed immediately after addition of NaBH4 . This shift was may be due to alkaline conditions after addition of NaBH4, which caused the formation of 4-nitrophenolate ions [51, 52]. After the addition of the biosynthesized AgNPs into the reaction mixture, the reduction process was monitored by UV-Vis spectroscopy of the reaction solution. As observed in UV-Vis spectrum, the absorption peak at 400 nm was reduced in a time dependent manner in the presence of the biosynthesized catalyst and the absorption peak at 297 simultaneously appeared and gradually increased Page | 14

with time which was ascribed to the 4-AP product with increasing the reaction time [31, 32]. In addition, the yellow color of 4-nitrophenol solution vanished and ultimately became colorless (4-AP). Considering the above results, the reduction has occurred only in the presence of the catalyst and no reduction has happened in the absence of the catalyst i.e. biosynthesized AgNPs in the present study. (Fig.S.5.a.) As reported earlier, when the AgNPs was used as the catalyst for the reduction of 4-NP, AgNPs could facilitate electron transfer from BH4¯ ion to the 4-NP, leading to 4-AP production. [29, 53] In other words, faster electron transfer can occur in presence of catalyst, which gives rise to the faster reaction process.

Fig.7.UV Vis spectra showing catalytic activities of AgNPs a)reduction of 4-nitrophenol b) reduction of methylene blue

3.6.2. Catalytic reduction of methylene blue

Methylene Blue dye degradation was carried out in the presence of UV–Vis light with and without AgNPs. The degradation of dye without AgNPs exhibited a stable absorbance peak. Methylene Blue showed a main maximum peak at 664 nm was recognized to the azo bond of the dye. In presence of biosynthesized AgNPs with the increase of time, the degradation was observed to be more which was Page | 15

indicated by the decrease of absorbance without any shift in peak as monitored by UV–Vis spectrophotometer. Maximum of dye was degraded in the presence of AgNPs within 30 minutes (Fig 7. b) while, in the absence of AgNPs the absorption remains constant for extract and AgNO3[31,32] (Fig.S.5.b.). It appears that photocatalytic degradation of methylene blue may be due to the cleavage of azo bond th.reby discoloring methylene blue as indicated by the reduced height of dye peak with less absorbance[45].Catalytic activities of biosynthesized silver nanoparticles were reported for the degradation of 4-nitrophenol and methylene blue by using the extracts of Bunium persicum seed [31], Plumeria alba flower [32] and Parkia roxburghii leaf [54]. Moreover, the ethanolic extract of D. indica mediated synthesized AgNPs were found to possess more effective catalytic properties in relation to time when it was compared with other biomediated AgNps (Table 1).

Catalytic degradation

Nanoparticle size and shape

Time(min)

Reference

26nm,spherical

15

[50]

28nm,spherical

30

10 nm ,spherical

22

[53]

29nm, spherical

11

Present work

36.19 nm, spherical

120

[32]

29nm,spherical

30

Present work

of

4-nitrophenol

Methylene blue

Table.1. Comparison of catalytic efficiency of synthesized AgNPs with size and shape 4. CONCLUSION Ethanolic extract from the bark of D. indica was found to be rich in phenolic content and possessed marked free radical scavenging activity and reducing ability. We were successful to synthesize stable silver nanoparticles using the bark extract from D. Indica. Synthesized AgNPs were spherical in shape, well dispersed with PDI 0.280 and crystalline in nature with a size variation from 15 to 35 nm with average size 29 nm. These nanoparticles were coated with some of the functional groups from an extract of D. indica bark which provided stability and enhanced antioxidant activity of the Page | 16

AgNPs. These biosynthesized silver nanoparticles were proven to be an efficient catalyst for the degradation of 4-nitrophenol and methylene blue dye. On the other hand, this work showed many remarkable advantages, some of which are listed below: •

A facile, eco-friendly and a cost effective method was employed for synthesis silver nanoparticle using D.indica bark extract Which is a natural and inexpensive valuable resource and environmentally benign support.

•

Rapid synthesis (within five to ten minutes) of stable spherical silver nanoparticles (~15-35 nm) was achieved in an aqueous medium.

•

Stability of synthesized nanoparticles was confirmed by UV-Vis spectral analysis at zero day and after 100 days and zeta potential analysis, which is -31.9 mV.

•

Ethanolic bark extract from D. indica was found to be reducing as well as the capping agent during synthesis of silver nanoparticles.

•

Synthesized silver nanoparticles showed enhanced radical scavenging activity than the D. indica bark extract alone so can be used for further pharmaceutical studies.

•

Moreover, results confirmed that the green synthesized silver nanoparticles may provide a new strategy in the design of new catalyst for reduction of environmental pollutant 4-nitrophenol and organic dye methylene blue.

•

Further, the synthesized AgNps were found to be more efficient as a biocatalyst when compared with other biomediated AgNPs.

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