Green synthesis and characterization of monodispersed silver nanoparticles using root bark aqueous extract of Annona muricata Linn and their antimicrobial activity
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In recent time, various phytosynthetic methods have been employed for the fabrication of silver nanoparticles; these unique metal nanoparticles are used in several applications which include pharmaceuticals and material engineering. The current research reports a rapid and simple synthetic partway for silver nanoparticles (AgNPs) using root bark aqueous extract of Annona muricata and the evaluation of its antimicrobial efficacy against pathogenic microorganisms. The root bark extract was treated with aqueous silver nitrate solution. Silver ions were reduced to silver atoms which on aggregation gave Silver nanoparticles; the biosynthesized AgNPs were characteristically spherical, discreet and stabilized by phytochemical entities and were characterized using ultraviolet visible spectroscopy, transmission electron microscope (TEM) and photon correlation microscopy. The aqueous plant extract-AgNPs suspension was subjected to Fourier transform infrared spectroscopy. TEM result for the average particle size is 22 ± 2 nm. The polydispersity index and zeta-potential were found to be 0.44 ± 0.02 and − 27.90 ± 0.01 mV, respectively (Zeta-Sizer). The antimicrobial evaluation result showed that the synthesized silver nanoparticles at different concentration were very active against the Gram-positive bacteria (B. subtilis, S. aureous) and Gram-negative bacteria (K. Pneumonia, E. Coli and Pseudomonas aeruginosa), P. aeruginosa being most susceptible to the anti microbial effect of the silver nanoparticles. Stable silver nanoparticles with antimicrobial activity were obtained through biosynthesis.
KeywordsCharacterization Antimicrobial Silver nanoparticle Photon correlation microscopy
The emergence of nanotechnology and nanoparticle research has gained ground among interdisciplinary research technologists. Metal nanoparticles have enjoyed great application in healthcare (Siddhartha and Debrata 2009), engineering (Narendra and Uday 2014), and water treatment. The biosynthesis of silver nanoparticles using plant extracts is an interesting area in nanobiotechnology, which is safe, simple, none toxic and eco-friendly with good pharmacological profile. The use in water treatment and purification cannot be overemphasized. In healthcare, silver nanoparticles have enjoyed wide application in areas such as bio-sensing, imaging and drug delivery (Sagar et al. 2011). High mortality rate due to bacterial drug resistance is recently on the increase and the overall result is high cost of medication and prolonged management period; this has recently been countered through the emergence of nano-biotechnology. Fabrication of silver metal nanoparticles employs both physical and chemical method (Saeid et al. 2017), but for interest of this research, the biological method using root bark aqueous extract of Annona muricata was employed in the synthesis of silver nanoparticles. This is a green synthetic method which is eco-friendly alternative to much toxic chemical synthesis (Ram 2014). Phyto-extracts are known to provide mediators for biocatalysis such as reducing and capping agents for nanoparticle synthesis. Product yield obtained using phyto-extracts is known to be more compared to those of bacterial synthesis, though the particle size range of the latter is known to be finer (Iravanip et al. 2014). Polyphenols are phytochemical compounds with anti-oxidant properties found abundantly in natural plants. There are over 7000 identified polyphenols found in plants (Mamta et al. 2013). Polyphenols play an important role in providing the reducing and capping agents needed in the synthesis of silver metal nanoparticles. In humans, they help in fighting free radicals (Florent et al. 2013). Flavonoids and lignins are example of polyphenols (Oscar et al. 2009). A. muricata has a long history of traditional uses; common names include graviola, guanabana or soursop. Ethnomedicinal uses include anticancer and anti-inflammatory purposes. The plant parts are known to be rich in polyphenols (Bora et al. 2004). To fabricate clinically acceptable and biodegradable nanoparticles, use of injurious chemicals as starting materials should be avoided. Hence, the current research aims at synthesizing silver nanoparticles using aqueous root bark extract of A. muricata, characterization of the synthesized nanoparticles using: UV–visible spectroscopy, transmission electron microscopy (TEM) and photon correlation microscopy (Zeta Sizer) and the evaluation of their antimicrobial activity against representative human pathogenic microorganism.
Silver nitrate, nutrient media, and other reagents used were of analytical grade obtained from Merck, Germany and Oxoid, Hampshire, UK. The medicinal plant, A. muricata employed in the synthesis of the AgNPs was procured from Pharmacognosy garden of the Faculty of Pharmacy, University of Port Harcourt, Nigeria.
The morphology and mean particle size of the AgNPs were characterized by transmission electron microscopy (TEM) (VEGAimu GmbH, Germany). Surface resonance plasmon absorption was determined using UV–visible spectrophotometer (Perkin Elmer Lambda 35). The polydispersity index and zeta potential were determined using photon correlation microscope (Mavern Nano ZS, ZS290, and UK). Infrared spectroscopy (FTIR) was performed using the (FTIR) (Shimadzu-8400).
Anonna muricata root extract preparation
Annona muricata root bark was collected, washed and boiled in double distilled water for 30 min at 100 °C. The extract was filtered through a cotton cloth sieve to remove insoluble fractions and macromolecules. The filtrate was further re-filtered using 0.45 µm sintered glass funnel and the resultant extract was stored in refrigerator at 5 °C until use. The extract provides basis for the reducing and stabilizing agent.
Synthesis of silver nanoparticles using Anonna muricata root bark aqueous extract
Silver nitrate in double distilled water afforded the silver ions for the reaction. 100 ml of the root bark aqueous extract was mixed with 150 ml of silver nitrate solution 1.00 mM. The reaction mixture was incubated at the temperature of 25 °C in the dark to avoid photochemical activation of silver nitrate. Silvery-brown colour was observed at the end of 20 min, indicating the formation of AgNPs. The formed product was washed, rinsed and centrifuged with double distilled water dried and stored away from light until used. A. muricata extract was used as control. All experiment was carried out in triplicate.
Characterization of synthesized silver nanoparticles
The absorption spectra of the silver nanoparticle sample were taken at 190–610 nm using a UV–visible spectrophotometer (Perkin Elmer Lambda 35) to determine the maximum point of production of silver nanoparticles. Double distilled water was used as blank.
The morphology and size of silver nanoparticles were determined by transmission electron microscopy (TEM). Sample preparation for TEM analysis involves depositing a drop of aqueous silver nanoparticle suspension on a carbon-coated copper grid and allowed to dry at room temperature; the transmission electron micrographs were produced and studied for particle size and morphology.
The average particle size, size distribution by intensity as well as polydispersity index were determined by injecting 1:20 dilution of aqueous silver nanoparticle solution into the U-shaped glass cuvette of the photon correlation microscope. FTIR studies were carried out on the A. muricata extract-silver nanoparticle suspension by employing KBr pellet technique. The FTIR spectra were generated at a resolution of 4 cm−1 in a transmission mode (4000–400 cm−1).
Antimicrobial activity of synthesized silver nanoparticles
Bacterial and fungal isolates were procured from the Department of Microbiology and Biotechnology of the Faculty of Pharmaceutical Sciences, University of Port Harcourt, Nigeria. The antimicrobial activity experiments were carried out under high ascetic condition.
The method of agar well diffusion assay was employed to investigate the antimicrobial activity of silver nanoparticles (Mukunthan et al. 2011). The tested micro-organisms were seeded in the nutrient agar plates, and then six 4-mm diameter paper discs were saturated with 5, 10 µg/ml AgNPs aqueous solution, Chloramphenicol (0.2 mg/ml), ketoconazole (0.5 mg/ml), A. muricata aqueous extract and double distilled water (control), respectively. The paper disc was placed on the solidified agar plates and was allowed to incubate at 37 °C for 24 and 48 h for bacteria and yeast cultures, respectively. The inhibition zone diameter was measured.
Result and discussion
Physichochemical characterization of AgNPs
Result of TEM micrograph of AgNPs
Droplet size, polydispersity index and zeta-potential result
Average size: 392.10 ± 18.56
PDI: 0.44 ± 0.02
ZP: − 27.90 ± 0.10
FTIR analysis of aqueous root extract of Anonna muricata-AgNPs
Result of AgNPs antibacterial efficacy
In the reported research work, AgNPs have been synthesized using A. muricata root bark extract. Synthesized AgNPs showed remarkable stability. The UV–visible spectroscopy, TEM and photon correlation spectroscopy analysis confirm the existence of elemental silver and its spherical form. The synthesized AgNPs have been confirmed to show profound activity against pathogenic bacteria. The present research is a simple, rapid, eco-friendly and non-toxic protocol for the synthesis of silver nanoparticles.
The authors do extend their profound appreciation to management of Department of Chemistry, Rhodes University, Grahamstown, South Africa for granting the research team access to their state of the art Nanotechnology centre.
Compliance with ethical standards
Conflict of interest
The authors report no conflicts of interest.
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