The Open Biotechnology Journal




ISSN: 1874-0707 ― Volume 13, 2019
RESEARCH ARTICLE

Biosynthesis and Characterization of Silver Nanoparticles Using Phoenix dactylifera Fruits Extract and their In Vitro Antimicrobial and Cytotoxic Effects



Sarvat Zafar1, *, Aiman Zafar2
1 Department of Chemistry, University College Samtah, Jazan University, Jazan, Saudi Arabia
2 Department of Oral and Maxillofacial Pathology, Saveetha Dental College and Hospital, P.O. Box 600077, Chennai, India

Abstract

Background:

In this article, silver nanoparticles (AgNPs) were synthesized by the biological green technique, using the aqueous extracts obtained from fruits of Phoenix dactylifera (date palm). This method is simple, rapid, non-toxic, and sustainable, and substitutes for the conventional physical/chemical methods. The cytotoxic activities of AgNPs derived from date fruit extract have not been mentioned in the earlier studies.

Methods:

The biosynthesized AgNPs are analyzed by Fourier transform infrared spectroscopy (FT-IR), UV-visible spectroscopy (UV-vis), and Transmission Electron Microscopy (TEM) methods. The assessment of antimicrobial effect towards human pathogenic microbial strains and their potential cytotoxicity against human breast cancer cell lines (MCF-7) were also evaluated.

Results:

FT-IR spectral studies showed that phytomolecules such as carbohydrates, phenolic acids and flavonoids present in date fruits extract are involved in the reduction and capping of the AgNPs. UV-vis spectrum revealed Surface Plasmon Resonance (SPR) at 425 nm which attributes the presence of AgNPs in aqueous extract. TEM micrographs showed that AgNPs particle diameter is ranged from 20 nm to 100 nm with spherical morphology. The biosynthesized AgNPs exhibited significant antimicrobial activity towards human microbial strains. Phytosynthesized NPs also induce cytotoxicity via necrosis, apoptosis and mitodepressive mechanisms that can disturb the cellular components at various stages of cell cycle.

Conclusion:

The present study concludes that biologically synthesized AgNPs using Phoenix dactylifera is cost-effective, rapid, non-toxic, and sustainable and can be effectively used as an adjunct for the treatment of breast carcinoma.

Keywords: : Bioreduction, Silver nanoparticles, Phoenix dactylifera fruit extract, Characterization, Antimicrobial, Cytotoxicity, MCF-7.


Article Information


Identifiers and Pagination:

Year: 2019
Volume: 13
First Page: 37
Last Page: 46
Publisher Id: TOBIOTJ-13-37
DOI: 10.2174/1874070701913010037

Article History:

Received Date: 09/01/2019
Revision Received Date: 14/03/2019
Acceptance Date: 15/03/2019
Electronic publication date: 30/04/2019
Collection year: 2019

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© 2019 Zafar and Zafar.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Chemistry, College of Science, Samtah Jazan University, Postal Code 45 142, Fax: 073211052 Jazan, Saudi Arabia; Tel: +966-543776627; Email: sarvatzafar@gmail.com




1. INTRODUCTION

Plant sources have been considered as one of the most essential resources for bio-green approaches focused on the formation of nanoparticles (NPs) [1Mittal AK, Chisti Y, Banerjee UC. Synthesis of metallic nanoparticles using plant extracts. Biotechnol Adv 2013; 31(2): 346-56.[http://dx.doi.org/10.1016/j.biotechadv.2013.01.003] [PMID: 23318 667] -10Kaler A, Nankar R, Bhattacharyya MS, Banerjee UC. Extracellular biosynthesis of silver nanoparticles using aqueous extract of Candida viswanathii. J Bionanoscience 2011; 5(1): 53-8.[http://dx.doi.org/10.1166/jbns.2011.1040] ]. Numerous strategic approaches have been employed for the biosynthesis of NPs from metal ions of the analogous metals [11Luangpipat T, Beattie IR, Chisti Y, Haverkamp RG. Gold nanoparticles produced in a microalga. J Nanopart Res 2011; 13(12): 6439-45.[http://dx.doi.org/10.1007/s11051-011-0397-9] -13Meyers MA, Mishra A, Benson DJ. Mechanical properties of nanocrystalline materials. Prog Mater Sci 2006; 51(4): 427-556.[http://dx.doi.org/10.1016/j.pmatsci.2005.08.003] ]. Among this, biological synthesis is advantageous not only because of eco-friendlier approaches compared with some of the physicochemical strategies but however, it can also produce a huge amount of NPs [14Anastas PT, Zimmerman JB. Green nanotechnology Why we need a green nano award and how to make it happen Washington DC 2007.-18Khatami M, Alijani H, Nejad M, Varma R. Core@ shell nanoparticles: Greener synthesis using natural plant products. Appl Sci (Basel) 2018; 8(3): 411.[http://dx.doi.org/10.3390/app8030411] ]. Therefore, biological methods employing aqueous extracts derived from plant materials have emerged as a simple and single step protocol and a viable substitute for chemical and physical synthetic procedures [19Lokina S, Stephen A, Kaviyarasan V, Arulvasu C, Narayanan V. Cytotoxicity and antimicrobial activities of green synthesized silver nanoparticles. Eur J Med Chem 2014; 76: 256-63.[http://dx.doi.org/10.1016/j.ejmech.2014.02.010] [PMID: 24583606] -24Syed DN, Afaq F, Mukhtar H, Eds. Pomegranate derived products for cancer chemoprevention Seminars in cancer biology 2007.]. Due to the immense potential of phytochemical synthesis of AgNPs, its application and development are ever growing in the area of advanced technologies [25Osonga FJ, Kariuki VM, Yazgan I, et al. Synthesis and antibacterial characterization of sustainable nanosilver using naturally-derived macromolecules. Sci Total Environ 2016; 563-564: 977-86.[http://dx.doi.org/10.1016/j.scitotenv.2015.12.064] [PMID: 26765510] -27Mallikarjuna N, Dionysios D, Rajender S. Synthesis of silver and gold nanoparticles using antioxidants from blackberry, blueberry, pomegranate, and turmeric extracts. ACS sustainable chemistry 2014.]. AgNPs are found to be widely used in various fields such as antibacterial, antiviral, anti-inflammatory, catalytic, sensing and optical properties [28Manivasagan P, Venkatesan J, Senthilkumar K, Sivakumar K, Kim S-K. Biosynthesis, antimicrobial and cytotoxic effect of silver nanoparticles using a novel Nocardiopsis sp. MBRC-1. BioMed research international 2013; 2013-31Ahmad N, Sharma S, Alam MK, et al. Rapid synthesis of silver nanoparticles using dried medicinal plant of basil. Colloids Surf B Biointerfaces 2010; 81(1): 81-6.[http://dx.doi.org/10.1016/j.colsurfb.2010.06.029] [PMID: 20656463] ]. Several works on the preparation of Ag nanoparticles utilizing plant-mediated extract materials have also been used such as Medicago sativa [32Chandran SP, Chaudhary M, Pasricha R, Ahmad A, Sastry M. Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract. Biotechnol Prog 2006; 22(2): 577-83.[http://dx.doi.org/10.1021/bp0501423] [PMID: 16599579] ], Aloe barbadensis [33Huang J, Li Q, Sun D, et al. Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora leaf. Nanotechnology 2007; 18(10)105104[http://dx.doi.org/10.1088/0957-4484/18/10/105104] ], Emblica officinalis [34Jain D, Daima HK, Kachhwaha S, Kothari S. Synthesis of plant-mediated silver nanoparticles using papaya fruit extract and evaluation of their anti microbial activities. Dig J Nanomater Biostruct 2009; 4(3): 557-63.], Hibiscus rosasinensis [12Bar H, Bhui DK, Sahoo GP, Sarkar P, De SP, Misra A. Green synthesis of silver nanoparticles using latex of Jatropha curcas. Colloids Surf A Physicochem Eng Asp 2009; 339(1-3): 134-9.[http://dx.doi.org/10.1016/j.colsurfa.2009.02.008] ], Capsicum annum [35Song JY, Kim BS. Biological synthesis of bimetallic Au/Ag nanoparticles using Persimmon (Diopyros kaki) leaf extract. Korean J Chem Eng 2008; 25(4): 808-11.[http://dx.doi.org/10.1007/s11814-008-0133-z] ], Momordica charanga [36Malaikozhundan B, Vaseeharan B, Vijayakumar S, Sudhakaran R, Gobi N, Shanthini G. Antibacterial and antibiofilm assessment of Momordica charantia fruit extract coated silver nanoparticle. Biocatal Agric Biotechnol 2016; 8: 189-96.[http://dx.doi.org/10.1016/j.bcab.2016.09.007] ], Olea europaea [37Sadeghi B. Green synthesis of silver nanoparticles using seed aqueous extract of Olea europaea. Int J Nanodimens 2014; 5: 575-81.], Mangifera indica [38Philip D. Mangifera indica leaf-assisted biosynthesis of well-dispersed silver nanoparticles. Spectrochim Acta A Mol Biomol Spectrosc 2011; 78(1): 327-31.[http://dx.doi.org/10.1016/j.saa.2010.10.015] [PMID: 21030295] ] and Citrus sinensis [39Kaviya S, Santhanalakshmi J, Viswanathan B, Muthumary J, Srinivasan K. Biosynthesis of silver nanoparticles using citrus sinensis peel extract and its antibacterial activity. Spectrochim Acta A Mol Biomol Spectrosc 2011; 79(3): 594-8.[http://dx.doi.org/10.1016/j.saa.2011.03.040] [PMID: 21536485] ].

Date fruit (Phoenix dactylifera) is a tropical and subtropical tree and belongs to family Arecaceae (40). It is widely found in Saudi Arabia, Iran, Iraq, Egypt, UAE, Algeria, Sudan, Oman, and Pakistan for its edible sweet fruit. The Phoenix dactylifera is regarded as an inexpensive fruit and rich in phytomolecules and antioxidants. The major biomolecules extracted from date fruit are carbohydrates, phenolics, sterols, carotenoids, anthocyanins, and flavonoids [41Baliga MS, Baliga BRV, Kandathil SM, Bhat HP, Vayalil PK. A review of the chemistry and pharmacology of the date fruits (Phoenix dactylifera L.). Food Res Int 2011; 44(7): 1812-22.[http://dx.doi.org/10.1016/j.foodres.2010.07.004] ]. The fruit-mediated AgNPs obtained from the bioreduction of date extract have been reported [42Farhadi S, Ajerloo B, Mohammadi A. Green biosynthesis of spherical silver nanoparticles by using date palm (Phoenix Dactylifera) fruit extract and study of their antibacterial and catalytic activities. Acta Chim Slov 2017; 64(1): 129-43.[http://dx.doi.org/10.17344/acsi.2016.2956] [PMID: 28380222] , 43Biglari F, AlKarkhi AF, Easa AM. Antioxidant activity and phenolic content of various date palm (Phoenix dactylifera) fruits from Iran. Food Chem 2008; 107(4): 1636-41.[http://dx.doi.org/10.1016/j.foodchem.2007.10.033] ]. However, cytotoxic activities of AgNPs in human cancer cell lines (MCF-7) by using date palm fruits extract are not reported in earlier studies. Moreover, the rate of bioreduction by using date fruit extract is extremely fast and produced more stable nanoparticles as compared to NPs derived from other plant resources, microbes and seaweeds [42Farhadi S, Ajerloo B, Mohammadi A. Green biosynthesis of spherical silver nanoparticles by using date palm (Phoenix Dactylifera) fruit extract and study of their antibacterial and catalytic activities. Acta Chim Slov 2017; 64(1): 129-43.[http://dx.doi.org/10.17344/acsi.2016.2956] [PMID: 28380222] ]. This study is cost-effective because it does not require any additional solvents, emulsifiers as well as consumed less energy for the synthesis of NPs. Moreover, Phoenix dactylifera is cheap and widely available fruit in Saudi Arabia. The above-mentioned procedure only requires the purification of nanoparticles through centrifugation method. Hence, the overall cost for biologically synthesized AgNPs is reduced to 60% compared to the synthetic drugs used for the treatment of breast carcinoma.

The objective of this article is to report one-step biosynthesis of AgNPs using aqueous fruit extract from Phoenix dactylifera. Generation of silver NPs are analyzed by using UV-visible and FT-IR spectroscopy, and particle size was confirmed through TEM analysis. The assessment of antimicrobial effect towards human pathogenic microbial strains and their potential cytotoxicity assay in MCF-7 cell lines were also evaluated.

2. MATERIALS AND METHODS

Silver nitrate (AgNO3) of analytical grade was purchased from Sigma-Aldrich Chemicals and was used without any further purification. Fresh Phoenix dactylifera (Fig. 1) fruits were purchased from local market in Jazan, Saudi Arabia. All of the solutions and chemical were prepared in the de-ionized water.

2.1. Preparation of Aqueous Phoenix dactylifera Fruit Extract

The fruits of Phoenix dactylifera were repeatedly washed with de-ionized water to remove dust and soil particles and were also surface sterilized by using 0.1% mercurous chloride (HgCl2) for 1 min to remove microbial stains [44Tyagi VSA, Chauhan P, Kumari P, Kaushal S. Identification and prevention of bacterial contimination on explant used in plant tissue culture labs. Alcohol 2011; 3: 0.722.]. 20 gm surface sterilized fruits were cut into small pieces and add 100 ml of double distilled-ionized water in it and stirred for 30 mins at 60oC. The aqueous fruit extract was cooled to ambient temperature and filtered twice by using Whatman No.1 filter paper. The obtained light yellow extract was collected as a stock solution stored at 40C in the refrigerator used for further studies.

2.2. Preparation of AgNPs

1mM AgNO3 solution was prepared in the de-ionized water. AgNPs were biosynthesized by mixing 3 ml of aqueous Phoenix dactylifera fruit extract with 9 ml of 1mM AgNO3 solution. Reaction products were heated for 20 mins at 60°C under constant stirring. The color of the aqueous mixture was changed from yellow to brown. This is due to the bioreduction in the aqueous solution, which indicates the formation of AgNPs and was identified through UV-vis spectra. The similar procedure was adopted for biosynthesis of AgNPs at different time intervals (5, 10 and 15 mins). The obtained AgNPs solution was centrifuged at 3000 rpm for 20 mins and the final product was redispersed in 10ml distilled water. The procedure of centrifugation and redispersion was done twice. AgNPs were then dried in an oven at 60°C for one hour. Finally, the obtained product was ground into powder for further studies.

Fig. (1)
Phoenix dactylifera fruits.


3. CHARACTERIZATIONS

3.1. UV-visible Spectroscopy (UV)

UV-vis absorption spectrophotometry was used to analyze the optical properties of AgNPs. The UV-visible absorption spectra of aqueous AgNPs were recorded on a double beam UV-visible spectrometer (6800, JENWAY) with a resolution of 2 nm and path length of 1 cm in 300–600 nm range.

3.2. Fourier Transform Infrared Spectroscopy (FT-IR)

FT-IR spectra were recorded in FT-IR spectroscopy with wavelength region from 4000 cm-1 to 400 cm-1 at a resolution of 4cm-1. Infrared spectra were obtained using a Thermo Scientific Nicolet iS10 FT-IR spectrophotometer [32Chandran SP, Chaudhary M, Pasricha R, Ahmad A, Sastry M. Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract. Biotechnol Prog 2006; 22(2): 577-83.[http://dx.doi.org/10.1021/bp0501423] [PMID: 16599579] ].

3.3. Transmission Electron Microscope (TEM)

The particle size and morphology of biosynthesized nanoparticles were analyzed by using Morgagni 268-D TEM, FEI, USA operated at an accelerating voltage between 80 and 200 kV. For TEM analysis, samples of AgNPs were prepared by placing a small volume of the aqueous solution onto the carbon coated copper TEM grids and vacuum dried.

3.4. In Vitro Antimicrobial Studies

The antimicrobial studies of the biosynthesized silver NPs were assessed using strains against four different pathogenic bacteria namely Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 424), Staphylococcus aureus (MTCC 96), Enterococcus faecalis (MTCC 439) and one yeast fungi Candida albicans (MTCC 227) by well diffusion methods [45Holder IA, Boyce ST. Agar well diffusion assay testing of bacterial susceptibility to various antimicrobials in concentrations non-toxic for human cells in culture. Burns 1994; 20(5): 426-9.[http://dx.doi.org/10.1016/0305-4179(94)90035-3] [PMID: 7999271] ]. Mueller Hinton Agar (MHA) obtained from Himedia (Mumbai) was used in In vitro antimicrobial studies. The MHA plates were prepared by using 25 ml of molten media into sterile Petri plates. These petri plates were then solidified and grown about 18 h (OD adjusted to 0.6). 100 µl of microbial strains cultures were placed onto a plate and made culture lawn. After five minutes, a sterile cork borer was used to make wells of 6 mm in size on the agar puncture. Different concentrations of aqueous silver nanoparticles (20, 40, 60, 80, 100 lg/ml) are placed into the agar well with positive control Streptomycin for bacterial strains and Clotrimazole for fungal strains. The petri dishes were kept for the incubation for 24 hours at 370C, and the inhibitory zone was measured in millimeter.

3.5. Cytotoxicity Assay (MTT)

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) method was used to study the cytotoxicity assays of silver nanoparticles obtained from date fruit extract on MCF-7 cell lines [46Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65(1-2): 55-63.[http://dx.doi.org/10.1016/0022-1759(83)90303-4] [PMID: 6606682] ]. In the colorimetric method, MTT is reduced to purple formazan by mitochondrial Succinate dehydrogenase. MCF-7 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum solution. The cells were seeded with 24 well culture plate along with different concentrations of AgNPs (12.5, 25, 50, 75, and 100, 125, 150, 175, and 200 µg/ml) and incubated at 37°C in a humidified CO2 incubator with 5% CO2. After 48 h incubation, 100µl MTT was poured into each well and incubated for 3h in room temperature until a purple product formazan was formed. The obtained formazan crystals were dissolved by using 100μl SDS in DMSO and absorbance was measured in Lark LIPR-9608 microplate reader at 540 nm wavelength. Absorbance for the untreated cells absorbance is considered as a control reference. The percentage of cell viability was calculated as follows [47Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res 1987; 47(4): 936-42.[PMID: 3802100] , 48Hayon T, Dvilansky A, Shpilberg O, Nathan I. Appraisal of the MTT-based assay as a useful tool for predicting drug chemosensitivity in leukemia. Leuk Lymphoma 2003; 44(11): 1957-62.[http://dx.doi.org/10.1080/1042819031000116607] [PMID: 14738150] ]

Percentage of cell viability = OD sample / OD control X 100

4. RESULTS AND DISCUSSION

4.1. Visual and UV-Visible Absorption Studies

Fig. (2) shows the visual appearance of date palm fruit-mediated AgNPs. The appearance of color change from yellow to brown during reaction attributes the generation of silver nanoparticles. The aqueous date extracts rich in polyphenols, antioxidants, and flavonoids. The hydroxyl groups present in biomolecules are responsible for the bioreduction of Ag+ ions as shown in the mechanism (Fig. 3)

The phyto-reduction of silver ions to AgNPs was analyzed by UV-vis spectroscopy. Fig. (4) shows the spectra of AgNPs prepared at different time intervals (5-20 minutes). As seen in the spectra, the intensity of absorbance bands increases as the reaction time of the reacting mixtures was increased. The maximum absorption appeared in the visible range at 425 nm after 20 minutes. This is due to SPR in silver NPs solution, which attributes the formation of silver nanoparticles. This showed that Ag+ ions were completely reduced to nanoparticles within 20 minutes in the presence of date fruit extract. Several researchers reported the SPR bands of AgNPs in the wavelength of 425-460 nm [49Udayasoorian C, Kumar KV, Jayabalakrishnan M. Extracellular synthesis of silver nanoparticles using leaf extract of Cassia auriculata. Dig J Nanomater Biostruct 2011; 6(1): 279-83.-52Mock JJ, Smith DR, Schultz S. Local refractive index dependence of plasmon resonance spectra from individual nanoparticles. Nano Lett 2003; 3(4): 485-91.[http://dx.doi.org/10.1021/nl0340475] ].

4.2. FT-IR Studies

FT-IR spectroscopy analysis was used to identify the functional groups of the biomolecules present in the fruit extract. As reported in the previous studies, date fruit is rich in phytomolecules such as carbohydrates, phenolic acids, sterols, carotenoids, anthocyanins, procyanidins and flavonoids [41Baliga MS, Baliga BRV, Kandathil SM, Bhat HP, Vayalil PK. A review of the chemistry and pharmacology of the date fruits (Phoenix dactylifera L.). Food Res Int 2011; 44(7): 1812-22.[http://dx.doi.org/10.1016/j.foodres.2010.07.004] ]. The FT-IR spectrum of date extract Fig. (5A), shows characteristic stretching bands for phenolic O–H, C=O, and C–OH at 3450, 1639, and 1011 cm–1, respectively. Absorption bands for C–H stretching and –OH bend for polyphenols also appeared at 2918 and 1420 cm-1 [53Nasrollahzadeh M, Sajadi SM, Rostami-Vartooni A, Alizadeh M, Bagherzadeh M. Green synthesis of the Pd nanoparticles supported on reduced graphene oxide using barberry fruit extract and its application as a recyclable and heterogeneous catalyst for the reduction of nitroarenes. J Colloid Interface Sci 2016; 466: 360-8.[http://dx.doi.org/10.1016/j.jcis.2015.12.036] [PMID: 26752431] ] that confirms the presence of aromatic compounds like flavonoids and terpenoids in fruit extract, which consist of many C-H bonds. The band at 1011 cm-1 were attributed to C-O stretching and secondary –OH which depicts the presence of phytomolecules in the aqueous date fruit extract [54Kumar B, Smita K, Cumbal L, Debut A. Green synthesis of silver nanoparticles using Andean blackberry fruit extract. Saudi J Biol Sci 2017; 24(1): 45-50.[http://dx.doi.org/10.1016/j.sjbs.2015.09.006] [PMID: 28053570] ].

Fig. (2)
Visual appearance of AgNPs after addition of date fruit extract to AgNO3 solution.


Fig. (3)
Bioreduction and stability of AgNPs using date fruit extract.


Fig. (4)
UV-Visible spectra of AgNPs at different reaction time.


In the spectrum of AgNPs (Fig. 5B), a strength of bands decreases at 3450, 1639, 1420 and 1011 cm–1 that clearly indicates the adsorption of natural polyphenols on the surface of the AgNPs. The band at 550 cm-1 for AgNPs is also observed in Fig. (5B), which corresponds to the bioreduction of Ag+ ions [55Bello BA, Khan SA, Khan JA, et al. Anticancer, antibacterial and pollutant degradation potential of silver nanoparticles from Hyphaene thebaica. Biochem Biophys Res Commun 2017; 490(3): 889-94.[http://dx.doi.org/10.1016/j.bbrc.2017.06.136] [PMID: 28648600] ].

4.3. TEM Analysis

The particle size and morphology of the phytosynthesized silver nanoparticles obtained from date extract were determined using TEM. From the TEM micrographs, it was depicted that AgNPs particle diameter is ranged from 20 nm to 100 nm with spherical morphology. As can be seen in Fig. (6), the morphology of the NPs is almost homogeneous and some are in aggregated form. Nanomaterials have very high surface energy and small particle diameter, they can be easily aggregated. The average particle diameter of the nanoparticles is found to be correlative to the range of AgNPs derived from fruit extracts of H. undatus, S. nigra, and M. domestica [19Lokina S, Stephen A, Kaviyarasan V, Arulvasu C, Narayanan V. Cytotoxicity and antimicrobial activities of green synthesized silver nanoparticles. Eur J Med Chem 2014; 76: 256-63.[http://dx.doi.org/10.1016/j.ejmech.2014.02.010] [PMID: 24583606] -21David L, Moldovan B, Vulcu A, et al. Green synthesis, characterization and anti-inflammatory activity of silver nanoparticles using European black elderberry fruits extract. Colloids Surf B Biointerfaces 2014; 122: 767-77.[http://dx.doi.org/10.1016/j.colsurfb.2014.08.018] [PMID: 25174985] ].

4.4. In Vitro Antimicrobial Studies of AgNPs

Biosynthesized silver NPs were investigated for their potential antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and one yeast fungi Candida albicans by agar well diffusion method. As seen in Table (1), the diameters of inhibition zones showed varied responses against all the tested human pathogenic strains. It has been analyzed that antimicrobial studies were in a dose-dependent manner [56Dipankar C, Murugan S. The green synthesis, characterization and evaluation of the biological activities of silver nanoparticles synthesized from Iresine herbstii leaf aqueous extracts. Colloids Surf B Biointerfaces 2012; 98: 112-9.[http://dx.doi.org/10.1016/j.colsurfb.2012.04.006] [PMID: 22705935] , 57Swamy MK, Akhtar MS, Mohanty SK, Sinniah UR. Synthesis and characterization of silver nanoparticles using fruit extract of Momordica cymbalaria and assessment of their In vitro antimicrobial, antioxidant and cytotoxicity activities. Spectrochim Acta A Mol Biomol Spectrosc 2015; 151: 939-44.[http://dx.doi.org/10.1016/j.saa.2015.07.009] [PMID: 26186612] ]. Silver nanoparticles phytosynthesized using date palm extract revealed highest inhibition zone (34.0 mm) at a concentration (100 µl) against C. albicans and followed by E. coli (30.0 mm), P. aeruginosa (29.0 mm), E. faecalis (27.0 mm) and S. aureus (29.0 mm), respectively. The result showed low inhibitory activity against E. faecalis (22.0 mm) at a concentration (25 µl) (Fig. 7). It was shown that the inhibition region is increased with increase in the concentration of nanoparticles against all the tested microbial strains.

Silver nanoparticles were found to be non-toxic in human cells and showed significant growth inhibitory activities on microbial pathogens because of their small dimensions and greater surface area [58Singh M, Singh S, Prasad S, Gambhir I. Nanotechnology in medicine and antibacterial effect of silver nanoparticles. Dig J Nanomater Biostruct 2008; 3(3): 115-22., 59Carlson C, Hussain SM, Schrand AMK, et al. Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species. J Phys Chem B 2008; 112(43): 13608-19.[http://dx.doi.org/10.1021/jp712087m] [PMID: 18831567] ]. Biosynthesized nanoparticles attached to the microbial cell membrane causing plasmolysis and finally inhibit the membrane synthesis [60Morones JR, Elechiguerra JL, Camacho A, et al. The bactericidal effect of silver nanoparticles. Nanotechnology 2005; 16(10): 2346-53.[http://dx.doi.org/10.1088/0957-4484/16/10/059] [PMID: 20818017] , 61Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res 2000; 52(4): 662-8.[http://dx.doi.org/10.1002/1097-4636(20001215)52:4<662::AID-JBM10>3.0.CO;2-3] [PMID: 11033548] ]. Silver NPs interact with functional moieties of the microbial cell such as thiol, hydroxyl, and a carboxyl group and disturbs the respiratory protease enzyme [62Hajipour MJ, Fromm KM, Ashkarran AA, et al. Antibacterial properties of nanoparticles. Trends Biotechnol 2012; 30(10): 499-511.[http://dx.doi.org/10.1016/j.tibtech.2012.06.004] [PMID: 22884769] -64Shemetov AA, Nabiev I, Sukhanova A. Molecular interaction of proteins and peptides with nanoparticles. ACS Nano 2012; 6(6): 4585-602.[http://dx.doi.org/10.1021/nn300415x] [PMID: 22621430] ]. Furthermore, nanoparticles entered into bacterial cells and release silver ions that inhibit DNA replication, cell division, and finally leads to the death of bacterial cell [65Li WR, Xie XB, Shi QS, Zeng HY, Ou-Yang YS, Chen YB. Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli. Appl Microbiol Biotechnol 2010; 85(4): 1115-22.[http://dx.doi.org/10.1007/s00253-009-2159-5] [PMID: 19669753] , 66Li WR, Xie XB, Shi QS, Duan SS, Ouyang YS, Chen YB. Antibacterial effect of silver nanoparticles on Staphylococcus aureus. Biometals 2011; 24(1): 135-41.[http://dx.doi.org/10.1007/s10534-010-9381-6] [PMID: 20938718] ]. However, the mechanism of nanoparticles with various human pathogens are not yet completely known.

Fig. (5)
FT-IR spectra of (A) date palm aqueous fruit extract and (B) biosynthesized silver nanoparticles.


Table 1
In vitro lntimicrobial effects of AgNPs using date palm extract against microbial pathogens


Fig. (6)
TEM micrographs of Silver nanoparticles obtained using date fruit extract.


Fig. (7)
Antimicrobial activity of AgNPs against different microbial pathogen.


4.5. Cytotoxicity Assay

Cytotoxic activities of biosynthesized AgNPs were studied on MCF-7 breast cancer cell lines using the MTT method. MTT data showed cytotoxicity of NPs towards MCF-7 cell lines in dose-dependent manner (12.5, 25, 50, 75, 100, 125, 150, 175, and 200 µg/ml). Significant cytotoxic activity was observed in biosynthesized nanoparticles with IC50 90.8 µg/ml. Nanoparticles have the ability to kill 50% of the cells and this indicates the effects of cytotoxicity. As can be seen in Fig. (8), the cell viability decreases with increase in the concentration of silver NPs. The highest inhibitory effect was observed at a concentration of 200µg/ml of AgNPs (Figs. 8 and 9). Cytotoxicity of fruit-mediated AgNPs against MCF-7 cell lines is also reported in different plant species [19Lokina S, Stephen A, Kaviyarasan V, Arulvasu C, Narayanan V. Cytotoxicity and antimicrobial activities of green synthesized silver nanoparticles. Eur J Med Chem 2014; 76: 256-63.[http://dx.doi.org/10.1016/j.ejmech.2014.02.010] [PMID: 24583606] , 67Ramar M, Manikandan B, Marimuthu PN, et al. Synthesis of silver nanoparticles using Solanum trilobatum fruits extract and its antibacterial, cytotoxic activity against human breast cancer cell line MCF 7. Spectrochim Acta A Mol Biomol Spectrosc 2015; 140: 223-8.[http://dx.doi.org/10.1016/j.saa.2014.12.060] [PMID: 25613692] -69Chokkalingam M, Singh P, Huo Y, et al. Facile synthesis of Au and Ag nanoparticles using fruit extract of Lycium chinense and their anticancer activity. J Drug Deliv Sci Technol 2019; 49: 308-15.[http://dx.doi.org/10.1016/j.jddst.2018.11.025] ]. Several mechanisms have been mentioned in the previous studies that nanoparticles induce cytotoxicity via Reactive Oxygen Species (ROS) that can damage all cellular moieties and finally lead to cell death through the necrosis [70Xia T, Kovochich M, Brant J, et al. Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano Lett 2006; 6(8): 1794-807.[http://dx.doi.org/10.1021/nl061025k] [PMID: 16895376] ]. This also increases the intracellular oxidative stress that results in cell death by the process of apoptosis [71Ott M, Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria, oxidative stress and cell death. Apoptosis 2007; 12(5): 913-22.[http://dx.doi.org/10.1007/s10495-007-0756-2] [PMID: 17453160] ]. Silver NPs have anti-angiogenic properties that have the ability to block the activity of abnormally expressing signal proteins and prevent the formation of new fine capillaries that are needed for tumor growth and metastasize [72Song JY, Kim BS. Rapid biological synthesis of silver nanoparticles using plant leaf extracts. Bioprocess Biosyst Eng 2009; 32(1): 79-84.[http://dx.doi.org/10.1007/s00449-008-0224-6] [PMID: 18438688] ]. The present study showed the mitodepressive and cytotoxic effect of nanoparticles. This is achieved by DNA inhibition at S-phase. Induction of AgNPs increases mitotic abnormalities which disturb both metaphase and anaphase. Finally, this effects both the impairment of mitotic spindles and disturbs the orientation of chromosomes at various stages of the cell cycle [73Kuriyama R, Sakai H. Role of tubulin-SH groups in polymerization to microtubules. Functional-SH groups in tubulin for polymerization. J Biochem 1974; 76(3): 651-4.[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a130609] [PMID: 44 74165] ]. Chromosome stickiness is also induced by AgNPs [74McGill M, Pathak S, Hsu TC. Effects of ethidium bromide on mitosis and chromosomes: A possible material basis for chromosome stickiness. Chromosoma 1974; 47(2): 157-66.[http://dx.doi.org/10.1007/BF00331803] [PMID: 4474934] , 75Klásterská I, Natarajan AT, Ramel C. An interpretation of the origin of subchromatid aberrations and chromosome stickiness as a category of chromatid aberrations. Hereditas 1976; 83(2): 153-62.[http://dx.doi.org/10.1111/j.1601-5223.1976.tb01581.x] [PMID: 977 379] ]. The induction of chromosomal breaks and micronuclei shows the clastogenic potential that results in loss of genetic material [76Brück I, Raun K, Synnestvedt B, Greve T. Follicle aspiration in the mare using a transvaginal ultrasound-guided technique. Equine Vet J 1992; 24(1): 58-9.[http://dx.doi.org/10.1111/j.2042-3306.1992.tb02780.x] [PMID: 155 5542] ]. Thus, the above-mentioned factors suggest that AgNPs possess mitodepressive, mitoclassic and clastogenic properties and can be effectively used as an adjunct for the treatment of breast carcinoma.

Fig. (8)
Cytotoxicity of AgNPs towards MCF-7 cell lines.


Fig. (9)
Morphology of control (A), biosynthesized AgNPs 100 µg/ml (B) and 200 µg/ml against MCF-7 cell lines.


CONCLUSION

AgNPs were prepared from aqueous fruit extract of Phoenix dactylifera and AgNO3 solution via. biological method. Phytoreduction of silver ions was analyzed by UV-visible spectra that showed a broad absorption band at 425 nm. Phytomolecules present in aqueous fruit extract responsible for bioreduction and capping of nanoparticles was evident from FT-IR studies. Particle size was analyzed by TEM which is ranged from 20 -100 nm with spherical morphology. The biosynthesized AgNPs exhibited significant antimicrobial activity towards human microbial strains. Phytosynthesized NPs also induce cytotoxicity via necrosis, apoptosis and mitodepressive mechanisms that can disturb the cellular components at various stages of the cell cycle. The present study concludes that biologically synthesized AgNPs using Phoenix dactylifera are cost-effective, rapid, non-toxic, and sustainable and can be effectively used as an adjunct for the treatment of breast carcinoma.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No animals/humans were used for these studies.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

Not applicable.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Declared none.

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Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


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