silver nanoparticles literature review

• Open access
• Published: 16 February 2018

A review on biosynthesis of silver nanoparticles and their biocidal properties

• Khwaja Salahuddin Siddiqi 1 ,
• Azamal Husen 2 &
• Rifaqat A. K. Rao 3  

Journal of Nanobiotechnology volume  16 , Article number:  14 ( 2018 ) Cite this article

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Use of silver and silver salts is as old as human civilization but the fabrication of silver nanoparticles (Ag NPs) has only recently been recognized. They have been specifically used in agriculture and medicine as antibacterial, antifungal and antioxidants. It has been demonstrated that Ag NPs arrest the growth and multiplication of many bacteria such as Bacillus cereus , Staphylococcus aureus , Citrobacter koseri , Salmonella typhii , Pseudomonas aeruginosa , Escherichia coli , Klebsiella pneumonia, Vibrio parahaemolyticus and fungus Candida albicans by binding Ag/Ag + with the biomolecules present in the microbial cells. It has been suggested that Ag NPs produce reactive oxygen species and free radicals which cause apoptosis leading to cell death preventing their replication. Since Ag NPs are smaller than the microorganisms, they diffuse into cell and rupture the cell wall which has been shown from SEM and TEM images of the suspension containing nanoparticles and pathogens. It has also been shown that smaller nanoparticles are more toxic than the bigger ones. Ag NPs are also used in packaging to prevent damage of food products by pathogens. The toxicity of Ag NPs is dependent on the size, concentration, pH of the medium and exposure time to pathogens.

Introduction

Nanoparticles exhibit novel properties which depend on their size, shape and morphology which enable them to interact with plants, animals and microbes [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ]. Silver nanoparticles (Ag NPs) have shown excellent bactericidal properties against a wide range of microorganisms [ 8 , 9 , 10 , 11 ]. They are prepared from different perspectives, often to study their morphology or physical characteristics. Some authors have used chemical method [ 12 ] and mistaken it with green synthesis, although they have done it inadvertently. The Ag NPs and their application in electronics, catalysis, drugs and in controlling microorganism development in biological system have made them eco-friendly [ 1 , 8 , 9 , 13 ]. Biogenic synthesis of Ag NPs involves bacteria, fungi, yeast, actinomycetes and plant extracts [ 1 , 10 , 11 , 13 , 14 , 15 ]. Recently, a number of parts of plants such as flowers, leaves and fruits [ 1 ], besides enzymes, have been used for the synthesis of gold and silver nanoparticles. The size, morphology and stability of nanoparticles depend on the method of preparation, nature of solvent, concentration, strength of reducing agent and temperature [ 1 , 6 , 10 , 11 ].

Of all the nanoparticles developed and characterized thus far, Ag NPs assume a significant position owing to their inherent characteristic of acting as an antimicrobial agent even in solid state. Although, its significance was recognized much earlier, it was not well exploited except for its use in oriental medicine and in coins. It is estimated that nearly 320 tons of Ag NPs are manufactured every year and used in nanomedical imaging, biosensing and food products [ 16 , 17 ].

There is a continuous increase in the number of multi-drug resistant bacterial and viral strains due to mutation, pollution and changing environmental conditions. To circumvent this predicament scientists are trying to develop drugs for the treatment of such microbial infections. Many metal salts and metal nanoparticles have been found to be effective in inhibiting the growth of many infectious bacteria. Silver and Ag NPs occupy a prominent place in the series of such metals which are used as antimicrobial agents from time immemorial [ 18 , 19 ]. Silver salts are used to inhibit the growth of a variety of bacteria in human system. They are used in catheters, cuts, burns and wounds to protect them against infection [ 20 , 21 ]. Das et al. [ 22 ] have reported that small sized Ag NPs are excellent growth inhibitors of certain bacteria. Ag NPs synthesized from silk sericin (SS), a water-soluble protein extracted from silk worms at pH 11, contain hydrophilic proteins with highly polar groups like hydroxyl, carboxyl and amino functional groups. Molecules containing the above functional groups act as reducing agents for AgNO 3 to produce elemental silver. Aramwit et al. [ 23 ] have suggested that the hydroxyl groups of SS are supposed to form complex with silver ions and prevent their aggregation or precipitation [ 24 , 25 ]. Ag NPs in elemental state may be segregated due to large molecules present in the solvent but may not be complexed as both of them are neutral. The antibacterial activity of SS-capped Ag NPs against gram positive and gram negative bacteria has been screened. It was found that MIC falls between 0.001 and 0.008 mM for both types of microorganisms namely Staphylococcus aureus , Bacillus subtilis , Pseudomonas aeruginosa , Acinetobacter baumannii and Escherichia coli.

Although, several reviews have been published on the fabrication and characterization of silver nanoparticles, very limited reports are available on their green synthesis, biocidal properties and mechanism of action [ 8 , 9 , 13 , 16 , 23 ]. Thus, in this review, we have attempted to give a comprehensive detail of the biosynthesis of Ag NPs from herbal extracts, fungi and bacteria. Their potential as antimicrobial agent and the mechanism of their action has also been discussed.

Synthesis and characterization of silver nanoparticles

In general, metallic nanoparticles are produced by two methods, i.e. “bottom-up” (buildup of a material from the bottom: atom by atom, molecule by molecule or cluster by cluster) and “top-down” (slicing or successive cutting of a bulk material to get nano-sized particle) [ 1 ]. The “bottom-up” approach is usually a superior choice for the nanoparticles preparation involving a homogeneous system wherein catalysts (for instance, reducing agent and enzymes) synthesize nanostructures that are controlled by the catalyst itself. However, the “top-down” approach generally works with the material in its bulk form, and the size reduction to nanoscale is achieved by specialized ablations, for instance thermal decomposition, mechanical grinding, etching, cutting, and sputtering. The main demerit of the top-down approach is the surface structural defects. Such defects have significant impact on the physical features and surface chemistry of metallic nanoparticles. Several methodologies are available for the synthesis of Ag NPs namely, chemical methods [ 26 , 27 , 28 , 29 ]; physical methods [ 30 , 31 , 32 ] and biological methods [ 1 , 10 , 11 ]. Chemical method of synthesis can be subdivided into chemical reduction, electrochemical, irradiation-assisted chemical and pyrolysis methods [ 33 ]. Ag NPs synthesis in solution requires metal precursor, reducing agents and stabilizing or capping agent. Commonly used reducing agents are ascorbic acid, alcohol, borohydride, sodium citrate and hydrazine compounds. Sotiriou and Pratsinis [ 28 ] have shown that the Ag NPs supported on nanostructured SiO 2 were obtained by flame aerosol technology, which allows close control of silver content and size. Also, silver/silica nanoparticles with relatively narrow size distribution were obtained by flame spray pyrolysis [ 29 ]. However, physical methods do not require lethal and highly reactive chemicals and generally have a fast processing time. These methods include arc-discharge [ 31 ], physical vapor condensation [ 30 ], energy ball milling method [ 34 ] and direct current magnetron sputtering [ 32 ]. Physical methods have another advantage over chemical methods in that the Ag NPs have a narrow size distribution [ 32 ], while the main demerits are consumption of high energy [ 32 ]. Thus, biological synthesis of Ag NPs from herbal extract and/or microorganisms has appeared as an alternative approach as these routes have several advantages over the chemical and physical methods of synthesis. It is also a well-established fact that these routes are simple, cost-effective, eco-friendly and easily scaled up for high yields and or production [ 1 , 2 , 3 ]. Biosynthesis of metal and metal oxide nanoparticles using biological agents such as bacteria, fungi, yeast, plant and algal extracts has gained popularity in the area of nanotechnology [ 1 , 2 , 3 , 5 , 6 , 10 , 11 ].

Plants and their parts contain carbohydrates, fats, proteins, nucleic acids, pigments and several types of secondary metabolites which act as reducing agents to produce nanoparticles from metal salts without producing any toxic by-product. The details have been provided in Table  1 . Similarly, biomolecules such as enzymes, proteins and bio-surfactants present in microorganisms serve as reducing agents. For instance, in many bacterial strains, bio-surfactants are used as capping and/or stabilizing agents (Table  2 ).

Extracellular synthesis of Ag NPs comprises of the trapping of metal ions on the outer surface of the cells and reducing them in the presence of enzymes or biomolecules, while intracellular synthesis occurs inside the microbial cells. It has been suggested that the extracellular synthesis of nanoparticles is cheap, favors large-scale production and requires simpler downstream processing. Thus, the extracellular method for the synthesis of nanoparticles is preferable [ 164 ] in comparison to the intracellular method. Ganesh Babu and Gunasekaran [ 165 ] and Kalimuthu et al. [ 166 ] have demonstrated that the intracellular synthesis requires additional steps for instance, ultrasound treatment or reactions with suitable detergents to release the synthesized silver nanoparticles. Further, the rate of biosynthesis of Ag NPs and their stability is a significant part in industrial production. Therefore, a proper monitoring of reaction conditions is also important (Fig.  1 ).

Biosynthesis of silver nanoparticles and their optimization techniques

From bacteria

In recent years, the potential of biosynthesis of Ag NPs using bacteria has been realized [ 15 , 153 , 156 , 157 , 158 , 159 ]. For instance, Pseudomonas stutzeri AG259—isolated from silver mine was used to produce Ag NPs inside the cells [ 167 ]. In addition, several bacterial strains (gram negative as well as gram positive) namely A. calcoaceticus, B. amyloliquefaciens, B. flexus, B. megaterium and S. aureus have been used for both extra- and intracellular biosynthesis of Ag NPs [ 168 , 169 , 170 , 171 , 172 , 173 , 174 ]. These Ag NPs are spherical, disk, cuboidal, hexagonal and triangular in shape. They have been fabricated using culture supernatant, aqueous cell-free extract or cells (Table  3 ). Saifuddin et al. [ 14 ] have demonstrated an extracellular biosynthesis of Ag NPs ( ∼ 5–50 nm) using a combination of culture supernatant of B. subtilis and microwave irradiation in water. Shahverdi et al. [ 15 ] have reported rapid biosynthesis of Ag NPs (within 5 min) using the culture supernatants of K. pneumonia , E. coli and Enterobacter cloacae . Saravanan et al. [ 172 ] have also reported an extracellular synthesis of Ag NPs using B. megaterium cultured supernatant, within minutes in presence of aqueous solutions of Ag + ions.

Rapid synthesis of Ag NPs has been achieved by the interaction of a bacterial strain S-27, belonging to Bacillus flexus group and 1 mM AgNO 3 in aqueous medium [ 173 ]. The colourless supernatant solution turned yellow and finally brown. Its UV–vis spectrum exhibited a sharp peak at 420 nm due to the surface plasmon resonance (SPR) of silver nanoparticles. Anisotropic nanoparticles of 12 and 65 nm size were stable in the dark for 5 months at room temperature although their slow degradation cannot be prevented. They were crystalline with a face centered cubic structure. These nanoparticles were found to be effective against multidrug resistant gram positive and gram negative bacteria. The colour intensity and rate of interaction depend on the concentration of the reacting components.

Das et al. [ 174 ] have reported extracellular biosynthesis of Ag NPs from the Bacillus strain (CS11). The interaction of 1 mM AgNO 3 with the bacteria at room temperature yielded nanoparticles within 24 h which showed a peak at 450 nm in UV–vis spectrum. Their size from TEM analysis was found to range between 42 and 92 nm (Table  3 ).

Biosynthesis of Ag NPs from both pathogenic and nonpathogenic fungi has been investigated extensively [ 10 , 164 , 213 , 214 , 215 ] (Table  4 ). It has been reported that silver ions are reduced extracellularly in the presence of fungi to generate stable Ag NPs in water [ 214 , 216 ].

Syed et al. [ 224 ] have also reported the extracellular synthesis of Ag NPs from thermophilic fungus Humicola sp. All manipulations were done in aqueous medium at room temperature. Mycelia were suspended in 100 mL of 1 mM AgNO 3 solution in an Erlenmeyer flask at 50 °C and the mixture was left in a shaker for 96 h at pH 9 and monitored for any change in colour. The solution showed a change in colour from yellow to brown due to the formation of Ag NPs [ 222 ]. It is a simple process for the extracellular synthesis of Ag NPs from Humicola sp. TEM micrograph showed nicely dispersed nanoparticles mainly of spherical shape ranging between 5 and 25 nm. They are crystalline with a face centered cubic structure [ 236 ]. IR spectrum of Ag NPs in the suspension showed peaks at 1644 and 1523 cm −1 assigned to amide I and amide II bands of protein corresponding to –C=O and N–H stretches. Owaid et al. [ 237 ] have reported the biosynthesis of Ag NPs from yellow exotic oysters mushroom, Pleurotus cornucopiae var. citrinopileatus . The dried basidiocarps were powdered, boiled in water and the supernatant was freeze dried. Different concentrations of hot water extract of this lyophilized powder were mixed with 1 mM AgNO 3 at 25 °C and incubated for 24, 48 and 72 h. Change in colour from yellow to yellowish brown exhibited an absorption peak at 420 and 450 nm in UV–vis region which is the characteristic of spherical silver nanoparticles. The width of the absorption peak suggests the polydispersed nature of nanoparticles [ 221 ]. IR spectrum of Ag NPs exhibited absorption peaks at 3304, 2200, 2066, 1969, 1636, 1261, 1094 and 611 cm −1 for different groups. Although, authors have indicated the presence of polysaccharide and protein in the mushroom they have ignored their stretching frequencies in the IR spectrum. However, the peak at 3304 has been assigned to υ (OH) of carboxylic acid and those at 2200 and 1969 cm −1 have been attributed to unsaturated aldehydes. The other peaks below 1500 cm −1 are due to unsaturated alkaloids. The field emission scanning electron and high-resolution transmission electron micrograph suggested that the Ag NPs are spherical with average size ranging between 20 and 30 nm.

Very recently, Al-Bahrani et al. [ 230 ] reported biogenic synthesis of Ag NPs from tree oyster mushroom Pleurotus ostreatus . Dried aqueous extract of mushroom (1–6 mg/mL) and 1 mM AgNO 3 were mixed and incubated in the dark for 6–40 h. The colour change from pale yellow to dark brownish yellow indicated the formation of silver nanoparticles. The UV–vis spectrum showed a sharp and broad absorption band at 420 nm. They are polydispersed nanoparticles of 10–40 nm with an average size of 28 nm. Several fungi namely, Aspergillus flavus , A. fumigates , Fusarium oxysporum, Fusarium acuminatum , F. culmorum , F. solani , Metarhizium anisopliae, Phoma glomerate, Phytophthora infestans, Trichoderma viride, Verticillium sp. have been used for both extra- and intracellular biosynthesis of Ag NPs [ 10 , 164 , 216 , 217 , 218 , 219 , 222 ]. These nanoparticles are of various sizes and shapes (Table  4 ).

From plants

Plant related parts such as leaves, stems, roots, shoots, flowers, barks, seeds and their metabolites have been successfully used for the efficient biosynthesis [ 1 , 238 ] of nanoparticles (Fig.  1 ). Very recently, Beg et al. [ 128 ] have reported green synthesis of Ag NPs from seed extract of Pongamia pinnata . The formation of nanoparticles was confirmed by an absorption max at 439 nm. The well dispersed nanoparticles with an average size of 16.4 nm had zeta potential equal to − 23.7 mV which supports dispersion and stability. Interaction of Ag NPs with human serum albumin was investigated and showed negligible change in α helics. In a very recent publication Karatoprak et al. [ 137 ] have reported green synthesis of Ag NPs from the medicinal plant extract Pelargonium endlicherianum . The plant containing gallic acid, apocyanin and quercetin act as reducing agents to produce silver nanoparticles. Phytomediated synthesis of spherical Ag NPs from Sambucus nigra fruit extract has been reported by Moldovan et al. [ 144 ]. XRD analysis showed them to be crystalline. The in vivo antioxidant activity was investigated against Wistar rats which showed promising activity. It suggests that functionalization of Ag NPs with natural phytochemicals may protect the cell proteins from ROS production. Ag NPs have also been synthesized from aqueous leaf extract of Artocapus altilis . They were moderately antimicrobial and antioxidant. Thalictrum foliolosum root extract mediated Ag NPs synthesis has been confirmed on the basis of the appearance of a sharp peak at 420 nm in UV–vis region of the spectrum [ 239 ]. The monodispersed spherical nanoparticle of 15–30 nm had face centered cubic geometry. Shape and size dependent controlled synthesis of Ag NPs from Aloe vera plant extract and their antimicrobial efficiency has been reported by Logaranjan et al. [ 35 ]. The UV–vis peak at 420 nm confirmed the formation of silver nanoparticles. After microwave irradiation of the sample, Ag NPs of 5–50 nm with octahedral geometry was obtained. Nearly two to fourfold antibacterial activity of Ag NPs was observed compared to commonly available antibiotic drugs. Biosynthesis of Ag NPs from the aqueous extract of Piper longum fruit extract has been also achieved [ 240 ]. The nanoparticles were spherical in shape with an average particle size of 46 nm determined by SEM and dynamic light scattering (DLS) analyser. The polyphenols present in the extract are believed to act as a stabilizer of silver nanoparticles. The fruit extract and the stabilized nanoparticles showed antioxidant properties in vitro. The nanoparticles were found to be more potent against pathogenic bacteria than the flower extract of P. longum . Ag NPs have been fabricated from leaf extract of Ceropegia thwaitesii and formation was confirmed from absorption of SPR at 430 nm. The nanoparticles of nearly 100 nm diameter were crystalline in nature [ 139 ]. Plant extract of Ocimum tenuiflorum , Solanum tricobatum , Syzygium cumini , Centella asiatica and Citrus sinensis have been used to synthesize Ag NPs of different sizes in colloidal form [ 249 ]. The size of all nanoparticles was found to be 22–65 nm. They were all stable and well dispersed in solution. Niraimathi and co-workers [ 140 ] have reported biosynthesis of Ag NPs from aqueous extract of Alternanthera sessilis and showed that the extract contains alkaloids, tannins, ascorbic acid, carbohydrates and proteins which serve as reducing as well as capping agents. Biomolecules in the extract also acted as stabilizers for silver nanoparticles. Ag NPs from seed powder extract of Artocarpus heterophyllus have been synthesized [ 138 ]. The morphology and crystalline phase of the nanoparticles were determined by SEM, TEM and SAED, EDAX and IR spectroscopy. They were found to be irregular in shape. The extract was found to contain amino acids, amides etc. which acted as reducing agents for AgNO 3 to produce silver nanoparticles. The quantity of phenols, anthocyanins and benzoic acid were determined in the berry juices and were responsible for the transformation of silver ions to Ag NPs [ 241 ]. UV–vis spectra displayed an absorbance peak at 486 nm for lingonberry and 520 nm for cranberry containing silver nanoparticles. Since the two absorption peaks are different they cannot be assigned only to Ag NPs but also partly to different quantities of the reducing chemicals present in the juices. However, the spectra indicated the presence of polydispersed silver nanoparticles. Puiso et al. [ 241 ] have proposed that due to irradiation of water by UV rays, strong oxidants and reductants as photolysis products are formed. They reduce silver ions to Ag NPs or silver oxide. The photolysis products may produce oxidant and reductant but it depends upon the quantum of radiation and exposure time which may not be enough to produce a sufficient quantity of redox chemicals to reduce Ag + to Ag NPs or Ag 2 O. This hypothesis is conceptually incorrect because Ag 2 O cannot be formed as it requires a very strong oxidizing agent. On the other hand, AgNO 3 itself is slowly reduced in water, but in the presence of reducing agents the reaction proceeds at a rapid rate. The SPR is dependent on the size, shape and agglomeration of Ag NPs which is reflected from the UV–vis spectra [ 242 ]. Mock et al. [ 243 ] have found different scattered colors in hyperspectral microscopic images which are mainly due to the different shape and size of silver nanoparticle in the colloidal solution. The blue, green, yellow and red colors have been attributed to spherical, pentagonal, round-triangle and triangle shapes, respectively.

Zaheer and Rafiuddin [ 12 ] have reported the synthesis of Ag NPs using oxalic acid as reducing agent and mistook it as green synthesis. Formation of nanoparticles was confirmed by a change in color of the solution which showed an absorption peak at 425 nm (Fig.  2 a) in the UV–visible region. It was also noted that a scattered silver film was formed on the wall of the container that shines and reflects light (Fig.  2 b) which is the characteristic of monodispersed spherical Ag NPs [ 244 , 245 ]. Since the size of nanoparticles varies between 7 and 19 nm the silver film is not uniform. It is different from regular silver mirror due to irregular shape and size of nanoparticles (Fig.  2 c). Actually, very small size nanoparticles can be obtained when AgNO 3 is exposed to a reducing agent for a longer duration of time [ 246 ]. The kinetics and mechanism proposed for the formation of Ag NPs by oxalic acid is not convincing [ 12 ] because oxalic acid in no case can produce CO 2 unless it reacts with any carbonate salt or heated at a very high temperature. The authors [ 12 ] have proposed following reactions to prove that the colour of Ag NPs in solution is due to Ag 2+ 4 formation that absorbs at 425 nm (Scheme  1 ). The formation of Ag 2+ 4 is highly improbable even if the above reaction is kinetically very fast. Also, the stabilization of Ag 2+ 4 is questionable (Scheme  1 ). This hypothesis of Ag 2+ 4 formation is beyond imagination and does not carry any experimental evidence in its support. Absorbance of Ag NPs in solution varies between 400 and 445 nm depending on the nature of reducing agent used for their fabrication. The SPR band in UV–vis spectrum is due to electron oscillation around the surface of nanoparticles. The reduction process is instantaneous and no further spectral change occurs after 60 min. Indicating the completion of redox process. Ag NPs are circular, triangular, hexagonal and polydispersed at 70 °C. The EDAX and XRD spectra support each other.

a UV–visible spectra of yellow color silver solution. b and c SEM images of the self-assembled silver nanoparticle mirror like illumination on the walls of the glass. Reaction conditions: [Ag + ] = 20.0 × 10 −4  mol dm −3 ; [oxalic acid] = 4.0 × 10 −4 mol dm −3 ; [CTAB] = 10.0 × 10 −4 mol dm −3 ; temperature = 30 °C [ 12 ]

Reduction of Ag + ions by oxalic acid [ 12 ]

Synthesis of Ag NPs from aqueous extract of Cleistanthus collinus and their characterization by UV–vis, FTIR, SEM, TEM and XRD has been reported by Kanipandian et al. [ 247 ]. The crystalline Ag NPs of 20–40 nm showed significant free radical scavenging capacity. Tippayawat et al. [ 27 ] have reported a green and facile synthesis of Ag NPs from Aloe vera plant extract. They were characterized by UV–vis, SEM, TEM and XRD. Fabrication of Ag NPs was confirmed on the basis of the appearance of a sharp peak at 420 nm in UV–vis region of the spectrum. In addition, they have reported that the reaction time and temperature markedly influence the fabrication of silver nanostructures. Ag NPs were spherical in shape and particle size ranged from 70.70 ± 22 to 192.02 ± 53 nm. Their size changes with time and temperature of the reaction mixture used during fabrication (Fig.  3 ).

SEM images of silver nanoparticles were obtained at a 100 °C for 6 h, b 150 °C for 6 h, c 200 °C for 6 h, d 100 °C for 12 h, e 150 °C for 12 h and f 200 °C for 12 h [ 36 ]

Green synthesis of Ag NPs from Boerhaavia diffusa plant extract has been reported by Vijay Kumar et al. [ 136 ] where the extract acted as both the reducing as well as capping agent. The colloidal solution of Ag NPs showed an absorption maximum at 418 nm in the UV–vis spectrum. The XRD and TEM analyses revealed a face centered cubic structure with an average particle size of 25 nm. Ag NPs of 5–60 nm have been synthesized from Dryopteris crassirhizoma rhizome extract in presence of sunlight/LED in 30 min [ 235 ]. XRD studies showed face centered cubic structure of silver nanoparticles.

Green synthesis of Ag NPs using 1 mM aqueous AgNO 3 and the leaf extract of Musa balbisiana (banana), Azadirachta indica (neem) and Ocimum tenuiflorum (black tulsi) has been done [ 248 ]. They were characterized by UV–vis, SEM, TEM, DLS, EDS and FTIR spectroscopy. They were found to accelerate the germination rate of Vigna radiata (Moong Bean) and Cicer arietinum (Chickpea). It is therefore, believed that Ag NPs are not toxic to such crops at germination level. Stable and capped Ag NPs from aqueous fruit extract of Syzygium alternifolium of 5–68 nm have been synthesized [ 92 ]. Nearly 12.7% of silver was detected from EDAX. The polydispersed spherical nanoparticles were capped and stabilized by the phenols and proteins present in the fruit extract. Biosynthesis of Ag NPs from methanolic leaf extract of Leptadenia reticulate has been done [ 142 ]. They were crystalline, face centred and spherical particles of 50–70 nm. They exhibited antibacterial activity and radical scavenging activity. Purple sweet potato ( Ipomoea batatas L.) root extract has been exploited to synthesize Ag NPs [ 143 ]. Organic components in the extract acted both as reducing and capping agents. Ag NPs have shown remarkable antibacterial activity against four clinical and four aquatic pathogens. Sweet potato root extract is known to contain glycoalkaloids, mucin, dioscin, choline, polyphenols and anthocyanins which function as antioxidant, free radical scavenger, antibacterial agent and reducing agents. In presence of Ag NPs these functions are further enhanced.

Cytotoxicity of silver nanoparticles

Cytotoxicity of nanomaterials depends on their size, shape, coating/capping agent and the type of pathogens against which their toxicity is investigated. Nanoparticles synthesized from green method are generally more toxic than those obtained from the non-green method. Some pathogens are more prone to nanomaterials, especially Ag NPs than others due to the presence of both the Ag ions released and Ag NPs. They slowly envelop the microbes and enter into the cell inhibiting their vital functions. It is clear that the fabrication and application of nanoparticles has resulted in public awareness of their toxicity and impact on the environment [ 249 , 250 ]. Nanoparticles are relatively more toxic than bulk materials. They are toxic at cellular, subcellular and biomolecular levels [ 251 ]. Oxidative stress and severe lipid peroxidation have been noticed in fish brain tissue on exposure to nanomaterials [ 252 ]. The cytotoxicity by Ag NPs is believed to be produced through reactive oxygen species (ROS) as a consequence of which a reduction in glutathione level and an increase in ROS level occur. From in vitro studies on animal tissue and cultured cells, Kim and Ryu [ 253 ] have observed an increase in oxidative stress, apoptosis and genotoxicity when exposed to silver nanoparticles. Since such studies have been made with varying sizes of Ag NPs and coatings under different conditions a direct correlation cannot be made. Hackenberg and coworkers [ 254 ] reported reduced viability at a dose of 10 µg/mL of Ag NPs of over 50 nm size in human mesenchymal cells whereas some people reported no toxicity [ 255 ] even at a higher dose (100 µg/mL). Besides, stability and aging of the sample are also important factors as an increase in toxicity has been reported by aged Ag NPs stored in water for 6 months which is related to the release of silver ions [ 256 ]. It seems that the toxicity is a cumulative effect of Ag NPs and silver ions. Some workers have shown that the toxicity of Ag NPs is due to released Ag ions [ 257 ] while others have attributed the toxicity to Ag NPs [ 258 ].

Vijay Kumar et al. [ 136 ] obtained Ag NPs from B. diffusa plant extract and tested them against three fish bacterial pathogens. It was found that Ag NPs were most effective against Flavobacterium branchiophilum . Ag NPs fabricated from P. longum fruit extract exhibited cytotoxic effect against MCF-7 breast cancer cell lines with an IC 50 of 67 μg/mL/24 h [ 240 ]. They also exhibited antioxidant and antimicrobial effects. Ag NPs were produced by using P. endlicherianum plant extract; and have shown that the inhibitory activity was increased against gram positive and gram negative bacteria when they were exposed to Ag NPs at a very low dose of 7.81 to 6.25 ppm [ 137 ]. Latha et al. [ 89 ] have fabricated Ag NPs from leaf extract of Adathoda vasica and studied their antimicrobial activity against Vibrio parahaemolyticus in agar medium. The nanoparticles were found to be significantly active against V. parahaemolyticus but were nontoxic to Artemia nauplii. V. parahaemolyticus is a prevalent sea food borne enteropathogen which is closely associated with mortality in Siberian tooth carps, milk fish [ 259 ], abalone [ 260 ] and shrimps [ 251 ]. Vibrio infection in cultured fish and shrimps causes large scale mortality. Quite often, the whole population perishes. The use of antibiotic has made them resistant. Under such conditions, Ag NPs have appeared as an effective remedy which saves shrimps from perishing. Ag NPs from seed powder extract of A. heterophyllus have also exhibited antibacterial activity against gram positive and gram negative bacteria [ 138 ].

Ag NPs fabricated from leaf extract of C. thwaitesii have shown antibacterial efficacy against Salmonella typhi , Shigella flexneri and Klbsiella pneumoniae indicating them to be significant. Niraimathi and co-workers [ 140 ] have also fabricated Ag NPs from aqueous extract of A. sessilis and showed significant antibacterial and antioxidant activities. Ag NPs from Ocimum tenuiflorum , Solanum tricobatum , Syzygium cumini , Centella asiatica and Citrus sinensis have also shown antibacterial activity against S. aureus , P. aeruginosa , E. coli and K. pneumoniae . The highest activity of nanoparticles was observed against S. aureus and E. coli [ 261 ]. Antimicrobial activity of colloidal Ag NPs was found to be higher than the plant extract alone. Lee et al. [ 141 ] synthesized Ag NPs from Dryopteris crassirhizoma and found them to be highly effective against B. cereus and P. aeruginosa . Similarly, Ag NPs obtained from leaf extract of banana, neem and black tulsi were also active against E. coli and Bacillus sp. [ 248 ]. Hazarika et al. [ 239 ] have performed antimicrobial screening of Ag NPs obtained from T. foliolosum root extract against six bacteria and three fungi which showed morphological changes in the bacterial cells. Fabricated of Ag NPs from Millettia pinnata flower extract and their characterization together with anti-cholinesterase, antibacterial and cytotoxic activities have been reported by Rajakumar et al. [ 145 ]. Spherical shaped Ag NPs ranging from 16 to 38 nm exhibited excellent inhibitory efficacy against acetyl cholinesterase and butyl cholinesterase. They also exhibited cytotoxic effects against brine shrimp.

Ag NPs obtained from S. alternifolium have also exhibited high toxicity towards bacterial and fungal isolates [ 92 ]. Ag NPs fabricated from L. reticulate [ 142 ] were found to be toxic to HCT15 cancer cell line. Kanipandian et al. [ 247 ] have reported that Ag NPs obtained from C. collinus aqueous extract exhibit dose dependent effects against human lung cancer cell (A549) and normal cell (HBL-100). The IC 50 for cancer cells was very low (30 µg/mL) but since Ag NPs synthesized from C. collinus were toxic to normal cells they cannot be used in vivo. However, if the plant extract contains some antioxidants, the whole mixture may exhibit this property but the nanoparticles alone are incapable to do so. Ag NPs from Aloe vera plant extract have shown varying degrees of antibactericidal effects [ 36 ]. Ag NPs obtained at 100 °C for 6 h and 200 °C for 12 h (varying temperature and reaction time) exhibited change in bacterial cell membrane when contacted with the nanoparticles (Fig.  4 ). They were more effective for gram negative bacteria ( P. aeruginosa , ATCC27803). In addition, they have also shown minimal cytotoxicity to human peripheral blood mononuclear cells.

SEM images of the bacterial strains. a Staphylococcus epidermidis , Gram-positive, b Pseudomonas aeruginosa , Gram-negative, c S. epidermidis treated with 100-6 h silver nanoparticles (0.04 mg/mL), d P. aeruginosa treated with 100–6 h silver nanoparticles (0.04 mg/mL) [ 36 ]

The particle size, agglomeration and sedimentation are related to the cytotoxicity of silver nanoparticles. It has been demonstrated from Alamar Blue (AB) and Lactate dehydrogenase test (LDH) that Ag NPs of 10 nm coated with citrate and PVP separately, are toxic to human lung cells [ 262 ] when exposed for 24 h. AB test is a measure of cell proliferation and mitochondrial activity. However, the LDH measures the cytotoxicity of Ag NPs in terms of membrane damage from the cytoplasm. Both the citrate and PVP coated nanoparticles of 10 nm exhibited significant toxicity after 24 h at the highest dose of 50 µg/mL. Ag NPs of larger dimensions did not alter cell viability [ 263 , 264 ]. Cytotoxicity is related to enzyme inhibition which is correlated to the release of Ag ions because they inhibit the catalytic activity of LDH.

It has been observed that Ag NPs damaged DNA but they did not increase ROS when cells were exposed to them for 24 h at a dose of 20 µg/mL [ 263 ]. Gliga et al. [ 262 ] have suggested that silver ions from AgCl are released in the biological fluid and complexed. The formation of AgCl is possible only if the fluid is contaminated with Cl − ions, nevertheless it cannot ionize to Ag + and Cl − ions since AgCl is almost insoluble in aqueous medium [ 265 ]. The experiment with extracellularly released silver ions in cell medium did not exhibit toxicity, perhaps it would have reacted with Cl − ions to yield insoluble AgCl.

Cytotoxicity is related to the size of Ag NPs irrespective of the coating agent. Carlson et al. [ 266 ] have shown an increase in ROS production for 15 nm hydrocarbon coated Ag NPs relative to 55 nm. It has been reported by Liu et al. [ 267 ] that 5 nm Ag-nanoparticles were more toxic than 20 and 50 nm nanoparticles to four cell lines, namely, A549, HePG2, MCF-7 and SGC-7901. Wang et al. [ 268 ] have also reported that smaller nanoparticles (10–20 nm) induce greater cytotoxicity than the larger ones (110 nm), and citrate coated 20 nm Ag NPs produced acute neutrophilic inflammation in the lungs of mice compared to those with larger ones. The cell viability and DNA damage may be explained by ROS generation [ 269 ] which may be contradictory to findings by others in in vitro studies [ 253 ].

It is hypothesized that irreparable DNA damage is due to the interaction of Ag NPs with repair pathways. Since this work has been done in vitro, the DNA once damaged may not have the ability to repair. However, in living systems the cells have the ability to undergo repair and multiply but such experiments have seldom been done. It is however, unanimously agreed that both Ag NPs and silver ions are present at the subcellular level. The transformation of Ag to Ag + ions occurs due to their interaction with biomolecules in the cell membrane. The release of elemental silver is directly proportional to the size of nanoparticles in a non-linear fashion [ 270 ]. The size dependent toxicity is related to the intracellular release of silver ions. Although, agglomeration of nanoparticles reduces their release, the antibacterial effect was hindered under anaerobic condition, because in absence of oxygen, the oxidation process Ag → Ag + ceases to continue. Ag NPs exhibited excellent activity against Y. enterocolitica , P. vulgaris , E. coli , S. aureus and S. faecalis . Since the nanoparticles are smaller than the bacterial cell they may stick to their cell walls disallowing permeation of essential nutrients leading to the death of microorganisms [ 236 ]. Smaller size is related to greater surface area of nanoparticles and their agglomeration around the cell wall inhibits the cell division of microbes.

Besides their application in diverse areas, Ag NPs are extensively used as antioxidant and antimicrobial agents regardless of the process of their synthesis [ 271 , 272 ]. They are more toxic to microorganisms than human beings. Antibacterial and antifungal activities of Ag NPs were tested against B. cereus , S. aureus , C. koseri , P. aeruginosa bacteria and C. albicans fungus respectively. It has been proposed that Ag NPs penetrate into the bacterial cell and interact with the thiol, hydroxyl and carboxyl groups of the biomolecules present in them, eventually deactivating the vital functions by releasing Ag + ions. The authors have, however, not explained how the Ag + ions were produced. We firmly believe that silver ions must have been produced through a redox mechanism and subsequently complexed with electron donating thiol and phosphate groups inhibiting the cell replication of pathogens. It is well known that silver ions strongly bind with sulfur and oxygen containing electron donor groups in living system and arrest the functioning of vital organs that lead to the death of animal.

Ag NPs synthesized from lingonberry and cranberry juices [ 241 ] were tested for their activity against microbes commonly found in food and food products namely, S. aureus , S. typhi , L. monocytogenes , B. cereus , E. coli , B. subtillis and C. albicans . They observed that Ag NPs were more effective towards S. aureus , B. subtillis and B. cereus . Antibacterial activity was screened against B. cereus and S. aureus which produce toxins in food products [ 243 ]. A similar study has also been reported by Nanda and Saravanan [ 168 ] on other pathogens such as S. aureus , S. epidermidies and S. pyogens . The decrease in antimicrobial effect of Ag NPs against food borne bacteria has been ascribed to low pH or high NaCl content in food. The high concentration of NaCl may increase the toxicity towards bacteria because they may kill them. However, it is concluded that Ag NPs may be used in packaging to prevent infection in food products by microbes.

Zhao and Stevens [ 273 ] have studied antimicrobial effects of Ag salts on 12 species of bacteria and showed that they are highly effective against them. It has also been shown [ 274 ] that Ag NPs with amphiphilic hyperbranched macro molecules act as antimicrobial coating agents. Kim et al. [ 275 ] have thoroughly screened the antimicrobial effect of Ag NPs prepared from AgNO 3 and NaBH 4 as reducing agent. They examined the efficacy of a wide range of concentrations of Ag NPs starting from 0.2 to 33 nM. At a concentration of 33 nM of Ag NPs the growth inhibition of E. coli and E. aureus was almost comparable with the positive control, although at 13.2 nM concentration a significant effect was observed. However, the inhibitory effect of 1.6–6.6 nM of Ag NPs is nearly the same (~ 55% relative to control). It was observed that silver nanoparticle is most effective against E. coli and has a mild inhibitory effect on S. aureus . However, gold nanoparticles of the same concentration were ineffective against these microbes, although it also belongs to the same group of elements.

Ag NPs synthesized from fungus Humicola sp. were investigated for their cytotoxicity on NIH3T3 mouse embryonic fibroblast cell line and MDA-MB-231 human breast carcinoma cell line [ 224 ]. In both cell lines, the cell viability declined in a dose-dependent manner. Cytotoxicity of Ag NPs was recorded at a concentration of 250 µg/mL; the cell viability declined by 20 83% in the case of NIH3T3 and 42 18% for MDA-MB-231 cell line at 1000 µg/mL concentration. Very recently [ 269 ], it has been investigated that Ag NPs in conjugation with other metals such as TiO 2 @Ag nanoparticles act against leishmaniasis. These nanoparticles along with other drugs for leishmania, like neglumine antimoniate at nontoxic concentrations increase the efficacy of both drugs. This combination of drug led to the inhibition of L. tropica amastigotes at a very high rate of 80–95%. Also, it increased the metabolic activities 7–20-fold.

Owaid et al. [ 237 ] have produced Ag NPs from aqueous extract of P. cornucopiae var. citrinopileatus which served both as reducing and stabilizing agent. Their antimicrobial activity was investigated against four pathogenic Candida sp. namely C . albicans, C. glabrate, C. krusei and C. pseudotropicalis . Ag NPs at 60 µg/well showed a significant increase in inhibition of candida sp. However, pure extract was ineffective against all microbes at 20–40 µg/well. Mechanism of action has been ascribed to the interaction between the positive charge on silver ion and the negative charge on the cell membrane of microorganism [ 25 , 35 ]. Due to electrostatic attraction between the two the silver ions penetrate into the microbial cell via diffusion leading to their death. Ag NPs synthesized using fungus Trichoderma viride were examined for their antimicrobial activity in combination with various antibiotics (ampicillin, kanamycin, erythromycin and chloramphenicol) against both gram positive and gram negative bacteria [ 234 ]. Antibacterial activities of antibiotics were increased in the presence of Ag NPs against the tested strains and P. aeruginosa . The original aqueous extract of P. ostreatus was found to be ineffective against all bacterial strains at 25–75 µg/mL.

Allahverdiyev et al. [ 276 ] have reported that the combination of Ag NPs with antibiotics decreases the toxicity toward human cells by reducing the required dosage. Furthermore, these combinations restore the ability of the drug to kill bacteria that have acquired resistance to them [ 175 ]. Hence, a separate approach of using Ag NPs synthesized from bacterial strains alone and in combination can act as effective novel antimicrobials to sensitize resistant pathogens. Nevertheless, a study with E. coli has demonstrated that the bacteria could become resistant to Ag NPs on its regular exposure for 225 generations through genetic mutations [ 277 ]. Thus, a precaution should be taken to avoid the constant exposure of microorganisms against such types of nanoparticles. In addition, treatment with bacterial Ag NPs has shown the cell viability reduction in a dose-dependent manner in HeLa cervical cancer [ 278 , 279 ], MDA-MB-231breast cancer [ 280 ], A549 adenocarcinoma lung cancer [ 281 ] and HEP2 [ 282 ] cell lines. Ag NPs produced from bacterial strains exhibited cytotoxicity to cancer cells but their impact on normal healthy cells cannot be ignored.

Mechanism of antibacterial activity

As discussed previously, several reports are available which have shown that Ag NPs are effective against pathogenic organisms namely B. subtilis , Vibrio cholerae , E. coli , P. aeruginosa , S. aureus , Syphilis typhus etc. [ 10 , 11 , 109 , 145 ]. Ag NPs with larger surface area provide a better contact with microorganisms [ 283 ]. Thus, these particles are capable to penetrate the cell membrane or attach to the bacterial surface based on their size. In addition, they were reported to be highly toxic to the bacterial strains and their antibacterial efficiency is increased by lowering the particle size [ 284 ]. Many arguments have been given to explain the mechanism of growth inhibition of microbes by Ag NPs but most convincing is the formation of free radical which has also been supported by the appearance of a peak at 336.33 in the electron spin resonance (ESR) spectrum of Ag NPs [ 275 ]. The free radical generation is quite obvious because in a living system they can attack membrane lipids followed by their dissociation, damage and eventually inhibiting the growth of these microbes [ 285 ]. It is worth noting that the equal mass of silver Ag NPs and that of Ag ions exhibit identical growth inhibition of E. coli and S. aureus . In a study, the highly antibacterial activity has been ascribed to the release of silver cation from Ag NPs [ 173 ]. The Ag+ permeated into bacteria through the cell wall [ 286 , 287 ] as a consequence of which the cell wall ruptures leading to denaturation of protein and death. Since Ag ions are positively charged and much smaller than neutral Ag NPs they can easily interact with electron rich biomolecules in the bacterial cell wall containing S or P and N. Some researchers have reported that interaction between the positive charge on Ag NPs and negative charge on the cell membrane of the microorganisms is the key to growth inhibition of the microbes [ 286 , 287 ]. On the other hand, Sondi et al. [ 288 ] have reported that antibacterial activity of Ag NPs toward gram negative bacteria depends on its concentration. The nanoparticles form pits in the cell wall of microbes, get accumulated, and permeate into the bacterial cell leading to their death. It has been reported [ 289 , 290 ] that Ag free radical formation and antimicrobial property are inter related which has been confirmed by ESR [ 275 ]. They claim that such an antimicrobial study included both the positively charged silver ions and negatively charged silver nanoparticles.

The absorption of Ag NPs at 391 nm is the signature of spherical nanoparticles due to their surface plasmon resonance [ 291 ]. This absorption spectrum does not undergo any change even when the suspension of Ag NPs is diluted ten times indicating that they are not agglomerated. Besides Ag NPs and silver compounds, there are other inorganic ions which also possess antibacterial properties [ 241 , 287 , 292 ]. It is known that silver ions bind to the protein of the microorganisms preventing their further replication but the organisms also avoid interacting with these ions and produce cysts to become resistant.

Ag NPs may be oxidized to Ag + but cannot be reduced [ 287 , 289 ]. Silver is known to have 4 d 10 , 5 s 1 outermost electronic configuration and it cannot hold an extra electron to become Ag − anion. Silver salt of sulphathiazine is used in burn therapy to protect the skin from infection by pseudomonas species. Silver is released slowly from the salt which is sufficiently toxic to microorganisms. Since the salt is sparingly soluble the silver acts on the external cell structure. Silver salt and Ag NPs exhibit cytotoxicity against a broad range of microorganisms, although the toxicity depends on the quantum of silver ions released [ 275 ].

The monodispersed nanoparticles of uniform size are produced. Graphene oxide exhibits antibacterial activity against E. coli [ 293 , 294 ] but Ag NPs functionalized graphene based material show enhanced antibacterial activity [ 295 , 296 ]. Graphene oxide is nicely dispersed in polar solvents like water which allows the deposition of nanoparticle for its use in various fields. Antibacterial activity of both Ag NPs and Ag-graphene oxide composite has been tested in a wide range of concentration between 6.25 and 100 µg/mL against both gram positive and gram negative bacteria. It was noticed that both Ag NPs and Ag-graphene oxide composite were more effective against gram positive than gram negative bacterial strains. Ag-graphene oxide is a better growth inhibitor of S. Typhi , even at a very low concentration of 6.25 µg/mL, than Ag NPs of the same concentration. However, Ag NPs and Ag-graphene oxide do not show any inhibitory effect against gram positive bacteria, S. aureus and S. epidermis below 50 µg/mL. It was also noted that graphene oxide alone is ineffective against these bacteria even at a higher concentration of 100 µg/mL [ 293 , 296 ].

Silver ions released from Ag NPs may penetrate into bacterial cell components such as peptidoglycan, DNA and protein preventing them from further replication [ 297 , 298 ]. Release of Ag + ions means the oxidation of elemental silver which requires an oxidizing agent.

The organic groups like carbonyl and protein in the bacterial cell wall are electron donors rather than electron acceptors and hence they cannot produce Ag + ions from Ag atoms, nevertheless the Ag + ions are produced which confirms the presence of an oxidizing agent [ 296 , 299 ]. Ag + ions are thus bonded to the proteins of bacteria and inhibit their vital functions.

Tho et al. [ 300 ] have shown that spherical Ag NPs of 2.76–16.62 nm size fabricated from Nelumbo nucifera seed extract are highly toxic to Gram negative bacteria. The antibacterial property has been ascribed to the attachment of Ag NPs to the surface of cell membrane disallowing permeation and respiration of the cells.

The outer layer of gram negative bacteria is made up of a lipopolysaccharide layer and the inner layer is composed of a linear polysaccharide chain forming a three-dimensional network with peptides. Ag NPs get accumulated due to attraction between the negative charge on the polysaccharides and weak positive charge on the silver nanoparticles. It stops the cell replication of the microbes.

Toxicity by nanoparticles is generally triggered by the formation of free radicals, such as ROS [ 301 , 302 ]. If the ROS is produced it may cause membrane disruption and disturb the permeability. The mechanism of growth inhibition follows electrostatic interaction, adsorption and penetration of nanoparticles into the bacterial cell wall. Toxicity of nanoparticles also depends on composition, surface modification, intrinsic properties and type of microorganisms [ 9 , 303 , 304 , 305 , 306 ]. For instance, TiO 2 -nanoparticles can increase peroxidation of the lipid membrane disrupting the cell respiration [ 307 ]. The biogenic Ag NPs in combination with antibiotics like erythromycin, chloramphenicol, ampicilin and kanamycin enhance the toxicity against gram positive and gram negative bacteria [ 308 , 309 ]. A possible mechanism is presented in Fig.  5 . Besides, Ag NPs are also toxic to nitrifying bacteria [ 310 ]. The ROS include superoxide (O 2 − ), hydroxyl (·OH), peroxy (RCOO·) and hydrogen peroxide (H 2 O 2 ). RNS includes nitric oxide (NO·) and nitrogen dioxide (NO 2 − ) [ 311 , 312 ]. The cell replication and development of microbes in ROS containing atmosphere will cease to continue. However, this process may be delayed in presence of an antioxidant such as an enzyme or a non-enzymatic component which scavenges the free radicals [ 313 ].

Mechanism of action of silver nanoparticles against bacterial cells

Regardless of the method of fabrication, Ag NPs are used as an antimicrobial agent, electrochemical sensors, biosensors, in medicine, health care, agriculture and biotechnology. They have great bactericidal potential against both gram positive and gram negative pathogens. Since Ag NPs coupled with antibiotics are active against many drug resistant bacteria they can be used as easily accessible medicine for the treatment of several infections. Ag NPs in the drug delivery system, quite often increase the solubility, stability and bio-distribution enhancing their efficiency. In presence of nanoparticles the absorption of medicine increases several times therefore, Ag NPs may be used as a drug delivery system.

Although, the long-term effect of nanoparticles on human health and crops is not clear. A large number of nanoparticles are being explored in many areas of industry technology, biotechnology and agriculture. It is known that various forms of silver from laundry, paints, clothes etc. and biosolids reach the sewage and sludge. It has been reported that nano sized Ag 2 S are formed in the activated sludge as a consequence of the reaction between silver nanoparticles/Ag + ions and the sulfide produced in sewage. It is not possible for Ag NPs in the elemental form to react with evolved H 2 S. Only Ag + ions may react with H 2 S to yield Ag 2 S according to the reaction given below.

Ag 2 S or AgNO 3 may be ionized to give free Ag + ions which inhibit the bacterial growth. Besides many advantages of Ag NPs there are some disadvantages too. They inhibit the growth of nitrifying bacteria, thereby inhibiting the biological nitrogen removal. As little as 1–20 ppm Ag NPs have been reported to be effective against microbes. It is anticipated that Ag NPs may be used as an inexpensive broad spectrum antimicrobial agent to protect plant crops and infections in human beings.

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Azamal Husen

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Siddiqi, K.S., Husen, A. & Rao, R.A.K. A review on biosynthesis of silver nanoparticles and their biocidal properties. J Nanobiotechnol 16 , 14 (2018). https://doi.org/10.1186/s12951-018-0334-5

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• Published: 19 May 2022

Green synthesis and characterization of silver nanoparticles using Eugenia roxburghii DC. extract and activity against biofilm-producing bacteria

• Alok Kumar Giri 1 ,
• Biswajit Jena 1 ,
• Bhagyashree Biswal 1 ,
• Arun Kumar Pradhan 1 ,
• Manoranjan Arakha 1 ,
• Saumyaprava Acharya 2 &
• Laxmikanta Acharya   ORCID: orcid.org/0000-0002-8434-8500 1  

Scientific Reports volume  12 , Article number:  8383 ( 2022 ) Cite this article

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• Biotechnology
• Nanoscience and technology

The green synthesis of silver nanoparticles (AgNPs) and their applications have attracted many researchers as the AgNPs are used effectively in targeting specific tissues and pathogenic microorganisms. The purpose of this study is to synthesize and characterize silver nanoparticles from fully expanded leaves of Eugenia roxburghii DC., as well as to test their effectiveness in inhibiting biofilm production. In this study, at 0.1 mM concentration of silver nitrate (AgNO3), stable AgNPs were synthesized and authenticated by monitoring the color change of the solution from yellow to brown, which was confirmed with spectrophotometric detection of optical density. The crystalline nature of these AgNPs was detected through an X-Ray Diffraction (XRD) pattern. AgNPs were characterized through a high-resolution transmission electron microscope (HR-TEM) to study the morphology and size of the nanoparticles (NPs). A new biological approach was undertaken through the Congo Red Agar (CRA) plate assay by using the synthesized AgNPs against biofilm production. The AgNPs effectively inhibit biofilm formation and the biofilm-producing bacterial colonies. This could be a significant achievement in contending with many dynamic pathogens.

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Introduction.

Most of the plants found in the family Myrtaceae are medicinally important. The secondary metabolites found in these plants can be utilized to cure different diseases. Among the different genera, Eugenia is an important taxon in the family having active principles which have pharmaceutical importance. Eugenia species produce delicious edible fruits with high vitamin and mineral content. Eugenia roxburghii DC. is one such wild edible fruit-producing plant under the family Myrtaceae. It is also known as Roxburgh’s Cherry due to the deliciousness of its fruits. This plant species is mostly found in the coastal and tropical areas of India and Sri Lanka. This species, containing the various secondary metabolites, has anticancer and antibacterial activity 1 , 2 , 3 , 4 , 5 , 6 , 7 . Though there is no such systematic study on the medicinal utilization of the species, the plant is used to treat diseases associated with diabetes, arthritis, hypertension, etc., as revealed by the local people 8 , 9 .

The bioavailability of the active principle is drastically reduced when it is supplied in the form of crude extract, but this can be enhanced when the crude extract is supplied in a modified form like nanomaterial 10 . A nanoparticle (NP) is a microscopic particle having a high surface area. Synthesis of NPs has picked up most attention in recent years due to their vast application in areas like catalysis, optics, electronics 11 , 12 , 13 , antibacterial, and antimicrobial activity 14 , 15 , 16 . The physical and chemical properties of metallic nanoparticles are remarkably different from their corresponding bulk form and can be used as an anti-microbial agent 17 . Plant and plant parts can be used for the reduction of metal to prepare respective metal nanoparticles 18 , 19 . Among the different metallic NPs, silver nanoparticles (AgNPs) have enormous applications in the medical and biotechnological fields 20 . The synthesis of AgNPs can be achieved both chemically and physically. Physicochemical approaches, on the other hand, include drawbacks such as high running costs, the use of toxic chemicals, and increased energy limits. Physical operations are complex procedures that fail to regulate particle sizes in the nanoscale range. The biggest drawbacks are that they create irregularly sized particles and have a high manufacturing cost 21 . Chemically synthesized NPs are not cost-effective and harm the environment with high energy requirements 22 . This is when biological approaches employing less expensive sources are exploited as AgNPs precursors. The green synthesis of nanoparticles has gained a lot of attraction since it uses non-toxic phytochemicals and avoids the dangerous ingredients that would otherwise be used in chemical synthesis 23 . Green synthesis methods use extracts from diverse plant parts, microbial cells, and biopolymers, and are so classified as such. The nanoparticles created are biocompatible and have the correct level of efficacy for the purpose for which they were created 24 . Metallic NPs can be synthesized biologically using various plants and their extracts which are easily available in huge quantities. The plants and their extracts are safe to handle, less toxic and eco-friendly.

From the leaf extract of Eugenia jambolana, silver nanoparticle synthesis was carried out and their phytochemical screening was evaluated 25 . Earlier, reports are available regarding the formation of AgNPs and their biological applications from Syzygium cumini   26 , Eugenia caryophyllata 27 . From the leaf extract of Eugenia uniflora, silver nanoparticle formation was carried out and their antibacterial and antidiabetic potential were evaluated 28 .

Biofilm is a very fine extracellular polymer fibril that helps the bacteria adhere to the surface 29 . The bacterial community secrets an extracellular polymeric substance after adherence to a matrix or substratum which results in an alternation of phenotype and genetic change with the growth rate 30 . Bacteria forming biofilms possess great resistance to numerous stress conditions including some antibiotics, high salt concentration, acidic conditions, and many oxidizing agents, which results in increasing their pathogenicity 31 . Biofilm formation is seen in most medical devices, catheters, and other implants 32 .

Silver nanoparticle formation has already been reported from different plant extracts such as Azadirachta indica (Neem), Aloe vera , Emblica Officinalis (Amla), Cinnamomum camphora 19 , 33 , 34 , 35 , 36 . However, there is no such information about the synthesis of silver nanoparticles and any of their biological applications from the plant Eugenia roxburghii . Hence, in this study, an attempt was made to synthesize silver nanoparticles from the leaf extract and their activity against microbes. In our previous study, we found that leaf extract is highly effective in inhibiting the growth of microbes 37 . To enhance the antimicrobial activity of the leaf extract we have tried to prepare AgNPs from the extract to access the effect of the nanoparticles, we have used it to inhibit the growth of biofilms by S. aureus . As it has been seen that nanomaterial is better at combating microbes than normal crude extracts, our present investigation will help evaluate the antimicrobial effect of Eugenia AgNPs.

Characterization by UV–Vis spectrophotometer

UV–Vis spectrophotometric analysis was carried out for the primary investigation of silver nanoparticle synthesis. A color change has been observed in the mixture of plant extract and AgNPs. The color of the mixture gradually changes from green to yellowish-brown confirming the production of E. roxburghii AgNPs. The absorbance of the solution was investigated for one week. From the spectral analysis, it is observed that the AgNPs peak was obtained at 417 nm with the highest peak (Fig.  1 ) and was stable thereafter for a few days as there was no increase in the absorption.

UV–Vis absorption spectra of synthesized AgNPs from E. roxburghii leaf extract.

Characterization by XRD

The crystallinity of the synthesized silver nanoparticle using E. roxburghii leaf extract was examined through X-ray diffraction (XRD) (Fig.  2 ). The size of the nanoparticles was calculated based on the Debye–Scherrer equation: (D = kλ/βcosθ).

XRD pattern of AgNPs synthesized from E. roxburghii leaf extract.

In the above equation: D represents particle diameter size, K: a constant with a value of 0.9, λ: X-ray source wavelength (0.1541 nm), β and θ represent the FWHM (full width at half maximum), and diffraction angle concerning the (111) lattice planes respectively. The average crystalline size was found to be approximately 35 nm. The lattice parameters for the synthesized AgNPs were determined to be a = 0.4086 nm, b = 0.4086 nm, c = 0.4086 nm respectively. The calculated lattice value was 0.4086 nm, which was nearly identical to the normal lattice parameter of 0.4073 nm for silver 38 .

Characterization by HR-TEM

The resulted colloidal particles were characterized to determine their shape and size by high-resolution transmission electron microscopy (TEM). For the preparation of the TEM grid, a carbon-coated copper grid was used. A drop of the particle solution was placed over the grid and dried at room temperature. Different TEM micrograph images including SAED pattern and HR-TEM images of the synthesized AgNPs were obtained which are displayed in Fig.  3 a–d. The estimated average particle size was approximately 24 nm whereas particle sizes ranged from approximately 19–39 nm.

( a ) TEM micrographs image of the synthesized AgNPs, ( b ) TEM image of different sized AgNPs, ( c ) SAED image of AgNPs, ( d ) HR-TEM image of AgNPs.

Characterization by Zeta sizer

The surface potential of nanoparticles is the potential difference between the medium where nanoparticles are dispersed and the accessible surface of dispersed nanoparticles, which can be analyzed using a zeta sizer. Figure  4 demonstrates the zeta potential of the biosynthesized AgNPs which was found to be  − 37.8 mV. This shows that the AgNPs synthesized from the leaf extract of E. roxburghii are highly stable.

Zeta potential of synthesized AgNPs.

Analysis of antimicrobial activity

An antibacterial activity assay was carried out by using disc diffusion and the MIC method. It was observed that among all the bacteria taken, the AgNPs extract of E. roxburghii showed maximum effectiveness towards S. aureus (Fig.  5 a). So, the MIC experiment was continued with the selection of S. aureus bacterium and the MIC test revealed that there was a continuous increase in absorbance at 120 µg/ml concentration whereas, at 240 µg/ml concentration of extract, there was a continuous decrease in absorbance. However, there was no such change in absorbance observed in other concentrations of the extract (Fig.  5 b).

( a ) Antimicrobial activity test by Disc Diffusion Method against different bacterial strains, ( b ) Minimal Inhibitory Concentration test on S. aureus.

Examine the effect on biofilm

In this study, it was observed that bacteria changed their color in the control plate (CRA plate without AgNPs) whereas there was no change in the color of bacteria in AgNPs treated CRA plate (Fig.  6 ). This confirmed the direct inhibition of the biofilm production of bacteria by AgNPs.

Effect of AgNPs on biofilm production by S. aureus on Congo Red Agar plates. *Control = without AgNPs, Treated = With AgNPs.

The essential enzyme for nitrogen assimilation in a variety of species is nitrate reductase (NR) 39 , which catalyses the conversion of nitrate to nitrite in the cytoplasm of plant cells 40 . An enzymatic pathway involving NADPH-dependent reductase was shown to be responsible for the bioreduction of silver ions. Silver ions exposed to nitrate reductase resulted in the formation of very stable silver NPs and NADPH was found to be the cofactor of the nitrate reductase enzyme 41 . From a previous study, the absorption spectra of synthesized AgNPs from Syzygium Jambola was found to be 460 nm and its particle size from TEM analysis was found to be ranging from 6 to 23 nm 42 , similarly in Syzygium cumini the UV spectra of synthesized nanoparticle was observed at ~ 450 nm with particle size 3.5 nm from the XRD analysis 43 and also in Eugenia uniflora UV spectra of synthesized nanoparticle was observed at 440 nm having its particle size was ranging from 25 to 50 nm 44 .

In this study, after mixing of extract and silver nitrate solutions a color change of extract was observed over the progression of time which may be due to the reduction of the silver ions leading to the excitation of Surface Plasmon Resonance (SPR) of the AgNPs 45 . To confirm this, UV spectra analysis was carried out and a peak was observed at 417 nm which showed a stable range for nanoparticle formation.

From the XRD pattern of the silver nanoparticle, the structure obtained to be a face-centered cubic one 46 . Four Bragg’s reflections conforming to (111), (200), (220), and (311) planes of metallic silver with FCC crystal structures are understood clearly from the XRD plot (JCPDS No. 89-3722) 47 . So, in the present study, the average crystalline nanoparticle size was measured to be approximately 35 nm. The extra peak obtained at 2θ nearly equal to 28 may be due to the bio-organic phase crystallization over silver nanoparticles surface 48 .

From the resulting images of HR-TEM analysis of synthesized AgNPs, it was observed that there was a presence of few agglomerated AgNPs in some places (Fig.  3 a) which may be an indication of further sedimentation. Mostly spherical-shaped particles were observed with variations in their size (Fig.  3 b). The average particle size was measured to be approximately 24 nm and the overall particle size ranged between 19 and 39 nm. The electron beam was directed perpendicular to one of the spheres to obtain the SAED (selected area electron diffraction) pattern and the crystallinity of the synthesized AgNO 3 was confirmed through this pattern (Fig.  3 c) which was recorded from one of the nanoparticles. The morphology of a single AgNO 3 was obtained from the HR-TEM (high-resolution transmission electron microscopy) image and found to be spherical (Fig.  3 d).

The antimicrobial activity of silver nanoparticle extract of E. roxburghii was tested against four different types of bacteria viz . E. coli, P. aeruginosa, V. cholera and S. aureus. In the disc diffusion method, the nanoparticle extract showed a significant effect towards S. aureus among the above four bacteria for that reason MIC experiment was conducted by taking S. aureus bacteria against which different concentrations (120 µg/ml, 160 µg/ml, 200 µg/ml and 240 µg/ml) of nanoparticle extract were treated. In this experiment, while measuring OD, a continuous increase in absorbance at 120 µg/ml concentration of extract may suggest that at a low concentration the bacteria get dominant over the activity of the extract while a continued decrease in absorbance at 240 µg/ml concentration of extract may suggest that at this concentration the extract is efficient enough to remove the bacterial colony.

As the bacteria, S. aureus itself is a biofilm-producing bacterium, confirmed biofilm production was observed in the control plate as the plate contains Congo Red media turns into a back color. It was reported that the biofilm-producing capacity of pathogenic bacteria was due to the secretion of exopolysaccharides (EPS) 49 . The change of color from red to black in CRA plates is due to EPS secretion by bacteria 50 . Because of the clinical approach, nowadays biofilm production by the microbes and their growth on the surfaces of medicating instruments and disposable products are the major paths through which microbes enter into the body 51 , 52 . The biofilms are extremely resistant to host defense mechanisms and also to antibiotic treatment. Adhesion or attachment of microorganisms to a substrate is the first step towards colonization and this strategy has been used for microbial biofilm production 53 . In this study, a new approach was undertaken by synthesizing nanoparticles from biomaterial and using them against biofilm-producing microorganisms to test their effects on them.

Material and methods

Preparation of plant extract.

Fresh (disease-free) and fully expanded leaves of Eugenia roxburghii were collected with the permission of local authorities from the coastal area of Konark, Odisha (latitude 19.878 and longitude 86.101). The plant was taxonomically identified and authenticated by Dr. Laxmikanta Acharya (Associate professor, Centre for Biotechnology, Siksha ‘O’ Anusandhan University, Odisha, India) and a voucher specimen (SOAU/CBT/2020/ER/01) was retained in the department for future reference and the plant has been maintained in an environmentally controlled greenhouse. Experimental research on the plant used for the study complies with relevant institutional, national, and international guidelines and legislation. For the experiment, fresh and healthy leaves were taken and washed three times with distilled water. After washing, the methanolic extract was prepared by finely grinding 25 g of leaves with liquid nitrogen in a mortar and pestle followed by the addition of 250 ml of methanol. The debris from the leaf extract was separated with filter paper (Whatman No 1). The filtrate was collected and preserved at − 20 °C.

Preparation of AgNPs with Eugenia roxburghii leaf extract

One molar silver nitrate (AgNO 3 ) stock solution was prepared. From that stock solution, 0.1 mM AgNO3 solution was taken along with the leaf extract in a 5:1 proportion for the preparation of AgNPs. 20 ml of leaf extract was mixed with 100 ml of 0.1 mM AgNO 3 solution and incubated in a shaker incubator at 300 rpm at 37 °C for 48 h. Gradually the deep green color solution changed to yellowish-brown color which indicated the conversion of Ag + to Ag 0 (Fig.  7 ). The effect of this synthesis of silver nanoparticles was monitored in UV–VIS spectrophotometer. The spectrophotometric reading was taken at different time intervals.

Synthesis of AgNPs from E. roxburghii leaf extract.

Characterization of AgNPs

Various analytical techniques were used for the characterization of green synthesized silver nanoparticles from E. roxburghii leaf extract. Constant monitoring of the reaction for the reduction of Ag + ion by taking the OD (optical density) from 200–700 nm in a double beam UV–Vis spectrophotometer (Hitachi, UH5300). Further characterization of AgNPs was carried out through XRD (Rigaku, Ultima IV, Japan) equipped with Cu-Kα radiation; a crystal monochromator employing wavelengths of 0.1541 nm in a 2θ range from 20° to 80°. HR-TEM analysis of derived nanoparticles was carried out on a JEM 2100 (Jeol), operated at 200 kV.

Antimicrobial activity test

By disc diffusion method.

To check the antimicrobial activity of E. roxburghii AgNPs extract, a disc diffusion method was carried out. For this test, different strains of bacteria such as E. coli (ATCC-443) , P. aeruginosa (Clinically isolated from SCB medical college, Microbiology department, Cuttack, Odisha, India) , V. cholera (ATCC-3906), and S. aureus (ATCC-96) were used which were identified and confirmed at the Centre for Biotechnology, Siksha O’ Anusandhan (Deemed to be University), Odisha, India. Active bacterial cultures were revived by inoculating a loop full of bacterial culture in nutrient broth from the stock maintained at 4 °C and incubated overnight at 37 °C in a shaker incubator at 800 rpm. Nutrient agar plates were prepared and spreading of 60 µl of each bacterial culture was carried out. Different concentrations such as 120 µg/ml, 160 µg/ml, 200 µg/ml, and 240 µg/ml of AgNPs extract infused discs were prepared and placed over the bacterial spread plates followed by incubation overnight at 37 °C. the observed zone of inhibition was measured in mm against the commercially available antibiotic ciprofloxacin.

By minimal inhibitory concentration (MIC)

To evaluate the minimal inhibitory concentration, different concentrations (120 µg/ml, 160 µg/ml, 200 µg/ml and 240 µg/ml) of AgNPs extract were tested against S. aureus. For this experiment, 25 ml of nutrient broth was added to four different conical flasks containing different concentrations of the AgNPs extract mentioned above followed by the addition of 100 µl of bacterial culture. After the addition of bacterial culture, OD was measured at 600 nm in every 2 h interval of time from 0 to 12 h followed by incubation at 37 °C at 800 rpm.

Effect of AgNPs on Biofilm synthesis

In this study, the main target was to evaluate the effectiveness of AgNPs extract of E. roxburghii against biofilm production and this experiment was carried out by selecting the S. aureus bacterium. A Congo Red Agar (CRA) plate assay was carried out to investigate the activity of AgNPs on Biofilm production 54 . Two media plates named control and treated were taken in which the control plate was incorporated with Congo Red Dye mixed nutrient agar streaked with S. aureus bacteria and the treated plate was incorporated with a mixture of nutrient agar, Congo Red Dye, and AgNPs extract (0.065 g/ml) streaked with S. aureus . Then the plates were incubated at 34 °C for 3 days.

The silver nanoparticle prepared from E. roxburghii leaf extract were observed under UV–Vis Spectroscopy monitored at 417 nm and their crystallinity nature was confirmed from their XRD study. AgNPs are found to be very effective against biofilm production by bacteria. However, an experiment must be carried out to find the effect of the NPs on the animal model as well as on human beings for the evaluation of efficacy. Toxicological studies are also required to eradicate any kind of intoxication in a mouse model or human being. Once the NP is found nontoxic or safe in vivo studies, the nanoparticle can be utilized for the treatment of various diseases such as diabetes, arthritis, hypertension, etc. AgNPs play a major role in inhibiting bacterial colonies and biofilm formation. This study springs a new approach for synthesizing nanoparticles from the leaves of E. roxburghii which is found out to be inhibiting biofilm production and bacterial colonies can be a significant achievement in contending many dynamic pathogens. Other nanoparticles besides AgNPs can also be prepared from the leaf extract and their medicinal properties can be exploited for the remedy of various diseases. So, the present work can be considered an attempt to exploit the active principle present in the leaf of E. roxburghii to cure various ailments.

Abbreviations

Silver nanoparticles

Nanoparticles

Silver nitrate

Xray Diffraction

Transmission Electron Microscope

Selected Area Electron Diffraction

Full Width at Half Maximum

Face Centered Cubic

Congo Red Agar

Exopolysaccharide

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The President and Vice-Chancellor of SOA (Deemed to be University) are highly acknowledged for the infrastructure facility.

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Molecular Biology and Genetic Engineering Lab, Centre for Biotechnology, School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan (Deemed to Be University), Kalinga Nagar, Bhubaneswar, Odisha, 751003, India

Alok Kumar Giri, Biswajit Jena, Bhagyashree Biswal, Arun Kumar Pradhan, Manoranjan Arakha & Laxmikanta Acharya

Department of Nanotechnology, ITER, Siksha ‘O’ Anusandhan (Deemed to Be University) Jagmohan Nagar, Bhubaneswar, Odisha, 751030, India

Saumyaprava Acharya

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Sample collection and experiment were performed by G.A.K., J.B., and B.B. Data analysis was carried out by P.A.K., A.M., and A.S.. Final manuscript was written by G.A.K. Overall experiment was designed and guided by A.L. All authors have contributed to this study.

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Correspondence to Laxmikanta Acharya .

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Giri, A.K., Jena, B., Biswal, B. et al. Green synthesis and characterization of silver nanoparticles using Eugenia roxburghii DC. extract and activity against biofilm-producing bacteria. Sci Rep 12 , 8383 (2022). https://doi.org/10.1038/s41598-022-12484-y

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DOI : https://doi.org/10.1038/s41598-022-12484-y

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A review on plant-mediated synthesis of silver nanoparticles, their characterization and applications

Sandip Kumar Chandraker 1 , Mithun Kumar Ghosh 2 , Mishri Lal 1 and Ravindra Shukla 1

Published 31 May 2021 • © 2021 The Author(s). Published by IOP Publishing Ltd Nano Express , Volume 2 , Number 2 Citation Sandip Kumar Chandraker et al 2021 Nano Ex. 2 022008 DOI 10.1088/2632-959X/ac0355

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1 Laboratory of Bio-resource Technology, Department of Botany, Indira Gandhi National Tribal University, Amarkantak-484887, Madhya Pradesh, India

2 Department of Chemistry, Pandit S. N. Shukla University, Shahdol-484001, Madhya Pradesh, India

Sandip Kumar Chandraker https://orcid.org/0000-0001-9710-3652

Mithun Kumar Ghosh https://orcid.org/0000-0003-3686-1745

Ravindra Shukla https://orcid.org/0000-0002-3900-4653

• Received 23 January 2021
• Revised 14 May 2021
• Accepted 19 May 2021
• Published 31 May 2021

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Method : Single-anonymous Revisions: 1 Screened for originality? Yes

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For decades, silver has been used as a non-toxic inorganic antimicrobial agent. Silver has a lot of potential in a variety of biological/chemical applications, particularly in the form of nanoparticles (NPs). Eco-friendly synthesis approach for NPs are becoming more common in nanobiotechnology, and the demand for biological synthesis methods is growing, with the goal of eliminating hazardous and polluting agents. Cultures of bacteria, fungi, and algae, plant extracts, and other biomaterials are commonly used for NP synthesis in the 'green synthesis' process. Plant-based green synthesis is a simple, fast, dependable, cost-effective, environmentally sustainable, and one-step method that has a significant advantage over microbial synthesis due to the lengthy process of microbial isolation and pure culture maintenance. In this report, we focussed on phytosynthesis of silver nanoparticles (AgNPs) and their characterization using various techniques such as spectroscopy (UV–vis, FTIR), microscopy (TEM, SEM), X-Ray diffraction (XRD), and other particle analysis. The potential applications of AgNPs in a variety of biological and chemical fields are discussed.

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1. Introduction

Nanotechnology is the use of scientific expertise from the physical, chemical, and biological disciplines to manipulate and monitor the matter on a nanoscale. In 1971, Professor Norio Taniguchi from the University of Tokyo introduced the term 'nanotechnology' for the first time [ 1 ]. The idea of nanotechnology was first delivered by Richard Feynman in 1959, and explained by Drexler in 1986. In 1981, the cluster science and scanning tunneling microscope (STM) helped the growth of nanotechnology, whereas, fullerenes and carbon nanotubes were discovered concerning the developments of nanotechnology. Nanotechnology emerged from the industrial revolution in the 21 st century [ 1 ].

The breakthrough in nanotechnology has resulted in advances in material science and electronic devices. Fabrication of nanoparticles (NPs) was one of nanotechnology's early stages of growth. NPs are any materials with at least one dimension in the range of 1 to 100 nanometers. [ 2 , 3 ]. NPs have different physical and chemical properties than their bulk materials due to reduction at smaller scale. NPs may be formed by metals, minerals, and polymers [ 4 ]. Owing to the appearance of quantum effects, NPs vary from their corresponding bulk materials in terms of size and origin of phenomena such as coulomb blockade, superparamagnetism, plasmon resonance, and so on. The high surface-to-volume ratio of NPs is another consequence of their reduced size. NPs exhibit entirely new properties as a result of their precise characteristics, such as small size, large surface area, varying shape, and particle distribution [ 1 ].

1.1. Why silver nanoparticles?

One of the first and most obvious questions is that why we are interested in AgNPs and investigating their biological and chemical properties. Metal nanoparticles (MNPs) are being produced from the salts of various metals, including copper (Cu), iron (Fe), gold (Au), silver (Ag), platinum (Pt), palladium (Pd) among others. Amongst various metallic nanoparticles, AgNPs have several advantages. Although many are characterized as 'AgNPs', owing to large ratio of surface to bulk Ag atoms, some contain a high amount of Ag 2 O. In the last decade, AgNPs have been used in a wide range of electronic and medical devices, surgical instruments, bone cements, surgical masks, and other applications [ 5 – 8 ]. In addition, a particular amount of ionic silver is used to treat wounds. As a result, AgNPs are used to treat wounds and burns [ 9 – 11 ]. Since AgNPs have large scattering cross-sections and surface plasmon resonance, they are used in molecular labeling [ 12 ]. Thus, many AgNPs are now being recognized for their wide range of applications [ 13 – 15 ].

2. Methods involved in the synthesis of nanoparticles

On the basis of precursor used, the nanoparticles are synthesized by mainly two processes, (i) Top-down and (ii) Bottom-up approach. In the Top-down approach, bulk materials are converted to fine nanoparticles mostly by physical methods i.e. milling, grinding, and sputtering techniques. Whereas, in the Bottom-up approach, substances are synthesized from bottom level i.e. atom-by-atom, molecule-by-molecule, and cluster-by-cluster involving physical, chemical and biological processes as shown in figure 1 .

Figure 1.  Synthesis processes of nanoparticles.

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2.1. Physical methods

The physical approach is one of the common methods, where nanoparticles are synthesized by high energy ball milling (HEBM) [ 16 ], laser pyrolysis [ 17 ], electrospraying [ 18 ], laser ablation [ 19 ], and evaporation-condensation [ 20 ] . Some other physical techniques are also used for the synthesis of nanoparticles such as electromagnetic radation [ 21 ], annealing [ 22 ], arc-discharge technique [ 23 ], and atomization [ 24 ]. The physical approaches mostly based on Top-down phenomena are costly to carry out with more energy requirement i.e. high temperature and pressure. These physical processes are lengthy tasks, and the created NPs have a limited lifespan and poor thermal stability. However, physical modes of synthesis have many benefits over chemical approaches, including the absence of solvent contamination in the prepared thin films and the homogenous dispersion of synthesized nanoparticles [ 25 ].

2.2. Chemical methods

The hydrothermal, sol-gel method, microemulsion technique, chemical vapour synthesis, and polyol synthesis techniques are mainly used in chemical approaches. In chemical approaches, some reducing agents are used for reduction, which may be organic or inorganic compounds such as sodium citrate, NaBH 4 , hydroquinone, elemental hydrogen, and gallic acid. Chemical processes are usually carried out in the solution level, but the result can also be found as a precipitate, so these reactions are referred to as co-precipitation [ 26 ].

2.3. Biological methods

Biosynthetic routes are considered ecofriendly because the biological system, including bacteria, actinomycetes, fungi, yeasts, algae, and plants themselves or their active principles, act as a 'bio-laboratory' for the development of pure metal and metal oxide NPs using a biomimetic approach [ 27 ]. The main advantage of the biological method is that neither bulk machinery nor external sources of hazardous chemicals are required in the synthesis of nanoparticles, and thus there is no chance of environmental toxicity, in contrast to physico-chemical methods where reducing agents are highly reactive and toxic in nature. In most of the cases, the secondary metabolites of the biological system may act as capping, reducing, and stabilization agents. The deposition of biomolecules on the surface of NPs enables them biocompatible, and thus the bio-based nanostructure opens up a lot of possibilities in biomedicine and other fields. In addition, the biological procedures allow for the development of NPs with unique morphologies and sizes [ 27 ]. These bio-based protocols have been termed 'Green synthesis' in nanobiotechnology due to their focus on mild reaction conditions and nontoxic precursors to reduce generated waste and to implement environmental sustainability [ 28 ].

2.3.1. Fungi mediated

Various species of fungi like Phaenerocheate chrysosporium [ 29 ], Colletotrichum sp. [ 30 ], Fusarium oxysporum [ 31 ], Aspergillus clavatus [ 32 ], and Trichoderma longibrachiatum [ 33 ] have been used for the synthesis of nanoparticles due to having high binding and metal-accumulation ability. The synthesis of nanoparticles using fungi is facile and more advantageous than using other microorganisms because mycelial growth can easily be monitored in the laboratory than bacteria and actinomycetes.

2.3.2. Bacteria mediated

Silver, gold and other metallic nanoparticles have been synthesized from different bacterial species. A gram-positive Staphylococcus aureus [ 34 ], and gram-negative Acinetobacter calcoaceticus [ 35 ] were used to synthesize AgNPs with antimicrobial applications. A rod shaped bacterium Bacillus was preferred by the researchers, and its various species, like B. subtilis [ 36 ] B. amyloliqefaciens [ 37 ] B. megaterium [ 38 ], and B. flexus [ 39 ], were used for biosynthesis of the AgNPs. Psychrophilic and mesophilic bacteria were also used for the synthesis of antibacterial AgNPs [ 40 ]. The synthesized AgNPs have different shapes i.e., discoidal, spherical, triangular, hexagonal, and cuboidal. Saifuddin and his co-workers synthesized AgNPs from Bacillus subtilis under microwave conditions in the presence of water [ 41 ]. Shahverdi group synthesized AgNPs within 5 min by using Escherichia coli , Enterobacter cloacae, and Klebsiella pneumonia [ 42 ]. For the desired NPs, bacteria can be manipulated genetically without much difficulty and show high growth rate; however, sophisticated equipments are required to obtain clear filtrate from colloidal broth.

2.3.3. Algae mediated

The algae show fast propagation and capacity to accumulate and reduce inorganic metal ions. Therefore, NPs have also been synthesized by using microalgae, seaweeds and diatoms from the aquatic ecosystem [ 43 ]. The antibacterial and cytotoxic O-AgNPs was reported to be synthesized using a blue-green alga, Oscillatoria limnetica [ 44 ]. Jena et al synthesized stable AgNPs within 4–16 nm size range by using a microalga, Chlorococcum humicola [ 45 ]. Sargassum wightii , a brown marine alga was also reported for extracellular synthesis of AgNPS with antibacterial potential [ 46 ].

2.3.4. Plant mediated

The plant mediated green synthetic approach has been emerged a better alternative over abovementioned biological methods due to tedious process of procurement, isolation, purification, and maintenance of microbial culture with costly nutrient media [ 47 ]. Further, contamination of culture and less identified biocapping agents are another issues which render microbes and algae, less preferred than plants. Therefore, biosynthesis of AgNPs using plants and their by-products is most desirable among nano-biotechnologists due to easily available precursors, and cost-effective, rapid, eco-friendly, and non-pathogenic protocol. The phytomolecules like amino acids, proteins, terpenoides, polyphenols, favonoides, polysaccharides, vitamins, alkaloids, tannins, saponins, resins, fats and enzymes are playing a significant role as reducing and stabilizing agents in phytosynthesis of NPs [ 27 , 48 – 50 ]. Some examples of plant mediated green synthesized AgNPs are shown in table 1 .

Table 1.  List of some AgNPs with mediated plants and size.

3. Optimization of plant-mediated synthesis of AgNPs

Phytosynthesis of AgNPs depends upon various factors such as temperature, pH, reaction time, concentration of metal salts, and concentration of plant extracts.

3.1. Effect of reaction time

The reaction time influences the quality, morphology, and properties of silver nanoparticles [ 63 ]. For a long incubation period, aggregation and shrinkage can occur [ 64 ].

3.2. Effect of plant extracts concentration

The concentration of plant extracts plays a vital role to determine the size and shape of the synthesized silver nanoparticles. Dubey et al synthesized AgNPs and AuNPs by using different concentrations (0.5 ml, 1.0 ml, 2.8 ml and 4.8 ml) of Tanacetum vulgare fruit extract [ 65 ]. The absorption peaks of NPs were found to be increased by increasing the concentration of plant extracts [ 66 ]. Dwivedi et al synthesized AgNPs and AuNPs from leaf extract of the Chenopodium album , and they observed that particle size was decreased with increasing the concentration of plant extracts [ 67 ].

3.3. Effect of silver nitrate (AgNO 3 ) concentration

The concentration of metal salts plays an important role to determine the shape, rate of reduction process, and size of NPs. Dubey et al synthesized AgNPs and AuNPs by using tansy fruit extract with different concentrations of metal ions from 1 to 3 mM [ 65 ]. They observed that the large size of NPs was found at a higher concentration of the metal salt. Similarly, when Ag and AuNPs were synthesized by using leaf extract of Chenopodium album, the absorption peaks and particle size were increased with increasing the metal ion concentration. The yields of the synthesized NPs were also increased by increasing the concentration of metal ions [ 67 ].

3.4. Effect of pH

The formation of NPs also depends on the pH of the reaction medium [ 68 ]. From the literature review, lower pH values favoured the production of larger sized NPs [ 65 , 69 ]. The larger rod shapes AuNPs were synthesized using Avena sativa at pH 2, whereas smaller sized AuNPs were made at pH 3 and 4 [ 70 ]. Spherical shapes of AgNPs were reported by using Cinnamon zeylanicum at high pH 5 and above [ 71 ].

3.5. Effect of temperature

The shapes and sizes of the NPs are also affected by the temperature of the reaction mixture. when AuNPs were synthesized using the leaf extract of Cymbopogon flexosus , the resulting shape was nano-triangles at low temperature, whereas spherical shapes were formed at high temperature [ 72 ].

4. Applications of green synthesized AgNPs

AgNPs have been used in a number of different applications. Figure 2 depicts a few examples of applications in various fields.

Figure 2.  Multiple applications of AgNPs.

4.1. As a photocatalyst for the degradation of synthetic dyes

Degradation of dyes from contaminated water and soil using NPs has recently become a viable option. The photocatalytic activity of AgNPs have been investigated with different types of pollutant dyes such as Methylene blue (MB), Methyl orange (MO), Naphthol orange (NO), Methyl red (MR), and Malachite green (MG), etc in presence of sunlight. Jyoti et al synthesized AgNPs using leaf extracts of Zanthoxylum armatum and investigated their photocatalytic activity against synthetic dyes like MB, MO, MR, and safranine-O [ 73 ]. Kolya et al synthesized AgNPs from leaf extract of Amaranthus gangeticus and studied their antimicrobial activity and photocatalytic activity against congo red (CR), an anionic azo dye [ 74 ]. Arya et al synthesized AgNPs using leaf extract of Cicer arietinum and evaluated the photocatalytic activity against 4-nitrophenol, CR, and MB [ 75 ]. The dyes are degraded by both oxidation and reduction reactions. These dyes have characteristic absorption peaks at certain wavelengths (e.g. MB: 665nm; CR: 497nm; MG: 624nm). After interaction with NPs, gradual decrease in the intensity of absorption occurs due to reduction of dye and formation of intermediate product. The blue color of MB comes from its oxidized state, which becomes colorless when reduced to leuco methylene blue. The AgNPs have been found to serve as mediators in the transfer of electrons, thus degrading the dye [ 76 ]. Camellia japonica mediated AgNPs generate ROS, which oxidize Eosin-Y dye and produce intermediate products [ 77 ].

4.2. In Hydrogen peroxide (H 2 O 2 ) sensing

H 2 O 2 is a highly toxic chemical, and even a small amount of H 2 O 2 contamination in food can be harmful to living organisms. AgNPs are said to be able to detect very small amounts of H 2 O 2 . According to Mohan et al , when H 2 O 2 was added to AgNPs, free radicals were produced, which oxidized Ag 0 to Ag + and thus reduced the absorbance. Therefore, it is suggested that AgNPs be used as detectors of H 2 O 2 [ 47 ]. Raja et al reported phytosynthesis of AgNPs using leaf extract of Calliandra haematocephala and investigated their sensing properties against H 2 O 2 [ 78 ]. Aadil et al synthesized AgNPs from Acacia lignin and used as a sensor for the detection of H 2 O 2 [ 79 ]. Kumar et al also reported the detection of H 2 O 2 using Euphorbia hirta mediated AgNPs [ 80 ].

4.3. In heavy metal sensing

Researchers are concerned about heavy metal pollution in our environment. Various heavy metals like, As, Pb, Hg, Cd, Ni, Al, Va, etc are known to cause carcinogenesis, neurotoxicity, loss of cellular function and cell damage in humans [ 81 ]. Phytosynthesized AgNPs are being employed to detect toxic metal cations from contamination soil and industrial effluents. Silver and gold NPs were reported from L-tyrosine , and investigated for their sensing property against heavy toxic metals like Mn 2+ , Hg 2+ , and Pb 2+ [ 82 ]. Firdaus et al synthesized AgNPs from papaya fruit and used them as a sensor against Hg 2+ [ 83 ]. Tagad et al reported AgNPs using the root extract of Panax Ginseng and investigated their sensing property against heavy metals [ 84 ]. AgNPs are used as sensors for the detection of heavy metals in two ways. One is the redox reaction, in which AgNPs are oxidized and heavy metal is reduced. The second way is the formation of a chelation complex between heavy metal and functional groups of phytochemicals adsorbed on NP surface [ 47 ].

4.4. In DNA interaction studies

In vitro , AgNPs have been reported to bind with calf thymus DNA (CT-DNA), halting their activity and causing cell damage. Such interactions between nanostructures and DNA have been discussed in a number of papers. The possible interaction between NPs and DNA are electrostatic interaction, intercalation, and groove binding [ 85 ]. Ribeiro et al synthesized AgNPs by aqueous extract of black tea, and investigated the CT-DNA binding capacity with AgNPs using the electronic absorption and fluorescence spectroscopies [ 85 ]. Rahban et al reported the interaction between AgNPs and CT-DNA using electronic absorption spectroscopy [ 86 ]. Roy et al also reported the interaction of biosynthesized AgNPs with CT-DNA [ 87 ].

4.5. As cytotoxic agent

AgNPs are found to disrupt normal cell division in somatic cells, and thus used as an antimitotic agent. AgNPs interfere with mitotic events in cells, and as a result, cells die due to decrease in the progress of S-phase, blockage of the G2 phase, and arrest of M phase with reduced expression of cyclins/cdks. NPs cause structural or numerical changes in chromosomes, resulting in their deformation and reconstruction. The viscosity of cytosol is affected by AgNPs, resulting in irregular spindle activity and aberration of chromosomes [ 88 , 89 ].

Jacob et al reported AgNPs from leaf extract of Piper longum and studied their cytotoxic activity [ 90 ]. AgNPs, synthesized by using leaf extract of Rauvolfia tetraphylla , were investigated for their genotoxic effects showing chromosomal aberrations in the root cells of Allium cepa [ 91 ]. Researchers also reported cytotoxicity of same NPs against MCF7 and A549 cell lines following MTT assay. Suman et al reported AgNPs using the root of Morinda citrifolia and investigated the cytotoxic activity on the HeLa cells [ 92 ].

4.6. As antimicrobial agent

Silver metal ions are well known for their antiseptic properties, and some of the plant extracts have antimicrobial properties as well. AgNPs have significant antibacterial, antifungal, and antiviral effects. Owing to their small size, AgNPs and released Ag + have ability to penetrate the cell wall and plasma membrane of bacterial cell, inactivate respiratory enzymes, and disrupt ATP formation. Likewise, AgNPs interrupt the electron transport chain and produce ROS, bind to DNA and prevent replication, denature ribosomes and inhibit protein synthesis, lyse the plasma membrane, causing release of cytoplasm and organelles, and ultimately, cell death [ 93 ]. The antimicrobial effects of ampicillin and other antibiotics were found to be enhanced in combination with AgNPs [ 94 ]. Therefore, AgNPs have significantly more antimicrobial activity than their source materials (i.e. Ag salt and plant extract). Phytosynthesized AgNPs using plant extract of Chenopodium murale were investigated for their antibacterial activity against Staphylococcus aureus [ 59 ]. Similarly, Eclipta alba mediated AgNPs were evaluated for antibacterial activity against Pseudomonas aeruginosa , S. aureus , and Escherichia coli [ 95 ].

4.7. As anti-cancer agent

Cancer is a life-threatening illness that is responsible for the majority of deaths worldwide. As a result, one of the most ardent targets is the discovery of powerful and successful anticancer drugs. Now a day, scientists have paid close attention to AgNPs in cancer therapy because of their special properties. These AgNPs have shown to be effective against a variety of cancer cell lines. Kuppusamy et al synthesized and characterized AgNPs from the plant extract of Commelina nudiflora , and investigated their potential cytotoxic activity against HCT-116 colon-cancer cell line [ 96 ]. Plant mediated AgNPs using leaf extract of Plantago major , was reported for anti-cancer activity against human breast cancer cells (MCF-7) [ 97 ]. Kathiravan et al synthesized AgNPs from Melia dubai extract, and evaluated their anticancer properties against human breast cancer (KB) cells [ 98 ]. Our research groups, also synthesized BP-AgNPs by using aqueous leaf extract of Bryophyllum pinnatum , and found potential cytotoxicity against squamous cell carcinoma (A431) and melanoma (B16F10) cell lines [ 52 ].

Ag + released from AgNPs interferes with mitochondrial enzymes and interacts with –SH groups of proteins and glutathione (GSH), lowering GSH's ability to scavenge ROS and causing oxidative stress [ 99 ]. Thus, NPs increase the production of intracellular ROS, and activate the caspase-3 protein which in turn starts the apoptosis of the cell by capturing the G2/M phase. NP-generated ROS also trigger the damage of mitochondrial membrane. As a result, cell cycle is arrested and necrosis death occurs. [ 100 ].

4.8. As antioxidant agent

The antioxidant activity of AgNPs is well investigated by monitoring the scavenging of stable free radicals viz. 2, 2'-diphenyl-1-picryl hydrazyl (DPPH), and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS). Kharat and Mendhulkar synthesized AgNPs using aqueous leaf extract of Elephantopus scaber and evaluated significant antioxidant activity of Es-AgNPs, following DPPH assay [ 101 ]. Similarly, phytosynthesized AgNPs using root extract of Helicteres isora were investigated as better antioxidants than ascorbic acid and butylated hydroxytoluene (BHT), via DPPH, H 2 O 2 , and NO radical inhibition assays [ 102 ]. Ravichandran et al synthesized AgNPs using leaf extract of Artocarpus altilis and studied their antimicrobial and antioxidant activity [ 103 ].

AgNPs are recognized as antioxidant agents because silver favours two oxidation states (Ag +1 and Ag +2 ) that depend on the reaction conditions, thus, AgNPs may quench free radicals by accepting or donating electrons [ 104 ]. According to Bedlovikova et al the enhanced antioxidant potential of AgNPs than plant extract is due to the coating of phenolics, flavonoids, and terpenoids on surface which allow NPs to act as singlet oxygen quenchers, hydrogen donors, and reducing agents [ 105 ].

5. Characterization of AgNPs and analytical techniques

Characterization of the NPs is one of the crucial parts of the material science research, and without characterization we cannot be confirmed about the formation of nanostructures. Some basic analytical procedures like spectroscopy and microscopy are required to understand the synthesized material scientifically. Characterization includes methods for the exploring material properties and microscopic structures, i.e. processes involving material analyses as mechanical, thermal, and density analyses. Characterization helps to define the structure and composition of materials as well as enables us to evaluate whether or not the approach was successful. In this section, we described different characterization techniques such as Ultraviolet-Visible spectroscopy (UV–vis), Fourier Transform Infrared (FTIR) spectroscopy, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), x-ray Diffraction (XRD), and Zeta potential/particle analysis. Sophisticated instruments, used in such analyses are shown in figures 3 and 4 .

Figure 3.  A typical UV–visible Spectroscope (a), Fourier Transform Infrared (FTIR) Spectroscope (b), Transmission Electron Microscope (TEM) (c), and Scanning Electron Microscope (SEM) (d).

Figure 4.  A typical X-Ray Diffractometer (XRD)(a), and Zeta particle size analyzer (b).

5.1. UV–vis spectroscopy

The most popular method for determining extracellular green synthesis of NPs in reacting solution is UV–vis spectroscopy. Besides estimating the concentration of a NP suspension, UV–vis spectroscope can also be used to determine the color absorption patterns of metallic-NPs (through surface Plasmon resonance), together with sorption, diffusion and release properties of nanostructures. A typical UV-visible spectroscope is comprised of a tungsten or deuterium lamp, a detector, and a monochromator for ultraviolet and visible region wavelengths (figure 5 ) [ 106 ]. The UV spectra are formed when the sample is exposed to UV light. Ultraviolet-visible (UV-Vis) spectrum is the count of minimization of light beams after passing through a sample or after refraction from the surface of the sample. Cuvettes are used for carrying a sample, and are kept on the lighting path within the instrument. The cuvettes are made up of plastic, glass, quartz, or silica. Plastic and glass cuvettes absorb wavelength below 310 nm and create interference, therefore, they are not appropriate for studies of absorption under UV light. Since quartz is transparent above 180 nm wavelength, therefore quartz cuvettes are used for ultraviolet absorption measurements. The reference beam is passed from the light source to the detector without interacting the sample. The wavelength of UV light changes constantly as the beam interacts with the sample. The electron moves to a higher orbital by absorbing energy from the light source and releasing the corresponding wavelength. The detector is used to record the intensity ratio between the reference and sample beams. According to Beer–Lambert's law, the absorption by the sample is directly proportional to the concentration of the sample and the length of the path. The equation of Beer–Lambert is mentioned below [ 107 ].

Where, A = absorbance, ε  = absorptivity, c = concentration, and l = path length.

Figure 5.  A schematic diagram of UV–vis spectrophotometer.

The formation of AgNPs in reacting solution is initially confirmed by employing UV-visible spectroscopy. Metallic NPs have characteristic bands in the visible range due to the SPR effect. Therefore, the formation of AgNPs is confirmed by characteristic peak of Ag 0 , displayed by UV–vis spectroscopy.

Chandraker et al synthesised AgNPs by using leaf extracts of Bryophyllum pinnatum and Sonchus arvensis. Although, change of reaction mixture from colorless to reddish-brown gave a clue of NP synthesis, the formation of BP-AgNPs and SA-AgNPs was confirmed by observing SPR peak within 400–460. On the other hand, no peaks were assigned with plant extract and solution of silver salt as shown in figure 6 [ 47 , 52 ]. Moreover, H 2 O 2 sensing, heavy-metal sensing, and CT-DNA binding are also determined by using UV–vis. spectroscopy.

Figure 6.  UV-visible spectra of (a) SA-AgNPs and (b) BP-AgNPs (Images are taken from self publications 47, 52).

Figure 7.  FT-IR spectra of (a) SA-AgNPs and (b) BP-AgNPs (Images are taken from self publications 47, 52).

5.2. Fourier transform infrared (FTIR) spectroscopy

In FT-IR spectroscopy, an infrared spectrum is obtained from emission and absorption of samples like gas, liquid or solid. Each functional group and chemical bond can absorb a certain range of frequencies. Therefore, the characteristic peaks are observed for each functional group or part of the molecule. A typical FTIR spectroscope is consists of a source, detector, sample cell, A/D converter, amplifier, and a monitor. The radiation which is generated from the source reaches the detector from passing by an interferometer. The signal has been amplified by the A/D converter or deuterium lamp, a detector, and a monochromator, for ultraviolet and visible and amplifier, which then passes the signal to the computer where the fourier transforms. The absorbed radiation is converted to rotational or vibrational energy by the sample. The resulting signal is a spectrum of 4000 to 400 cm −1 typically reflecting the molecular fingerprint of the samples. Each molecule has a unique fingerprint that makes FTIR an invaluable tool to recognize phytochemicals [ 108 ]. Figures 7 (a) and (b) represent signature peaks (transmittance) of certain phytomolecules (coated on the surface of SA-NPs, and BP-NPs, respectively) at defined wavenumbers [ 47 , 52 ]. Figure 7 (a) demonstrates the existence of similar functional groups in Sonchus arvensis leaf extract, indicating that they coat NPs as capping and reducing agents. The interpretation of FTIR data in figure 7 (a) explains the attached functional groups on NP surface, i.e. wavenumbers 2358, 1694, 1520 indicate C–H, C=O, and O–C stretches, respectively; whereas, 637 and 534 indicate C–H bends. Figure 8 (a) represents the schematic diagram of FTIR.

Figure 8.  Schematic diagrams of FTIR (a), TEM (b) and SEM (c).

5.3. Transmission electron microscopy (TEM)

Transmission Electron Microscopy (TEM) is a technique in which beam of electrons are transferred through the sample and images are generated as a result of electronic interaction with the sample (figure 8 (b)) [ 109 ]. The images are focused on the image detecting fluorescent screens, photographic film and charge-coupled device [ 110 ]. TEM is used in various research fields such as materials science, nanotechnology, cancer research, etc In nanotechnology research, TEM is used for the determination of size and surface morphology of the NPs. TEM can supply images with atomic lattice resolution. In TEM, the images are created because of the difference in the electron waves spread by thin samples.

Since the observational area in TEM is small, the analyzed region may not be representative of the entire sample. In the case of biological materials, the electron beam poses a risk of causing harm to the sample. Despite these limitations, TEM is a preferred technique for obtaining visual information on scale, shape, dispersion, and structure due to the atomic level resolution.

5.4. Scanning electron microscopy (SEM)

Scanning Electron Microscope (SEM), shown in figure 3 (d), is a device that scans the surface of a sample with an electron beam, and creates images [ 111 ] . SEM can generate the surface topography and compositional information of the NPs by interacting the electrons and atoms of the NPs [ 112 ] . In field emission (FE) SEM, electrons are formed in an emission source and accelerated by strong electric fields. When an object is scanned, electromagnetic coils generate electrons that pass through a set of lenses to produce a focussed electron beam, and form secondary electrons after colliding with the surface of sample. These resulting electrons contain useful information that is used to rebuild a very detailed picture of the specimen surface topography. SEM is an excellent instrument for direct surface observations since it provides improved resolution and field depth as an electron microscope. The electron source and control convenience are two key components of SEM, shown in figure 8 (c). The electron source consists of an electron piston and two or more lenses that affect the paths of electrons moving through an evacuated tube. The control convenience consists of an electron beam control unit, display screen, and monitor. The electron guns have the purpose of providing a stable electron beam. In general, the electron gun is made by the tungsten or lanthanum hexaboride (LaB 6 ). The filament is heated to a resistant current to achieve a temperature between 2000–2700 K. The electron gun emits and speeds up the electrons in the 0.1–30 keV range towards the sample.

5.5. X-ray diffraction (XRD)

X-ray diffraction is a widely used technique for the structural characterization of AgNPs. This technique is also useful to calculate the average particle size. As monochromatic X-rays occur on a crystal, they are scattered through the atoms and these scattered rays interact constructively to give a diffracted ray as shown in figure 9 . The diffraction usually happens when the path difference is several wavelengths between the rays, scattered from successive planes. Every crystalline material has a unique atomic structure, and thus diffracts the x-rays in a separate pattern. The diffraction angle is measured by Bragg's equation [ 113 ], as mentioned below.

where, d  = pacing between the plane, θ  = angle of incidence, n  = integer and λ  = beam wavelength The crystalline size of the NPs is calculated from the Scherer equation by using XRD data. The Scherer equation is represented as follows.

where, K  = 0.9, D  = Crystal size (Å), λ  = Wavelength of Cu-K α radiation, and β  = Corrected half-width of the diffraction peak.

Figure 9.  X-ray diffraction pattern in XRD analysis.

The fundamental part of the geometry of this system is the source of radiation and the x-ray detector positioned around the graduated circle, which focuses on the powder specimen. Figure 10 shows the schematic diagram of XRD. Divergent slit lies between the sample and the ray source, and the detector and the sample. That reduces the background noise, helps to limit dispersed radiation, and there is collimated radiation.

Figure 10.  A schematic diagram of XRD.

5.6. Particle size and zeta potential analysis

Zeta particle analyzer is being employed to determine average size of NPs and their dispersion (figure 4 (b)). The distribution of NPs and zeta potential are also studied via 'dynamic light scattering' and 'laser doppler electrophoresis', respectively. Zeta potential magnitude provides details on the stability of the particles. Greater the magnitude, the more stable is NP due to increased electrostatic repulsion. The magnitudes like 0–5 mV, 5–20 mV, 20–40 mV, and above 40 mV indicate the aggregate, minimum stable, moderate stable and highly stable NPs, respectively. The pH of the solution is another important factor that can affect the magnitude of the charge on the surface of NPs. The surface charge, known as the isoelectric point, may be zero at a particular pH [ 114 , 115 ].

6. Comprehensive review

As discussed in the preceding sections, phytosynthesis of AgNPs have been given major attention due to their various properties. Some of the recent research outcomes on plant-mediated green synthesis of AgNPs, botanical and common names of synthesizing plants, belonging families, growth forms (habit), part used, shape-size of NPs, and their explored applications are mentioned in table 2 .

Table 2.  AgNPs synthesized using plant extracts (with names of the plants, their habit, part used, size and shape of NPs and their proposed applications).

7. Conclusion

Plant-mediated green synthesized AgNPs have incredible physico-chemical properties compared to other metal-NPs, thus make them most suitable agents in nanobiotechnology research. Plant-mediated AgNPs have been explored for diverse biomedical applications viz. antioxidant, anticancer, antimicrobial, photocatalysis, antimalarial, biosensors, etc. Growing knowledge of green chemistry, which aims to reduce the use of hazardous chemicals or radiations, as well as the release of toxic wastes and byproducts in physical and chemical approaches, has encouraged researchers to focus on plant-mediated green synthesis protocols, which are a one-step method that is easy, safe, quick, energy efficient, dependable, cost-effective, and environmentally friendly. In the field of nanomedicines, green synthesized AgNPs have extraordinary significance to function as therapeutics with wide range of proven clinical and pharmacological properties. Recent research findings on phytosynthesis of AgNPs are documented in this topical review. Significant variations in the size, shape, and applications of AgNPs have been identified, owing to the presence of different phytochemicals in different plant extracts, even from the same species collected from different locations. This report will aid researchers involved in the finding of novel AgNP-based nanomaterials and their use in green chemistry applications.

Data availability statement

The data that support the findings of this study are available upon reasonable request from the authors.

Author contributions

The Data was collected by SKC and manuscript was written and reviewed by SKC, MKG, ML, and RS. All authors contributed to the article and approved the submitted version Author contributions. All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

No funding to declare.

Conflict of interest

All authors have no conflict of interest to report.

RSC Advances

Green synthesis of silver nanoparticles using plant extracts and their antimicrobial activities: a review of recent literature.

* Corresponding authors

a Department of Botany, Mizoram University, Tanhril, Aizawl, Mizoram, India

b Department of Chemistry, Govt. Zirtiri Residential Science College, Aizawl, Mizoram, India

c Department of Chemistry, National Institute of Technology Silchar, Silchar, India E-mail: [email protected] , [email protected] , [email protected]

d Department of Chemistry, Faculty of Science, King Khalid University, Abha 61413, Saudi Arabia

e Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK

Synthesis of metal nanoparticles using plant extracts is one of the most simple, convenient, economical, and environmentally friendly methods that mitigate the involvement of toxic chemicals. Hence, in recent years, several eco-friendly processes for the rapid synthesis of silver nanoparticles have been reported using aqueous extracts of plant parts such as the leaf, bark, roots, etc. This review summarizes and elaborates the new findings in this research domain of the green synthesis of silver nanoparticles (AgNPs) using different plant extracts and their potential applications as antimicrobial agents covering the literature since 2015. While highlighting the recently used different plants for the synthesis of highly efficient antimicrobial green AgNPs, we aim to provide a systematic in-depth discussion on the possible influence of the phytochemicals and their concentrations in the plants extracts, extraction solvent, and extraction temperature, as well as reaction temperature, pH, reaction time, and concentration of precursor on the size, shape and stability of the produced AgNPs. Exhaustive details of the plausible mechanism of the interaction of AgNPs with the cell wall of microbes, leading to cell death, and high antimicrobial activities have also been elaborated. The shape and size-dependent antimicrobial activities of the biogenic AgNPs and the enhanced antimicrobial activities by synergetic interaction of AgNPs with known commercial antibiotic drugs have also been comprehensively detailed.

• This article is part of the themed collections: 2021 Reviews in RSC Advances , Chemistry in the battle against infections and Synthesis of nanomaterials

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Toxic implications of silver nanoparticles on the central nervous system: A systematic literature review

Affiliations.

• 1 Symbiosis School of Biological Sciences, Faculty of Health Sciences, Symbiosis International (Deemed) University, Pune, India.
• 2 Symbiosis Centre for Stem Cell Research, Symbiosis International (Deemed) University, Pune, India.
• PMID: 35285037
• DOI: 10.1002/jat.4317

Silver nanoparticles have many medical and commercial applications, but their effects on human health are poorly understood. They are used extensively in products of daily use, but little is known about their potential neurotoxic effects. A xenobiotic metal, silver, has no known physiological significance in the human body as a trace metal. Biokinetics of silver nanoparticles indicates its elimination from the body via urine and feces route. However, a substantial amount of evidence from both in vitro and in vivo experimental research unequivocally establish the fact of easier penetration of smaller nanoparticles across the blood-brain barrier to enter in brain and thereby interaction with cellular components to induce neurotoxic effects. Toxicological effects of silver nanoparticles rely on the degree of exposure, particle size, surface coating, and agglomeration state as well as the type of cell or organism used to evaluate its toxicity. This review covers pertinent facts and the present state of knowledge about the neurotoxicity of silver nanoparticles reviewing the impacts on oxidative stress, neuroinflammation, mitochondrial function, neurodegeneration, apoptosis, and necrosis. The effect of silver nanoparticles on the central nervous system is a topic of growing interest and concern that requires immediate consideration.

Keywords: blood-brain barrier; neurodegeneration; neuroinflammation; neurotoxicity; silver nanoparticles; toxicology.

© 2022 John Wiley & Sons, Ltd.

Publication types

• Systematic Review
• Blood-Brain Barrier
• Metal Nanoparticles* / toxicity
• Neurotoxicity Syndromes* / etiology
• Oxidative Stress
• Particle Size
• Silver / metabolism
• Silver / toxicity

Phytomediated synthesis of Fe 3 O 4 nanoparticles using Cannabis sativa root extract: photocatalytic activity and antibacterial efficacy

• Original Article
• Published: 06 June 2024

Cite this article

• Garima Rana   ORCID: orcid.org/0000-0001-6280-3915 1 ,
• Pooja Dhiman 1 ,
• Amit Kumar 1 ,
• Satheesh Selvaraj 2 ,
• Ankush Chauhan 2 , 3 &
• Gaurav Sharma 1  

This study examines the degradation of malachite green by utilizing Fe 3 O 4 nanoparticles. These nanoparticles were prepared using an extract derived from Cannabis sativa roots. The synthesized catalysts were characterized using X-ray diffraction, scanning electron microscopy, UV-visible spectroscopy, and vibrating sample magnetometry to assess their structural, optical, and magnetic properties. The results suggest that the CF2 nanoparticles demonstrate exceptional degrading efficiencies of 87.72% when exposed to UV-visible light. Furthermore, the mildly alkaline conditions were determined to be advantageous for enhancing the efficiency of catalyst degradation (97.55%), as evidenced by the pH variation experiment. The study also included an examination of the main active species and possible photocatalytic mechanism. Furthermore, there was a minimal decrease of just 20.95% in the efficiency of degradation after four rounds of the degradation reusability experiment. Therefore, the Fe 3 O 4 nanoparticles manufactured using Cannabis sativa roots show potential for use as very effective photocatalysts in the degradation of malachite green. Furthermore, anti-bacterial efficacy was checked against the bacterial strains Escherichia coli , Salmonella typhi , Shigella sonnei , Pseudomonas aeruginosa , and Staphylococcus aureus . This work presents a simple green method for producing innovative Fe 3 O 4 nanoparticles as a remarkable nanomaterial for water bodies to degrade hazardous pollutants by visible light photodegradation and as an antibacterial agent.

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International Research Centre of Nanotechnology for Himalayan Sustainability (IRCNHS), Shoolini University, Solan, India

Garima Rana, Pooja Dhiman, Amit Kumar & Gaurav Sharma

Faculty of Allied Health Sciences, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, 603103, Tamil Nadu, India

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Centre for Herbal Pharmacology and Environmental Sustainability, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, 603103, Tamil Nadu, India

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Garima Rana: writing original draft, investigation, and supervision. Pooja Dhiman: data curation and writing original draft. Amit Kumar: writing, review, and editing. Satheesh Selvaraj: writing, review, and editing. Ankush Chauhan: writing, review, and editing. Gaurav Sharma: writing, review, and editing.

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Rana, G., Dhiman, P., Kumar, A. et al. Phytomediated synthesis of Fe 3 O 4 nanoparticles using Cannabis sativa root extract: photocatalytic activity and antibacterial efficacy. Biomass Conv. Bioref. (2024). https://doi.org/10.1007/s13399-024-05785-x

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Received : 21 March 2024

Revised : 15 May 2024

Accepted : 21 May 2024

Published : 06 June 2024

DOI : https://doi.org/10.1007/s13399-024-05785-x

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• Int J Nanomedicine

Green nanotechnology: a review on green synthesis of silver nanoparticles — an ecofriendly approach

Shabir ahmad.

1 Department of Chemistry, Islamia College University, Peshawar, 25120, Pakistan

Sidra Munir

2 Department of Chemistry, Government Girls Degree College, Peshawar, Pakistan

Behramand Khan

3 Department of Chemistry, Kohat University of Science and Technology, Kohat, Pakistan

Muhammad Bilal

Muhammad omer.

4 Institute of Chemical Sciences, University of Swat, Swat, 19201, Pakistan

Muhammad Alamzeb

5 Department of Chemistry, University of Kotli 11100, Azad Jammu and Kashmir, Pakistan

Syed Muhammad Salman

Background: Nanotechnology explores a variety of promising approaches in the area of material sciences on a molecular level, and silver nanoparticles (AgNPs) are of leading interest in the present scenario. This review is a comprehensive contribution in the field of green synthesis, characterization, and biological activities of AgNPs using different biological sources.

Methods: Biosynthesis of AgNPs can be accomplished by physical, chemical, and green synthesis; however, synthesis via biological precursors has shown remarkable outcomes. In available reported data, these entities are used as reducing agents where the synthesized NPs are characterized by ultraviolet-visible and Fourier-transform infrared spectra and X-ray diffraction, scanning electron microscopy, and transmission electron microscopy.

Results: Modulation of metals to a nanoscale drastically changes their chemical, physical, and optical properties, and is exploited further via antibacterial, antifungal, anticancer, antioxidant, and cardioprotective activities. Results showed excellent growth inhibition of the microorganism.

Conclusion: Novel outcomes of green synthesis in the field of nanotechnology are appreciable where the synthesis and design of NPs have proven potential outcomes in diverse fields. The study of green synthesis can be extended to conduct the in silco and in vitro research to confirm these findings.

Introduction

Nanotechnology offers fields with effective applications, ranging from traditional chemical techniques to medicinal and environmental technologies. AgNPs have emerged with leading contributions in diverse applications, such as drug delivery, 31 ointments, nanomedicine, 37 chemical sensing, 41 data storage, 47 cell biology, 54 agriculture, cosmetics, 60 textiles, 17 the food industry, photocatalytic organic dye–degradation activity, 64 antioxidants, 66 and antimicrobial agents. 68

Despite the contradictions reported on the toxicity of AgNPs, 69 its role as a disinfectant and antimicrobial agent has been given considerable appreciation. The available documented data 73 , 74 and the interest of the community in this field prompted us to work on plant-mediated green synthesis and biological activities of AgNPs.

Different types of nanoparticles

Some distinctive reported forms of nanoparticles (NPs) are core–shell NPs, 76 photochromic polymer NPs, 78 polymer-coated magnetite NPs, 80 inorganic NPs, AgNPs, CuNPs, 82 AuNPs, 85 PtNPs, 86 PdNPs, 88 SiNPs, 89 and NiNPs, 91 while others are metal oxide and metal dioxide NPs, such as ZnONPs, 94 CuO NPs, 95 FeO, 97 MgONPs, 100 TiO 2 NPs, 102 CeO 2 NPs, 103 and ZrO 2 NPs. 104 Each of these has an exclusive set of characteristics and applications, and can be synthesized by either conventional or unconventional methods. An extensive classification of NPs is provided in Figure 1 . 105 – 111

Different approaches to nanomaterial (NM) classification.

Abbreviation: NPs, nanoparticles.

Nanoparticle synthesis

Comprehensive approaches available for NP synthesis are bottom-up and top-down. 112 The latter approach is immoderate and steady, whereas the former involves self-assembly of atomicsize particles to grow nanosize particles. This can be achieved by physical and chemical means, 113 as summarized in Table 1 . However, ecofriendly green syntheses are economical, and proliferate and trigger stable NP formation, as shown in Figure 2 .

Chemical and physical synthesis of AgNPs

Abbreviations: NPs, nanoparticles; TEM, transmission electron microscopy; FTIR, Fourier-transform infrared; XRD, X-ray diffraction; DSC, differential scanning calorimetry; TGA, thermogravimetric analysis; UV-vis, ultraviolet-visible (spectroscopy); EDS, energy-dispersive spectroscopy; DLS, dynamic light scattering; Fl-FFF, flow field-flow fractionation; EFTEM, energy-filtered TEM; NA, not applicable.

Various approaches to the synthesis of Ag nanoparticles (NPs).

Green approach (biological/conventional methods)

The surging popularity of green methods has triggered synthesis of AgNPs using different sources, like bacteria, fungi, algae, and plants, resulting in large-scale production with less contamination. Green synthesis is an ecofriendly and biocompatible process, 119 generally accomplished by using a capping agent/stabilizer (to control size and prevent agglomeration), 120 plant extracts, yeast, or bacteria. 121

Green synthesis using plant extracts

In contrast to microorganisms, plants have been exhaustively used,as apparent from Table 2 . This is because plant phytochemicals show greater reduction and stabilization. 122 Eugenia jambolana leaf extract was used to synthesize AgNPs that indicated the presence of alkaloids, flavonoids, saponins, and sugar compounds. 123 Bark extract of Saraca asoca indicated the presence of hydroxylamine and carboxyl groups. 124 AgNPs using leaves of Rhynchotechum ellipticum were synthesized, and the results indicated the presence of polyphenols, flavonoids, alkaloids, terpenoids, carbohydrates, and steroids. 125 Hesperidin was used to form AgNPs of 20–40 nm. 126 Phenolic compounds of pyrogallol and oleic acid were reported to be essential for the reduction of silver salt to form NPs. 127 Pepper-leaf extract acts as a reducing and capping agent in the formation of AgNPs of 5–60 nm. 128 Fruit extracts of Malus domestica acted as a reducing agent. Similarly, Vitis vinifera , 39 Andean blackberry, 129 Adansonia digitata , 130 Solanum nigrum , 131 Nitraria schoberi 132 or multiple fruit peels have also been reported for AgNP synthesis. 133 Combinations of plant extracts have also been reported. 134 Some other reductants used for AgNO 3 are polysaccharide, 135 soluble starch, 136 natural rubber, 137 tarmac, 138 cinnamon, 25 stem-derived callus of green apple, 25 red apple, 139 egg white, 140 lemon grass, 141 coffee, 142 black tea, 143 and Abelmoschus esculentus juice. 144 Besides these, an extensive diagram representing different parts of different plant leaves, eg, peel, seed, fruit, bark, flower, stem, and root, also used in nanoformulations, is given in Figure 3 . Green synthesis is economical and innocuous. 30 , 38 , 150

Plant-mediated synthesis of AgNPs

Abbreviations: CV, Cyclic voltammograms; ART, total reflectance technique; NPs, nanoparticles; UV-vis, ultraviolet-visible spectroscopy; TEM, transmission electron microscopy; SEM, scanning electron microscopy; FESEM, field-emission SEM; HREM, high-resolution transmission electron microscopy; XRD, X-ray diffraction; FTIR, Fourier-transform infrared spectroscopy; AFM, atomic force microscopy; HPLC, high-performance liquid chromatography; DLS, dynamic light scattering; EDX, energy-dispersive X-ray (spectroscopy); EDAX, ED X-ray analysis; SAED, selected-area electron diffraction; TGA, thermogravimetric analysis; NA, not available; CV, ; ART, .

Plant mediated synthesis of AgNPs.

Biosynthesis using microorganisms

Bacteria-mediated synthesis of AgNPs

Microorganisms like fungi, bacteria, and yeast are of huge interest for NP synthesis; however, the process is threatened by culture contamination, lengthy procedures, and less control over NP size. NPs formed by microorganisms can be classified into distinct categories, depending upon the location where they are synthesized. 183 Otari et al synthesized AgNPs intracellularly using Actinobacteria Rhodococcus sp. NCIM 2891. 184 Kannan et al biosynthesized AgNPs using Bacillus subtillus extracellularly. 185 Table 3 provides some illustrative examples of the synthesis of AgNPs using different bacterial strains.

Abbreviations: NPs, nanoparticles; UV-vis, ultraviolet-visible (spectroscopy); TEM, transmission electron microscopy; SEM, scanning electron microscopy; FESEM, field-emission SEM; HRSEM, high-resolution TEM; XRD, X-ray diffraction; FTIR, Fourier-transform infrared (spectroscopy); AFM, atomic force microscopy; HPLC, high-performance liquid chromatography; DLS, dynamic light scattering; EDX, energy-dispersive X-ray (spectroscopy); EDAX, ED X-ray analysis; SAED, selected-area electron diffraction; TGA, thermogravimetric analysis; NA, not available; TLC, thin-layer chromatography.

Alga-mediated synthesis of AgNPs

A diverse group of aquatic microorganisms, algae have been used substantially and reported to synthesize AgNPs. They vary in size, from microscopic (picoplankton) to macroscopic (Rhodophyta). AgNPs were synthesized using microalgae Chaetoceros calcitran s , C. salina , Isochrysis galbana , and Tetraselmis gracilis 199 Cystophora moniliformis marine algae were used by Prasad et al as a reducing and stabilizing agent to synthesize AgNPs. 200 Table 4 illustrates some examples of the micro and macro-algae used for AgNPs synthesis.

Abbreviations: NPs, nanoparticles; UV-vis, ultraviolet-visible (spectroscopy); TEM, transmission electron microscopy; SEM, scanning electron microscopy; XRD, X-ray diffraction; FTIR, Fourier-transform infrared (spectroscopy).

Fungus-mediated synthesis of AgNPs

Extracellular synthesis of AgNPs using fungi is also a viable alternative, because of their economical large-scale production. Fungal strains are chosen over bacterial species, because of their better tolerance and metal-bioaccumulation property. Table 5 gives some of the fungal strains used for AgNP synthesis.

Abbreviations: NPs, nanoparticles; UV-vis, ultraviolet-visible (spectroscopy); TEM, transmission electron microscopy; SEM, scanning electron microscopy; EDX, energy-dispersive X-ray (spectroscopy); XRD, X-ray diffraction; FTIR, Fourier-transform infrared (spectroscopy); AFM, atomic force microscopy; TLC, thin-layer chromatography.

Synthesis from miscellaneous sources

Nanotechnology has placed DNA on a recent drive to be used as a reducing agent. 215 Strong affinity of DNA bases for silver make it a template stabalizer 216 AgNPs were synthesized on DNA strands and found to be possibly located at N 7 guanine and phosphate. 217 Another attempt was made with calf-thymus DNA to synthesize AgNPs. 218 Similarly, silver-binding peptides were identified and selected using a combinatorial approach for NP synthesis. 219

Bioactivities

Antibacterial activity of agnps.

As a broad-spectrum antibiotic, silver is highly toxic to bacteria. It has been of great interest for the past couple of years, due to its wide spectrum of pharmacological activities, with applications in the fields of agriculture, textiles, and especially medicine. Some attributed contributions are given in Table 6 .

Antibacterial activities of AgNPs

Abbreviations: NPs, nanoparticles; NA, not available.

Antifungal activity of AgNPs

Resistant pathogenic activities of bacteria and fungi have increased invasive infections at an alarming rate. Ultimately, the subsequent need is to find more potent antifungal agents. Table 7 provides some examples from the literature that have reported antifungal properties of green synthesized AgNPs.

Antifungal properties of AgNPs

Abbreviations: NPs, nanoparticles; UV-vis, ultraviolet-visible (spectroscopy); TEM, transmission electron microscopy; SEM, scanning electron microscopy; XRD, X-ray diffraction; FTIR, Fourier-transform infrared (spectroscopy); DLS, dynamic light scattering; EDX, energy-dispersive X-ray (spectroscopy); EDAX, ED X-ray analysis; SAED, selected-area electron diffraction.

Anticancer activity of AgNPs

The paramount need of today is the synthesis of effective anticancer treatment, as cardiovascular at the top most; cancer is the second most leading cause of human dysphoria. Therefore the synthesis of anticancer agents is of the utmost necessity. AgNPs also possess substantial anticancer activities, 239 as shown in Table 8 .

Anticancer property of AgNPs

Abbreviations: NPs, nanoparticles; UV-vis, ultraviolet-visible (spectroscopy); TEM, transmission electron microscopy; SEM, scanning electron microscopy; FESEM, field-emission SEM; HRTEM, high-resolution TEM; XRD, X-ray diffraction; FTIR, Fourier-transform infrared (spectroscopy); AFM, atomic force microscopy; HPLC, high-performance liquid chromatography; DLS, dynamic light scattering; EDX, energy-dispersive X-ray (spectroscopy); EDAX, ED X-ray analysis; TGA, thermogravimetric analysis; PCCS, .

Anti-inflammatory activity of AgNPs

AgNPs of 20–80 nm were synthesized using Sambucus nigra (blackberry) extract. The NPs were characterized using ultraviolet-visible and Fourier-transform infrared spectroscopy and X-ray diffraction, and further investigations were carried out for anti-inflammatory effects, both in vitro and in vivo, against Wister rats. 177

Antiviral activity of AgNPs

Multidimensional biological activities of AgNPs provide significant antiviral potentiality. HEPES buffer was used to synthesize NPs of 5–20 nm. Postinfection antiviral activity of AgNPs was evaluated using Hut/CCR5 cells using ELISA. AgNPs inhibited HIV1 retrovirus 17%–187% more than the reverse-transcriptase inhibitor azidothymidine triphosphate 245 Polysulfone-incorporated AgNPs manifested antiviral and antibacterial activity. This was attributed to the release of sufficient silver ions from the membrane, acting as an antiviral agent. 246

Cardioprotection

The medicinal herb neem ( Millingtonia hortensis ) has been used to synthesize AgNPs, and showed significant cardioprotective properties in rats. 178

Wound dressing

anotechnology has contributed significantly in the area of wound healing, as healing is attributed to increased anti-inflammatory and antimicrobial activity. A cotton fabric treated with NPs of size 22 nm exhibited potent healing power. 247 Another advance in this area was made with the impregnation of AgNPs into bacterial cellulose for antimicrobial wound dressing. Acetobacter xylinum (strain TISTR 975) was used to produce bacterial cellulose, which was immersed in silver nitrate solution. It was effective against both Gram-positive and Gram-negative bacteria. 248 The performance of a polymer is increased by the introduction of inorganic NPs. In this regard, polyurethane solution containing silver ions was reduced by dimethylformamide using electrospinning. Collagen was introduced to increase its hydrophilicity. This collagen sponge incorporatingd AgNPs had enhanced wound-healing ability in an animal model. 249 Most recently, Jacob et al biosynthesized nanoengineered tissue impregnated with AgNPs, which significantly prevented borne bacterial growth on the surface of tissue and could help in controlling health-associated infections. 250

Nature has its own coaching manners of synthesizing miniaturized functional materials. Increasing awareness of green chemistry and the benefit of synthesis of AgNPs using plant extracts can be ascribed to the fact that it is ecofriendly, low in cost, and provides maximum protection to human health. Green synthesized AgNPs have unmatched significance in the field of nanotechnology. AgNPs cover a wide spectrum of significant pharmacological activities, and the cost-effectiveness provides an alternative to local drugs. Besides plant-mediated green synthesis, special emphasis has also been placed on the diverse bioassays exhibited by AgNPs. This review will help researchers to develop novel AgNP-based drugs using green technology.

Author contributions

All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

The authors report no conflicts of interest in this work.