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Green synthesis of anisotropic silver nanoparticles from the aqueous leaf extract of Dodonaea viscosa with their antibacterial and anticancer activities
Process Biochemistry 80 (2019) 80–88
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Green synthesis of anisotropic silver nanoparticles from the aqueous leaf extract of Dodonaea viscosa with their antibacterial and anticancer activities
T
M. Anandana, G. Pooranib, P. Boomic, K. Varunkumard, K. Anande, A. Anil Chuturgoone, ⁎ M. Saravananf, H. Gurumallesh Prabua, a
Department of Industrial Chemistry, School of Chemical Sciences, Alagappa University, Karaikudi, 630 003, India Kumaraguru College of Technology, Coimbatore, Tamil Nadu, 641049, India c Department of Bioinformatics, Alagappa University, Karaikudi, 630 003, India d Cancer Biology lab, Department of Biochemistry, Bharathidasan University, Trichirappalli, India e Discipline of Medical Biochemistry and Chemical Pathology, School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZuluNatal, Durban, South Africa f Department of Medical Microbiology and Immunology, Institute of Biomedical Science, College of Health Sciences, Mekelle University, Ethiopia b
ARTICLE INFO
ABSTRACT
Keywords: A549 NSCLC cancer cells Dodonaea viscosa MTT assay Silver nanoparticles Transmission electron microscopy
Distinct morphological silver nanoparticles were synthesized using Dodonaea viscosa leaves, extracted using different polar and non-polar solvents. Petroleum ether, methanol, acetone, acetonitrile and water were used for the extraction of active ingredients from the leaves of Dodonaea viscosa which attributed to obtaining nanoparticles with different physical, chemical, antibacterial and cytotoxic properties. The synthesized nanoparticles were characterized by UV–vis, FT-IR, XRD, HR-SEM with EDX and HR-TEM with SAED patterns. The XRD, HR-SEM and HR-TEM results reveal different nano sizes (15, 18, 12 and 20 nm) with different surface morphology (worm-like, irregular flower, spherical and dendritic structures) of the nanoparticles prepared using different solvent extracts (methanol, acetone, acetonitrile and water). The antibacterial results show significant zone of inhibition (20, 16, 13, 18 mm) against the test bacterium Streptococcus pyogenes for AgNPs synthesized by methanol, acetone, acetonitrile and water extracts, respectively. The cytotoxicity of synthesized silver nanoparticles in A549 NSCLC cells using the MTT assay were found to be 14, 3, 80, and 4 μg/mL for AgNPs synthesized using leaf extracts obtained from methanol, acetone, acetonitrile and water, respectively. The results revealed that the synthesized AgNPs were effective in inhibiting the growth of A549 NSCLC cells.
1. Introduction Nano sized materials find great interest in the emerging field of science and curative medicines. The optical, electronic, magnetic and catalytic properties of nanoparticles depend on their size, morphology and chemical environments [1,2]. Among others, silver nanoparticles (AgNPs) possess potential applications in the fields of agriculture, waste management, forensic science, pollution control, solar cells, and medicine [3]. Conventional methods for synthesis of AgNPs require hazardous chemicals as reducing agents with large quantities of external energy (heat) and also give rise to the formation of hazardous byproducts. Thus, there exists a huge thirst for developing energy efficient green chemical processes to evade the use of hazardous chemicals in the synthesis of nanoparticles [4,5]. To counteract these obstacles, plants present the best green resource suitable for large-scale synthesis of metal and metal oxide nanoparticles with distinct morphology and size [6]. The reason is that plant extracts are ably suited to synthesize silver nanoparticles due to the presence of biomolecules such as alkaloids, carbohydrates, terpenoids and phenolic compounds that can effectively reduce the
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metal ions into nanoparticles in a single-step process. The morphologies and sizes of the nanoparticles can be modified by adjusting the reducing and capping agents [7,8]. The plant, Dodonaea viscosa is a stiff bushy plant belonging to the family Sapindacea and traditionally used because of their medicinal diversity. It was administered orally to treat various disorders like head-ache, cold, sore throat, fever, malaria and rheumatism [9]. Dodonaea viscosa leaf extracts were evaluated for their antibacterial activity [10,11], antifungal property [12], antiviral activity [13] and antiplasmodial activity [14]. Kaempferol, sakuranetin, hautriwaic acid and flavonoids present in the leaf extract possess antimicrobial activity. The alcoholic extracts have been reported to have local anesthetic and muscle relaxant activity, anti-fertile activity (96.05%), anti-inflammation (68.42%) and early abortifacial activity (27.63%) in female rats [15]. Over the above stated medicinal value, this plant has been further utilized to synthesize silver nanoparticles because of their tremendous potential impact in medicine. Silver nanoparticles are mainly used in medicine as tropical ointments to prevent microbial infection against burns and wounds. Further these nanoparticles
Corresponding author. E-mail address: [email protected] (H. Gurumallesh Prabu).
https://doi.org/10.1016/j.procbio.2019.02.014 Received 4 December 2018; Received in revised form 29 January 2019; Accepted 14 February 2019 Available online 15 February 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
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2.6. Cell culture
were found to be highly toxic against different multidrug resistant human pathogens. The efficacy of silver nanoparticles in the killing of cancer cells was tested and found to be more effective in ovarian cancer cell lines [16]. The combinational treatment of the tumors by silver and plant extract may cause synergistic cytotoxicity on the cancer cells [17]. Thus, the researchers focus on the development of safe and effective therapeutic drugs to cure various cancers with the use of natural compounds as curative agents. In the present study, green synthesis and characterization of stable colloidal silver nanoparticles was carried out using leaf extracts of Dodonaea viscosa as reducing agents. It was aimed to determine the effect of AgNPs synthesized by various Dodonaea viscosa extracts individually in A549 NSCLC cell line.
A549 lung cancer cells were cultured in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum and 1% penicillin/streptomycin. The cultured cells were incubated in a 5% CO2 humidified atmosphere at 37 °C.
2.7. Cytotoxicity assay The cytotoxicity analysis was performed using the MTT (dimethyl thiazolyl tetrazolium bromide) assay as described by Yuan et al [5]. Cells in monolayer (˜1 × 104) were added to each well of a 96-well culture plate and incubated for overnight at 37 °C with 5% CO2. The A549 cancer cells were treated with a series of concentrations (1 to 200 μg/mL) of AgNPs synthesized using different solvent extracts of Dodonaea viscosa and control culture was treated with dimethyl sulfoxide (DMSO). DMSO by itself was found to be non-toxic to the cells. About 20 μL of MTT (5 mg/mL) was added to the cultures after 24 h of incubation and incubated further for 4 h. Thereafter, the MTT was aspirated and about 200 μL of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 590 nm (measurement) and 620 nm (reference) using a micro-plate reader (Bio-Rad, USA) [18,19]. Experiments were conducted in triplicate; the mean and standard deviation were calculated from the results obtained. The percentage of inhibition was calculated using the following formula:
2. Materials and methods 2.1. Materials Silver nitrate (AgNO3) and pH buffer capsules were purchased from Merck chemicals and solvents from SRL Chemicals, Chennai, India. The chemicals were of AR grade and that are used as such without any further purification. The A549 lung cancer cell line was obtained from the National Centre for Cell Science, Pune, India. Dulbecco's Modified Eagle's Medium (DMEM), bovine serum and penicillin/streptomycin were purchased from Hi-Media Laboratories Mumbai, India. Millipore water was used for all the experiments.
[Mean OD of untreated cells (control) Mean OD of treated cells] × 100 Mean OD of untreated cells (control)
2.2. Bioreductant preparation
Inhibition (%) =
Solvent screening of active components from Dodonaea viscosa leaves was employed by successive extraction with different solvents. Five different solvents such as petroleum ether, acetone, methanol, acetonitrile and water were used for the extraction of active ingredients from the leaves of Dodonaea viscosa. The fresh leaves were collected, washed and dried in absence of direct sunlight for 15 days and powdered well in a grinder. To the 5 g of powdered leaves, 25 mL of solvent were added and extracted using Soxhlet apparatus at boiling temperature for about 2 h. Then, the extract was filtered through Whatman filter paper and the residue was again suspended with the solvent. The above procedure was repeated several times with the residue to obtain utmost extraction.
A linear graph was plotted between the surviving fraction of A549 cancer cells and AgNPs concentrations to obtain the survival curve for the specified concentration of the tested compound. The curves were fitted linear and halfmaximal inhibition concentration (IC50) was calculated from the graph.
3. Results and discussion 3.1. UV–vis spectroscopy analysis The assessment of AgNPs synthesized by different solvent extracts of Dodonaea viscosa leaves was carried out by UV–vis analysis. The difference in intensities of the black color (confirmation for AgNPs formation) show the variation in effective reduction of metallic silver by different solvent extracts depending on their reducing capabilities. It is a well known phenomenon that AgNPs exhibit various colors with respect to their size and morphology [20]. The completion of the reduction process was monitored by analyzing the UV–vis spectrum of the reaction mixture at different time intervals. The results were presented in terms of the maximum absorbance values (λmax) of the SPR phenomenon. The UV–vis results reveal that the SPR absorption peak (λmax) of silver was observed from 441 to 564 nm as shown in Fig. 1(A–E). The intensity of SPR peaks for AgNPs synthesized from petroleum ether and methanol extracts was low compared to that of other extracts because of either incomplete reduction or larger sized particles. Further, it was observed that the nanoparticles obtained from the acetone extract (Fig. 1(C)) were reasonably larger in size distribution. From Fig. 1(E), the sharp and intense SPR peak was observed for silver nanoparticles synthesized using aqueous leaf extract of Dodonaea viscosa and the intensity of the absorption band increases due to the formation of a large number of highly dense nanoparticles [21]. It was noticed that the solvent used for the extraction of Dodonaea viscosa leaves was influential in the effective synthesis of silver nanoparticles and also controlled their size and distribution [22]. Thus, the solvent used in the extraction viz., that the metabolites present in the leaf extracts play a significant role in the size, shape and optical properties of silver nanoparticles.
2.3. Synthesis of silver nanoparticles A 5 mL of leaf extract (reductant) was added to AgNO3 (1 mM, 25 mL) solution with 2.5 mL of freshly prepared pH buffer at room temperature and allowed to stir for 2 h using magnetic stirrer. The colorless solution of AgNO3 turned to dark brown after the addition of extract and it was finally turned to black color due to the formation of AgNPs. The reduction of silver nitrate to AgNPs was confirmed by surface plasmon resonance (SPR) peak using UV–vis spectroscopy.
2.4. Characterization techniques The synthesized silver nanoparticles were characterized by UltravioletVisible (JASCO V-530), Fourier transform-infrared (JASCO FT-IR 4600), X-ray diffraction (X’Pert PRO analytical), High resolution-Scanning electron microscopy and Energy dispersive X-ray spectrum (QUANTA FEG-250) and High resolution-Transmission electron microscopy with selected area diffraction patterns (JEOL 3010).
2.5. Antibacterial activity analysis Antibacterial efficacy of synthesized AgNPs capped with extract was measured in terms of zone of inhibition against gram positive bacterium, Streptococcus pyogenes using agar well diffusion assay [Clinical and Laboratory Standards Institute, 2006] in Muller-Hinton agar (MHA) (Himedia Laboratories) medium. The sterile plates containing nutrient agar medium were spread with fresh bacterial culture by using sterile cotton buds. Wells (5 mm size) were made from agar plates by using a sterile cork borer, the wells were loaded with 200 μL of colloidal AgNPs (25–100 μg/mL). The plates were incubated at 37 °C for 25 h. The zone of inhibition was observed around the well and calculated by measuring the diameter of zone around the well.
3.2. FT-IR analysis The FT-IR spectra of synthesized AgNPs with the presence of studied extracts were analyzed and are displayed in Fig. 2(A–E). The dual role of the plant extract as a reductant and capping agent was confirmed by FT-IR analysis of synthesized AgNPs. The assignment of functional groups responsible for the wavenumber ranges present in the extract capped AgNPs are tabulated (Table 1). The similarities between the spectra of neat extract 81
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Fig. 1. UV–vis spectra of the synthesized AgNPs using (A) petroleum ether, (B) methanol, (C) acetone, (D) acetonitrile and (E) water extract of Dodonaea viscosa leaves. and extract with synthesized AgNPs show some marginal shifts in peak position and absence of some peaks that clearly demonstrate the presence of residual plant extract in the reduction mixture. Shifts in the peaks with decreased band intensity were observed in the frequencies near 2850, 1700, 1620 and 1035 cm−1 implying that alkyl, C]O, C = O(NH) and CeOeC groups respectively from the extracts were capped with silver nanoparticles [23,24]. Of the five studied solvent extracts, petroleum ether extract did not respond well in the synthesis of AgNPs. Hence, it was not considered for further studies.
the AgNPs synthesized by Dodonaea viscosa extracts using methanol, acetone, acetonitrile and water as solvents. The distinct peaks at 2θ values of 38.12°, 44.31°, 64.45° and 77.41° with respect of the planes (111), (200), (220) and (311) were indexed for face centered cubic (FCC) of silver as per the JCPDS file no. 01-087-0717. The wellresolved and intense XRD patterns in the figure clearly showed that the synthesized AgNPs were crystalline in nature. The appearance of low intense peaks at 64.45° and 77.41° of (220) and (311) planes were optional with respective of the solvents used in the synthesis of AgNPs. Thus, put forth the truth that, solvent screening and compounds in the different extracts were the responsible features in the effective reduction of Ag+ ions to Ag° particles [3,25].
3.3. X-ray diffraction (XRD) analysis X-ray diffraction pattern was performed to authenticate the phase angle and crystalline nature of the AgNPs synthesized by different solvent extracts of Dodonaea viscosa. The XRD results showed the Bragg’s reflections that may be indexed with the FCC structure of silver. Fig. 3 demonstrates the XRD patterns of
3.4. HR-SEM with EDX analysis The surface morphology and composition of the synthesized AgNPs were 82
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Fig. 3. XRD patterns of the synthesized AgNPs using (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa leaves. Fig. 2. FT-IR spectra of the synthesized AgNPs using (A) petroleum ether, (B) methanol, (C) acetone, (D) acetonitrile and (E) water extract of Dodonaea viscosa leaves.
The prominent XRD peak obtained for silver is observed as (111) plane and hence the growth mechanism is more feasible in the (111) direction than others [28]. Silver dendritic nanostructures are currently focused in the design of super hydrophobic materials [21]. Hence this approach could be a simple route to synthesize the silver dendritic nanostructures via simple reduction of AgNO3 by aqueous extract of Dodonaea viscosa plant leaves. The elemental composition and stoichiometry of the synthesized silver nanostructures were determined by energy dispersive X-ray spectroscopic analysis. EDX spectrum reveals that the presence of strong silver peak around 3.25 keV and other peaks for inorganic impurities from the bioactive molecules in the Dodonaea viscosa leaves extract [29]. The carbon peak present in the spectrum was majorly attributed to the carbon tape used in the spectrum analysis. The EDX data showed that the weight % of Ag was 46, 38.37, 57.7 and 40.7% for wormlike particles, irregularly shaped flower particles, spherical particles and dendrites structures respectively of AgNPs as shown in Fig. 4 (E–H).
analyzed by HR-SEM with EDX analyses. HR-SEM analysis of high density AgNPs synthesized by Dodonaea viscosa leaf extracts are shown in Fig. 4(A–D). From the micrograph images, the morphology and size were strongly dependent on the biomolecules which were effectively screened by different solvents [22]. This observation shows that the solvents used in the extraction of biomolecules from the leaves leads in the synthesis of morphologically different nanoparticles. The SEM images of AgNPs from different solvent extracts showed worm-like, irregular flower, spherical particles and dendrite like structures. The sizes are also dependent on the extracting solvents. The size of the nanostructures ranged between 20–50 nm for nano worms, 50–100 nm for flowers, 70–100 nm for spherical particles and micro-sized dendrites (with a diameter about 0.7–2.5 μm and length is about 3.3–30 μm). This observation can be accredited to the difference in concentrations and combinations of the molecules present in the extracts were the X-factor for the synthesis of different silver nanostructures. The formation of silver nanoparticles was due to the interactions of hydrogen bond and electrostatic interaction between the biomolecules capping with Ag° [26]. The nanoparticles were not in direct contact even in the aggregated condition, indicating the stabilization of nanoparticles by the capping agent. In this experiment, a typical nanostructure of silver i.e., dendrite structure was obtained with the aqueous extract of Dodonaea viscosa leaves. In dendrite structures, particles are attached one by one adjacent to the edge of the another at random positions to grow as a finite structure [27].
3.5. HR-TEM with SAED patterns The morphology of the synthesized AgNPs was also investigated by HRTEM and the micrograph images were shown in Fig. 5. The average particle size was found to be 15, 18, 12 and 20 nm for the AgNPs synthesized by Dodonaea viscosa leaves extract using methanol, acetone, acetonitrile and water solvents, respectively. The magnified images of the synthesized AgNPs showed a near spherical, pentagonal and hexagonal structures for leaf extract obtained using methanol, acetone, acetonitrile and water, respectively. The inter-planar distance of the synthesized AgNPs nanoparticles was obtained to be 2.36 Å and it corresponds to (111) plane of the FCC lattice structure
Table 1 Comparative FT-IR spectral data of colloidal AgNPs in different extracts and assignment of functional groups with respect to their wavenumbers. Functional groups
eOeH stretching (cm−1)
CH2OH (cm−1)
C]C (cm−1)
CeH stretching (cm−1)
C]O stretching (cm−1)
CeOeC stretching (sapogenins of saponins) (cm−1)
eCH3 (methyl) stretching (cm−1)
Petroleum Ether Methanol Acetone Acetonitrile Water
3406 3398 3432 3408 3384
1192 1207 – – –
1636 1646 1637 1615 –
2930 2929 2925 2924 2932
– – 1759 – –
1059 1056 1077 1043 1077
1384 1386 1383 1382 1381
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Fig. 4. SEM micrographs (inset: enlarged micrograph) of the synthesized AgNPs using (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa leaves; and E–H are their respective elemental compositions (EDX analysis).
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Fig. 5. TEM micrographs (inset: enlarged micrograph) of the synthesized AgNPs using (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa leaves with respective SAED patterns (E–H).
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Fig. 6. Antibacterial activity of AgNPs against Streptococcus pyogenes using (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa at different concentrations (25–100 μg/mL). of silver [30]. The formation of FCC structured AgNPs was confirmed using the XRD patterns. The SAED patterns of synthesized silver nanoparticles show the d-spacing values as 0.236, 0.204, 0.144, and 0.121 nm, corresponds to (111), (200), (220) and (311) planes respectively.
lines were treated with different concentrations (1–200 μg/mL) of synthesized AgNPs for 24 h. The cell viability results obtained by MTT assay for neat extracts and AgNPs synthesized by those extracts are shown in Fig. 7 and 8, respectively. The extracts of Dodonaea viscosa leaves (using different solvents and without AgNPs) do exhibit growth inhibition against A549 cells, but only at high concentrations. The percentage of cell death at a concentration of 200 μg/ mL was 74, 66.9, 79.1 and 86.9% for methanol, acetone, acetonitrile and water extracts, respectively (the acetone extract possessed the highest inhibitory effect compared to others). The results show that the biomolecules present in the ketone extract have good inhibitory effects. The AgNPs significantly increased cell mortality in the A549 cancer cells; death was observed to be 49.11, 52.30, 51.23 and 49.98% after 24 h treatment of AgNPs synthesized using methanol, acetone, acetonitrile and water extracts, respectively. The AgNPs induced cell death at significantly lower concentrations than the plant extracts only. Percentage of cell death was in direct correlation with the AgNPs concentration i.e, cell death increased gradually with the increase of AgNPs concentration [32,33]. The IC50 values were 14, 3, 80 and 4 μg/mL for worm-like, flower-like, spherical and dendrite structures of AgNPs. The most probable mechanism of cell death for these tumor cells may have been either by apoptosis or necrosis. The nanoparticles with varying sizes and surface properties had a great impact on cell membranes. It was noticed that the phase transition temperature of the lipid membrane increases by increasing the surface roughness of nanoparticles [19]. Thus, the results show that the synthesized AgNPs synergistically inhibit the proliferation of A549 NSCLC cancer cells.
3.6. Antibacterial activity analysis The antibacterial activity of the synthesized colloidal AgNPs with extract was studied and the results obtained were presented in Fig. 6. The zone of inhibition was obtained as 20, 16, 13, 18 mm for AgNPs synthesized by methanol, acetone, acetonitrile and water extracts, respectively. The AgNPs synthesized using methanol extract showed better antibacterial activity in terms of zone of inhibition against the test bacterium Streptococcus pyogenes when compared to other extracts. Increasing the concentration of the AgNPs increased their respective antibacterial activity. The AgNPs synthesized by acetone and water extracts have resulted moderate antibacterial activity against the bacterium. The obtained antibacterial activity is mainly depends upon the size and morphology of AgNPs synthesized using Dodonaea viscosa leaf extract. Furthermore, the plant extracts posses a variety of compounds in it and that also contributes little in the antibacterial activity due to the synergistic action. Scientific reports reveal that compounds such as tannins, flavones, glycosides, polyenes, saponins, alkaloids, quinines and anthraquinones have been found to have antimicrobial activity [31].
3.7. AgNPs induced growth inhibition of A549 NSCLC cells
4. Conclusions
The synthesized AgNPs were assessed for their antiproliferative activities in the A549 lung cancer cells using the MTT assay. It is a preliminary assessment of tumor inhibitory action that is used to determine the half minimal inhibition concentration (IC50) values at the specific time points. The A549 lung cancer cell
Morphologically distinct silver nanostructures were synthesized using the leaf extracts of Dodonaea viscosa as reductant and characterized by optical and microscopic methods. The formation of silver nanoparticles (from Ag+ to Ag°) 86
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Fig. 7. MTT response of the (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa leaves.
Fig. 8. MTT response of the synthesized AgNPs using (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa leaves.
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is confirmed by the surface plasmon resonance (λmax at 441–564 nm) in UV–vis spectra. The XRD results ascertained the crystalline nature of FCC silver with the respective planes of (111), (200), (220) and (311) at an angle of 38.12°, 44.31°, 64.45° and 77.41°, respectively. The HR-SEM analysis shows that the size of the particles are in the range of 20–50 nm for nano worms, 50–100 nm for flowers, 70–100 nm for spherical particles and micro sized dendrites (with a diameter about 0.7–2.5 μm and length is about 3.3–30 μm). The EDX spectrum confirms that the presence of a strong silver peak at 3.25 keV. The antibacterial results show that the colloidal AgNPs synthesized by different Dodonaea viscosa extracts has a potency to inhibit the growth of the test bacterium Streptococcus pyogenes. The cytotoxic property of the water extract and acetone extract mediated nanostructures showed IC50 values at very low concentrations than the others proved that the synthesized AgNPs along with the extracts comprise good inhibition against the cancerous A549 NSCLC cells.
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Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements The authors are thankful to the University Grants Commission (UGC-BSR: F.4-1/2006 (BSR)/7-188/2007 (BSR)), New Delhi, India for the financial assistance by the scheme of a basic scientific research fellowship. The author (M. Anandan) acknowledges the Department of Physics, Alagappa University, Karaikudi for providing X-Ray Diffraction analysis facility. The authors (P. Boomi) thankUGC, New Delhi, India for providing Assistant professor (No. F. 14-13/2013 (Inno/ASIST) dated 30.03.2013) under the Innovative scheme to carry out the teaching and research work. The authors (H. Gurumallesh Prabu) acknowledge the UGC-BSR, New Delhi, India for the financial assistance by the onetime grant scheme. The authors (P. Boomi and H. Gurumallesh Prabu) sincere thankful to the DST, New Delhi, India for the financial support in general and infrastructure facilities sponsored under PURSE 2nd phase (Order No: SR/PURSE 2/38 (G)) and MHRD-RUSA 2.0 schemes (Letter No: F.24-51/ 2014-U Policy (TN Multi-Gen), Dept. of Edn. Govt. of India, Dt. 09.10.2018).
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2019.02.014.
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Green synthesis of anisotropic silver nanoparticles from the aqueous leaf extract of Dodonaea viscosa with their antibacterial and anticancer activities
T
M. Anandana, G. Pooranib, P. Boomic, K. Varunkumard, K. Anande, A. Anil Chuturgoone, ⁎ M. Saravananf, H. Gurumallesh Prabua, a
Department of Industrial Chemistry, School of Chemical Sciences, Alagappa University, Karaikudi, 630 003, India Kumaraguru College of Technology, Coimbatore, Tamil Nadu, 641049, India c Department of Bioinformatics, Alagappa University, Karaikudi, 630 003, India d Cancer Biology lab, Department of Biochemistry, Bharathidasan University, Trichirappalli, India e Discipline of Medical Biochemistry and Chemical Pathology, School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZuluNatal, Durban, South Africa f Department of Medical Microbiology and Immunology, Institute of Biomedical Science, College of Health Sciences, Mekelle University, Ethiopia b
ARTICLE INFO
ABSTRACT
Keywords: A549 NSCLC cancer cells Dodonaea viscosa MTT assay Silver nanoparticles Transmission electron microscopy
Distinct morphological silver nanoparticles were synthesized using Dodonaea viscosa leaves, extracted using different polar and non-polar solvents. Petroleum ether, methanol, acetone, acetonitrile and water were used for the extraction of active ingredients from the leaves of Dodonaea viscosa which attributed to obtaining nanoparticles with different physical, chemical, antibacterial and cytotoxic properties. The synthesized nanoparticles were characterized by UV–vis, FT-IR, XRD, HR-SEM with EDX and HR-TEM with SAED patterns. The XRD, HR-SEM and HR-TEM results reveal different nano sizes (15, 18, 12 and 20 nm) with different surface morphology (worm-like, irregular flower, spherical and dendritic structures) of the nanoparticles prepared using different solvent extracts (methanol, acetone, acetonitrile and water). The antibacterial results show significant zone of inhibition (20, 16, 13, 18 mm) against the test bacterium Streptococcus pyogenes for AgNPs synthesized by methanol, acetone, acetonitrile and water extracts, respectively. The cytotoxicity of synthesized silver nanoparticles in A549 NSCLC cells using the MTT assay were found to be 14, 3, 80, and 4 μg/mL for AgNPs synthesized using leaf extracts obtained from methanol, acetone, acetonitrile and water, respectively. The results revealed that the synthesized AgNPs were effective in inhibiting the growth of A549 NSCLC cells.
1. Introduction Nano sized materials find great interest in the emerging field of science and curative medicines. The optical, electronic, magnetic and catalytic properties of nanoparticles depend on their size, morphology and chemical environments [1,2]. Among others, silver nanoparticles (AgNPs) possess potential applications in the fields of agriculture, waste management, forensic science, pollution control, solar cells, and medicine [3]. Conventional methods for synthesis of AgNPs require hazardous chemicals as reducing agents with large quantities of external energy (heat) and also give rise to the formation of hazardous byproducts. Thus, there exists a huge thirst for developing energy efficient green chemical processes to evade the use of hazardous chemicals in the synthesis of nanoparticles [4,5]. To counteract these obstacles, plants present the best green resource suitable for large-scale synthesis of metal and metal oxide nanoparticles with distinct morphology and size [6]. The reason is that plant extracts are ably suited to synthesize silver nanoparticles due to the presence of biomolecules such as alkaloids, carbohydrates, terpenoids and phenolic compounds that can effectively reduce the
⁎
metal ions into nanoparticles in a single-step process. The morphologies and sizes of the nanoparticles can be modified by adjusting the reducing and capping agents [7,8]. The plant, Dodonaea viscosa is a stiff bushy plant belonging to the family Sapindacea and traditionally used because of their medicinal diversity. It was administered orally to treat various disorders like head-ache, cold, sore throat, fever, malaria and rheumatism [9]. Dodonaea viscosa leaf extracts were evaluated for their antibacterial activity [10,11], antifungal property [12], antiviral activity [13] and antiplasmodial activity [14]. Kaempferol, sakuranetin, hautriwaic acid and flavonoids present in the leaf extract possess antimicrobial activity. The alcoholic extracts have been reported to have local anesthetic and muscle relaxant activity, anti-fertile activity (96.05%), anti-inflammation (68.42%) and early abortifacial activity (27.63%) in female rats [15]. Over the above stated medicinal value, this plant has been further utilized to synthesize silver nanoparticles because of their tremendous potential impact in medicine. Silver nanoparticles are mainly used in medicine as tropical ointments to prevent microbial infection against burns and wounds. Further these nanoparticles
Corresponding author. E-mail address: [email protected] (H. Gurumallesh Prabu).
https://doi.org/10.1016/j.procbio.2019.02.014 Received 4 December 2018; Received in revised form 29 January 2019; Accepted 14 February 2019 Available online 15 February 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
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2.6. Cell culture
were found to be highly toxic against different multidrug resistant human pathogens. The efficacy of silver nanoparticles in the killing of cancer cells was tested and found to be more effective in ovarian cancer cell lines [16]. The combinational treatment of the tumors by silver and plant extract may cause synergistic cytotoxicity on the cancer cells [17]. Thus, the researchers focus on the development of safe and effective therapeutic drugs to cure various cancers with the use of natural compounds as curative agents. In the present study, green synthesis and characterization of stable colloidal silver nanoparticles was carried out using leaf extracts of Dodonaea viscosa as reducing agents. It was aimed to determine the effect of AgNPs synthesized by various Dodonaea viscosa extracts individually in A549 NSCLC cell line.
A549 lung cancer cells were cultured in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum and 1% penicillin/streptomycin. The cultured cells were incubated in a 5% CO2 humidified atmosphere at 37 °C.
2.7. Cytotoxicity assay The cytotoxicity analysis was performed using the MTT (dimethyl thiazolyl tetrazolium bromide) assay as described by Yuan et al [5]. Cells in monolayer (˜1 × 104) were added to each well of a 96-well culture plate and incubated for overnight at 37 °C with 5% CO2. The A549 cancer cells were treated with a series of concentrations (1 to 200 μg/mL) of AgNPs synthesized using different solvent extracts of Dodonaea viscosa and control culture was treated with dimethyl sulfoxide (DMSO). DMSO by itself was found to be non-toxic to the cells. About 20 μL of MTT (5 mg/mL) was added to the cultures after 24 h of incubation and incubated further for 4 h. Thereafter, the MTT was aspirated and about 200 μL of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 590 nm (measurement) and 620 nm (reference) using a micro-plate reader (Bio-Rad, USA) [18,19]. Experiments were conducted in triplicate; the mean and standard deviation were calculated from the results obtained. The percentage of inhibition was calculated using the following formula:
2. Materials and methods 2.1. Materials Silver nitrate (AgNO3) and pH buffer capsules were purchased from Merck chemicals and solvents from SRL Chemicals, Chennai, India. The chemicals were of AR grade and that are used as such without any further purification. The A549 lung cancer cell line was obtained from the National Centre for Cell Science, Pune, India. Dulbecco's Modified Eagle's Medium (DMEM), bovine serum and penicillin/streptomycin were purchased from Hi-Media Laboratories Mumbai, India. Millipore water was used for all the experiments.
[Mean OD of untreated cells (control) Mean OD of treated cells] × 100 Mean OD of untreated cells (control)
2.2. Bioreductant preparation
Inhibition (%) =
Solvent screening of active components from Dodonaea viscosa leaves was employed by successive extraction with different solvents. Five different solvents such as petroleum ether, acetone, methanol, acetonitrile and water were used for the extraction of active ingredients from the leaves of Dodonaea viscosa. The fresh leaves were collected, washed and dried in absence of direct sunlight for 15 days and powdered well in a grinder. To the 5 g of powdered leaves, 25 mL of solvent were added and extracted using Soxhlet apparatus at boiling temperature for about 2 h. Then, the extract was filtered through Whatman filter paper and the residue was again suspended with the solvent. The above procedure was repeated several times with the residue to obtain utmost extraction.
A linear graph was plotted between the surviving fraction of A549 cancer cells and AgNPs concentrations to obtain the survival curve for the specified concentration of the tested compound. The curves were fitted linear and halfmaximal inhibition concentration (IC50) was calculated from the graph.
3. Results and discussion 3.1. UV–vis spectroscopy analysis The assessment of AgNPs synthesized by different solvent extracts of Dodonaea viscosa leaves was carried out by UV–vis analysis. The difference in intensities of the black color (confirmation for AgNPs formation) show the variation in effective reduction of metallic silver by different solvent extracts depending on their reducing capabilities. It is a well known phenomenon that AgNPs exhibit various colors with respect to their size and morphology [20]. The completion of the reduction process was monitored by analyzing the UV–vis spectrum of the reaction mixture at different time intervals. The results were presented in terms of the maximum absorbance values (λmax) of the SPR phenomenon. The UV–vis results reveal that the SPR absorption peak (λmax) of silver was observed from 441 to 564 nm as shown in Fig. 1(A–E). The intensity of SPR peaks for AgNPs synthesized from petroleum ether and methanol extracts was low compared to that of other extracts because of either incomplete reduction or larger sized particles. Further, it was observed that the nanoparticles obtained from the acetone extract (Fig. 1(C)) were reasonably larger in size distribution. From Fig. 1(E), the sharp and intense SPR peak was observed for silver nanoparticles synthesized using aqueous leaf extract of Dodonaea viscosa and the intensity of the absorption band increases due to the formation of a large number of highly dense nanoparticles [21]. It was noticed that the solvent used for the extraction of Dodonaea viscosa leaves was influential in the effective synthesis of silver nanoparticles and also controlled their size and distribution [22]. Thus, the solvent used in the extraction viz., that the metabolites present in the leaf extracts play a significant role in the size, shape and optical properties of silver nanoparticles.
2.3. Synthesis of silver nanoparticles A 5 mL of leaf extract (reductant) was added to AgNO3 (1 mM, 25 mL) solution with 2.5 mL of freshly prepared pH buffer at room temperature and allowed to stir for 2 h using magnetic stirrer. The colorless solution of AgNO3 turned to dark brown after the addition of extract and it was finally turned to black color due to the formation of AgNPs. The reduction of silver nitrate to AgNPs was confirmed by surface plasmon resonance (SPR) peak using UV–vis spectroscopy.
2.4. Characterization techniques The synthesized silver nanoparticles were characterized by UltravioletVisible (JASCO V-530), Fourier transform-infrared (JASCO FT-IR 4600), X-ray diffraction (X’Pert PRO analytical), High resolution-Scanning electron microscopy and Energy dispersive X-ray spectrum (QUANTA FEG-250) and High resolution-Transmission electron microscopy with selected area diffraction patterns (JEOL 3010).
2.5. Antibacterial activity analysis Antibacterial efficacy of synthesized AgNPs capped with extract was measured in terms of zone of inhibition against gram positive bacterium, Streptococcus pyogenes using agar well diffusion assay [Clinical and Laboratory Standards Institute, 2006] in Muller-Hinton agar (MHA) (Himedia Laboratories) medium. The sterile plates containing nutrient agar medium were spread with fresh bacterial culture by using sterile cotton buds. Wells (5 mm size) were made from agar plates by using a sterile cork borer, the wells were loaded with 200 μL of colloidal AgNPs (25–100 μg/mL). The plates were incubated at 37 °C for 25 h. The zone of inhibition was observed around the well and calculated by measuring the diameter of zone around the well.
3.2. FT-IR analysis The FT-IR spectra of synthesized AgNPs with the presence of studied extracts were analyzed and are displayed in Fig. 2(A–E). The dual role of the plant extract as a reductant and capping agent was confirmed by FT-IR analysis of synthesized AgNPs. The assignment of functional groups responsible for the wavenumber ranges present in the extract capped AgNPs are tabulated (Table 1). The similarities between the spectra of neat extract 81
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Fig. 1. UV–vis spectra of the synthesized AgNPs using (A) petroleum ether, (B) methanol, (C) acetone, (D) acetonitrile and (E) water extract of Dodonaea viscosa leaves. and extract with synthesized AgNPs show some marginal shifts in peak position and absence of some peaks that clearly demonstrate the presence of residual plant extract in the reduction mixture. Shifts in the peaks with decreased band intensity were observed in the frequencies near 2850, 1700, 1620 and 1035 cm−1 implying that alkyl, C]O, C = O(NH) and CeOeC groups respectively from the extracts were capped with silver nanoparticles [23,24]. Of the five studied solvent extracts, petroleum ether extract did not respond well in the synthesis of AgNPs. Hence, it was not considered for further studies.
the AgNPs synthesized by Dodonaea viscosa extracts using methanol, acetone, acetonitrile and water as solvents. The distinct peaks at 2θ values of 38.12°, 44.31°, 64.45° and 77.41° with respect of the planes (111), (200), (220) and (311) were indexed for face centered cubic (FCC) of silver as per the JCPDS file no. 01-087-0717. The wellresolved and intense XRD patterns in the figure clearly showed that the synthesized AgNPs were crystalline in nature. The appearance of low intense peaks at 64.45° and 77.41° of (220) and (311) planes were optional with respective of the solvents used in the synthesis of AgNPs. Thus, put forth the truth that, solvent screening and compounds in the different extracts were the responsible features in the effective reduction of Ag+ ions to Ag° particles [3,25].
3.3. X-ray diffraction (XRD) analysis X-ray diffraction pattern was performed to authenticate the phase angle and crystalline nature of the AgNPs synthesized by different solvent extracts of Dodonaea viscosa. The XRD results showed the Bragg’s reflections that may be indexed with the FCC structure of silver. Fig. 3 demonstrates the XRD patterns of
3.4. HR-SEM with EDX analysis The surface morphology and composition of the synthesized AgNPs were 82
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Fig. 3. XRD patterns of the synthesized AgNPs using (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa leaves. Fig. 2. FT-IR spectra of the synthesized AgNPs using (A) petroleum ether, (B) methanol, (C) acetone, (D) acetonitrile and (E) water extract of Dodonaea viscosa leaves.
The prominent XRD peak obtained for silver is observed as (111) plane and hence the growth mechanism is more feasible in the (111) direction than others [28]. Silver dendritic nanostructures are currently focused in the design of super hydrophobic materials [21]. Hence this approach could be a simple route to synthesize the silver dendritic nanostructures via simple reduction of AgNO3 by aqueous extract of Dodonaea viscosa plant leaves. The elemental composition and stoichiometry of the synthesized silver nanostructures were determined by energy dispersive X-ray spectroscopic analysis. EDX spectrum reveals that the presence of strong silver peak around 3.25 keV and other peaks for inorganic impurities from the bioactive molecules in the Dodonaea viscosa leaves extract [29]. The carbon peak present in the spectrum was majorly attributed to the carbon tape used in the spectrum analysis. The EDX data showed that the weight % of Ag was 46, 38.37, 57.7 and 40.7% for wormlike particles, irregularly shaped flower particles, spherical particles and dendrites structures respectively of AgNPs as shown in Fig. 4 (E–H).
analyzed by HR-SEM with EDX analyses. HR-SEM analysis of high density AgNPs synthesized by Dodonaea viscosa leaf extracts are shown in Fig. 4(A–D). From the micrograph images, the morphology and size were strongly dependent on the biomolecules which were effectively screened by different solvents [22]. This observation shows that the solvents used in the extraction of biomolecules from the leaves leads in the synthesis of morphologically different nanoparticles. The SEM images of AgNPs from different solvent extracts showed worm-like, irregular flower, spherical particles and dendrite like structures. The sizes are also dependent on the extracting solvents. The size of the nanostructures ranged between 20–50 nm for nano worms, 50–100 nm for flowers, 70–100 nm for spherical particles and micro-sized dendrites (with a diameter about 0.7–2.5 μm and length is about 3.3–30 μm). This observation can be accredited to the difference in concentrations and combinations of the molecules present in the extracts were the X-factor for the synthesis of different silver nanostructures. The formation of silver nanoparticles was due to the interactions of hydrogen bond and electrostatic interaction between the biomolecules capping with Ag° [26]. The nanoparticles were not in direct contact even in the aggregated condition, indicating the stabilization of nanoparticles by the capping agent. In this experiment, a typical nanostructure of silver i.e., dendrite structure was obtained with the aqueous extract of Dodonaea viscosa leaves. In dendrite structures, particles are attached one by one adjacent to the edge of the another at random positions to grow as a finite structure [27].
3.5. HR-TEM with SAED patterns The morphology of the synthesized AgNPs was also investigated by HRTEM and the micrograph images were shown in Fig. 5. The average particle size was found to be 15, 18, 12 and 20 nm for the AgNPs synthesized by Dodonaea viscosa leaves extract using methanol, acetone, acetonitrile and water solvents, respectively. The magnified images of the synthesized AgNPs showed a near spherical, pentagonal and hexagonal structures for leaf extract obtained using methanol, acetone, acetonitrile and water, respectively. The inter-planar distance of the synthesized AgNPs nanoparticles was obtained to be 2.36 Å and it corresponds to (111) plane of the FCC lattice structure
Table 1 Comparative FT-IR spectral data of colloidal AgNPs in different extracts and assignment of functional groups with respect to their wavenumbers. Functional groups
eOeH stretching (cm−1)
CH2OH (cm−1)
C]C (cm−1)
CeH stretching (cm−1)
C]O stretching (cm−1)
CeOeC stretching (sapogenins of saponins) (cm−1)
eCH3 (methyl) stretching (cm−1)
Petroleum Ether Methanol Acetone Acetonitrile Water
3406 3398 3432 3408 3384
1192 1207 – – –
1636 1646 1637 1615 –
2930 2929 2925 2924 2932
– – 1759 – –
1059 1056 1077 1043 1077
1384 1386 1383 1382 1381
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Fig. 4. SEM micrographs (inset: enlarged micrograph) of the synthesized AgNPs using (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa leaves; and E–H are their respective elemental compositions (EDX analysis).
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Fig. 5. TEM micrographs (inset: enlarged micrograph) of the synthesized AgNPs using (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa leaves with respective SAED patterns (E–H).
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Fig. 6. Antibacterial activity of AgNPs against Streptococcus pyogenes using (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa at different concentrations (25–100 μg/mL). of silver [30]. The formation of FCC structured AgNPs was confirmed using the XRD patterns. The SAED patterns of synthesized silver nanoparticles show the d-spacing values as 0.236, 0.204, 0.144, and 0.121 nm, corresponds to (111), (200), (220) and (311) planes respectively.
lines were treated with different concentrations (1–200 μg/mL) of synthesized AgNPs for 24 h. The cell viability results obtained by MTT assay for neat extracts and AgNPs synthesized by those extracts are shown in Fig. 7 and 8, respectively. The extracts of Dodonaea viscosa leaves (using different solvents and without AgNPs) do exhibit growth inhibition against A549 cells, but only at high concentrations. The percentage of cell death at a concentration of 200 μg/ mL was 74, 66.9, 79.1 and 86.9% for methanol, acetone, acetonitrile and water extracts, respectively (the acetone extract possessed the highest inhibitory effect compared to others). The results show that the biomolecules present in the ketone extract have good inhibitory effects. The AgNPs significantly increased cell mortality in the A549 cancer cells; death was observed to be 49.11, 52.30, 51.23 and 49.98% after 24 h treatment of AgNPs synthesized using methanol, acetone, acetonitrile and water extracts, respectively. The AgNPs induced cell death at significantly lower concentrations than the plant extracts only. Percentage of cell death was in direct correlation with the AgNPs concentration i.e, cell death increased gradually with the increase of AgNPs concentration [32,33]. The IC50 values were 14, 3, 80 and 4 μg/mL for worm-like, flower-like, spherical and dendrite structures of AgNPs. The most probable mechanism of cell death for these tumor cells may have been either by apoptosis or necrosis. The nanoparticles with varying sizes and surface properties had a great impact on cell membranes. It was noticed that the phase transition temperature of the lipid membrane increases by increasing the surface roughness of nanoparticles [19]. Thus, the results show that the synthesized AgNPs synergistically inhibit the proliferation of A549 NSCLC cancer cells.
3.6. Antibacterial activity analysis The antibacterial activity of the synthesized colloidal AgNPs with extract was studied and the results obtained were presented in Fig. 6. The zone of inhibition was obtained as 20, 16, 13, 18 mm for AgNPs synthesized by methanol, acetone, acetonitrile and water extracts, respectively. The AgNPs synthesized using methanol extract showed better antibacterial activity in terms of zone of inhibition against the test bacterium Streptococcus pyogenes when compared to other extracts. Increasing the concentration of the AgNPs increased their respective antibacterial activity. The AgNPs synthesized by acetone and water extracts have resulted moderate antibacterial activity against the bacterium. The obtained antibacterial activity is mainly depends upon the size and morphology of AgNPs synthesized using Dodonaea viscosa leaf extract. Furthermore, the plant extracts posses a variety of compounds in it and that also contributes little in the antibacterial activity due to the synergistic action. Scientific reports reveal that compounds such as tannins, flavones, glycosides, polyenes, saponins, alkaloids, quinines and anthraquinones have been found to have antimicrobial activity [31].
3.7. AgNPs induced growth inhibition of A549 NSCLC cells
4. Conclusions
The synthesized AgNPs were assessed for their antiproliferative activities in the A549 lung cancer cells using the MTT assay. It is a preliminary assessment of tumor inhibitory action that is used to determine the half minimal inhibition concentration (IC50) values at the specific time points. The A549 lung cancer cell
Morphologically distinct silver nanostructures were synthesized using the leaf extracts of Dodonaea viscosa as reductant and characterized by optical and microscopic methods. The formation of silver nanoparticles (from Ag+ to Ag°) 86
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Fig. 7. MTT response of the (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa leaves.
Fig. 8. MTT response of the synthesized AgNPs using (A) methanol, (B) acetone, (C) acetonitrile and (D) water extract of Dodonaea viscosa leaves.
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is confirmed by the surface plasmon resonance (λmax at 441–564 nm) in UV–vis spectra. The XRD results ascertained the crystalline nature of FCC silver with the respective planes of (111), (200), (220) and (311) at an angle of 38.12°, 44.31°, 64.45° and 77.41°, respectively. The HR-SEM analysis shows that the size of the particles are in the range of 20–50 nm for nano worms, 50–100 nm for flowers, 70–100 nm for spherical particles and micro sized dendrites (with a diameter about 0.7–2.5 μm and length is about 3.3–30 μm). The EDX spectrum confirms that the presence of a strong silver peak at 3.25 keV. The antibacterial results show that the colloidal AgNPs synthesized by different Dodonaea viscosa extracts has a potency to inhibit the growth of the test bacterium Streptococcus pyogenes. The cytotoxic property of the water extract and acetone extract mediated nanostructures showed IC50 values at very low concentrations than the others proved that the synthesized AgNPs along with the extracts comprise good inhibition against the cancerous A549 NSCLC cells.
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Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements The authors are thankful to the University Grants Commission (UGC-BSR: F.4-1/2006 (BSR)/7-188/2007 (BSR)), New Delhi, India for the financial assistance by the scheme of a basic scientific research fellowship. The author (M. Anandan) acknowledges the Department of Physics, Alagappa University, Karaikudi for providing X-Ray Diffraction analysis facility. The authors (P. Boomi) thankUGC, New Delhi, India for providing Assistant professor (No. F. 14-13/2013 (Inno/ASIST) dated 30.03.2013) under the Innovative scheme to carry out the teaching and research work. The authors (H. Gurumallesh Prabu) acknowledge the UGC-BSR, New Delhi, India for the financial assistance by the onetime grant scheme. The authors (P. Boomi and H. Gurumallesh Prabu) sincere thankful to the DST, New Delhi, India for the financial support in general and infrastructure facilities sponsored under PURSE 2nd phase (Order No: SR/PURSE 2/38 (G)) and MHRD-RUSA 2.0 schemes (Letter No: F.24-51/ 2014-U Policy (TN Multi-Gen), Dept. of Edn. Govt. of India, Dt. 09.10.2018).
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2019.02.014.
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