- Open Access
- Published: 30 October 2018
‘Green’ synthesis of metals and their oxide nanoparticles: applications for environmental remediation
- Jagpreet Singh 1 ,
- Tanushree Dutta 2 ,
- Ki-Hyun Kim 3 ,
- Mohit Rawat 1 ,
- Pallabi Samddar 3 &
- Pawan Kumar ORCID: orcid.org/0000-0003-0712-8763 4
Journal of Nanobiotechnology volume 16 , Article number: 84 ( 2018 ) Cite this article
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In materials science, “green” synthesis has gained extensive attention as a reliable, sustainable, and eco-friendly protocol for synthesizing a wide range of materials/nanomaterials including metal/metal oxides nanomaterials, hybrid materials, and bioinspired materials. As such, green synthesis is regarded as an important tool to reduce the destructive effects associated with the traditional methods of synthesis for nanoparticles commonly utilized in laboratory and industry. In this review, we summarized the fundamental processes and mechanisms of “green” synthesis approaches, especially for metal and metal oxide [e.g., gold (Au), silver (Ag), copper oxide (CuO), and zinc oxide (ZnO)] nanoparticles using natural extracts. Importantly, we explored the role of biological components, essential phytochemicals (e.g., flavonoids, alkaloids, terpenoids, amides, and aldehydes) as reducing agents and solvent systems. The stability/toxicity of nanoparticles and the associated surface engineering techniques for achieving biocompatibility are also discussed. Finally, we covered applications of such synthesized products to environmental remediation in terms of antimicrobial activity, catalytic activity, removal of pollutants dyes, and heavy metal ion sensing.
Introduction
Over the last decade, novel synthesis approaches/methods for nanomaterials (such as metal nanoparticles, quantum dots (QDs), carbon nanotubes (CNTs), graphene, and their composites) have been an interesting area in nanoscience and technology [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. To obtain nanomaterials of desired sizes, shape, and functionalities, two different fundamental principles of synthesis (i.e., top down and bottom up methods) have been investigated in the existing literature (Fig. 1 ). In the former, nanomaterials/nanoparticles are prepared through diverse range of synthesis approaches like lithographic techniques, ball milling, etching, and sputtering [ 10 ]. The use of a bottom up approach (in which nanoparticles are grown from simpler molecules) also includes many methods like chemical vapor deposition, sol–gel processes, spray pyrolysis, laser pyrolysis, and atomic/molecular condensation.
Different synthesis approaches available for the preparation of metal nanoparticles
Interestingly, the morphological parameters of nanoparticles (e.g., size and shape) can be modulated by varying the concentrations of chemicals and reaction conditions (e.g., temperature and pH). Nevertheless, if these synthesized nanomaterials are subject to the actual/specific applications, then they can suffer from the following limitation or challenges: (i) stability in hostile environment, (ii) lack of understanding in fundamental mechanism and modeling factors, (iii) bioaccumulation/toxicity features, (iv) expansive analysis requirements, (v) need for skilled operators, (vi) problem in devices assembling and structures, and (vii) recycle/reuse/regeneration. In true world, it is desirable that the properties, behavior, and types of nanomaterials should be improved to meet the aforementioned points. On the other hand, these limitations are opening new and great opportunities in this emerging field of research.
To counter those limitations, a new era of ‘green synthesis’ approaches/methods is gaining great attention in current research and development on materials science and technology. Basically, green synthesis of materials/nanomaterials, produced through regulation, control, clean up, and remediation process, will directly help uplift their environmental friendliness. Some basic principles of “green synthesis” can thus be explained by several components like prevention/minimization of waste, reduction of derivatives/pollution, and the use of safer (or non-toxic) solvent/auxiliaries as well as renewable feedstock.
‘Green synthesis’ are required to avoid the production of unwanted or harmful by-products through the build-up of reliable, sustainable, and eco-friendly synthesis procedures. The use of ideal solvent systems and natural resources (such as organic systems) is essential to achieve this goal. Green synthesis of metallic nanoparticles has been adopted to accommodate various biological materials (e.g., bacteria, fungi, algae, and plant extracts). Among the available green methods of synthesis for metal/metal oxide nanoparticles, utilization of plant extracts is a rather simple and easy process to produce nanoparticles at large scale relative to bacteria and/or fungi mediated synthesis. These products are known collectively as biogenic nanoparticles (Fig. 2 ).
Key merits of green synthesis methods
Green synthesis methodologies based on biological precursors depend on various reaction parameters such as solvent, temperature, pressure, and pH conditions (acidic, basic, or neutral). For the synthesis of metal/metal oxide nanoparticles, plant biodiversity has been broadly considered due to the availability of effective phytochemicals in various plant extracts, especially in leaves such as ketones, aldehydes, flavones, amides, terpenoids, carboxylic acids, phenols, and ascorbic acids. These components are capable of reducing metal salts into metal nanoparticles [ 11 ]. The basic features of such nanomaterials have been investigated for use in biomedical diagnostics, antimicrobials, catalysis, molecular sensing, optical imaging, and labelling of biological systems [ 12 ].
Here, we summarized the current state of research on the green synthesis of metal/metal oxide nanoparticles with their advantages over chemical synthesis methods. In addition, we also discussed the role of solvent systems (synthetic materials), various biological (natural extracts) components (like bacteria, algae, fungi, and plant extracts) with their advantages over other conventional components/solvents. The main aim of this literature study is to provide detailed mechanisms for green synthesis and their real world environmental remediation applications. Overall, our goal is to systematically describe “green” synthesis procedures and their related components that will benefit researchers involved in this emerging field while serving as a useful guide for readers with a general interest in this topic.
Biological components for “green” synthesis
Innumerable physical and chemical synthesis approaches require high radiation, highly toxic reductants, and stabilizing agents, which can cause pernicious effects to both humans and marine life. In contrast, green synthesis of metallic nanoparticles is a one pot or single step eco-friendly bio-reduction method that requires relatively low energy to initiate the reaction. This reduction method is also cost efficient [ 13 , 14 , 15 , 16 , 17 , 18 , 19 ].
Bacterial species have been widely utilized for commercial biotechnological applications such as bioremediation, genetic engineering, and bioleaching [ 20 ]. Bacteria possess the ability to reduce metal ions and are momentous candidates in nanoparticles preparation [ 21 ]. For the preparation of metallic and other novel nanoparticles, a variety of bacterial species are utilized. Prokaryotic bacteria and actinomycetes have been broadly employed for synthesizing metal/metal oxide nanoparticles.
The bacterial synthesis of nanoparticles has been adopted due to the relative ease of manipulating the bacteria [ 22 ]. Some examples of bacterial strains that have been extensively exploited for the synthesis of bioreduced silver nanoparticles with distinct size/shape morphologies include: Escherichia coli , Lactobacillus casei , Bacillus cereus , Aeromonas sp. SH10 Phaeocystis antarctica , Pseudomonas proteolytica , Bacillus amyloliquefaciens , Bacillus indicus , Bacillus cecembensis , Enterobacter cloacae , Geobacter spp., Arthrobacter gangotriensis , Corynebacterium sp. SH09, and Shewanella oneidensis . Likewise, for the preparation of gold nanoparticles, several bacterial species (such as Bacillus megaterium D01, Desulfovibrio desulfuricans , E. coli DH5a, Bacillus subtilis 168, Shewanella alga , Rhodopseudomonas capsulate , and Plectonema boryanum UTEX 485) have been extensively used. Information on the size, morphology, and applications of various nanoparticles is summarized in Table 1 .
Fungi-mediated biosynthesis of metal/metal oxide nanoparticles is also a very efficient process for the generation of monodispersed nanoparticles with well-defined morphologies. They act as better biological agents for the preparation of metal and metal oxide nanoparticles, due to the presence of a variety of intracellular enzyme [ 23 ]. Competent fungi can synthesize larger amounts of nanoparticles compared to bacteria [ 24 ]. Moreover, fungi have many merits over other organisms due to the presence of enzymes/proteins/reducing components on their cell surfaces [ 25 ]. The probable mechanism for the formation of the metallic nanoparticles is enzymatic reduction (reductase) in the cell wall or inside the fungal cell. Many fungal species are used to synthesize metal/metal oxide nanoparticles like silver, gold, titanium dioxide and zinc oxide, as discussed in Table 1 .
Yeasts are single-celled microorganisms present in eukaryotic cells. A total of 1500 yeast species have been identified [ 26 ]. Successful synthesis of nanoparticles/nanomaterials via yeast has been reported by numerous research groups. The biosynthesis of silver and gold nanoparticles by a silver-tolerant yeast strain and Saccharomyces cerevisiae broth has been reported. Many diverse species are employed for the preparation of innumerable metallic nanoparticles, as discussed in Table 1 .
Plants have the potential to accumulate certain amounts of heavy metals in their diverse parts. Consequently, biosynthesis techniques employing plant extracts have gained increased consideration as a simple, efficient, cost effective and feasible methods as well as an excellent alternative means to conventional preparation methods for nanoparticle production. There are various plants that can be utilized to reduce and stabilize the metallic nanoparticles in “one-pot” synthesis process. Many researchers have employed green synthesis process for preparation of metal/metal oxide nanoparticles via plant leaf extracts to further explore their various applications.
Plants have biomolecules (like carbohydrates, proteins, and coenzyme) with exemplary potential to reduce metal salt into nanoparticles. Like other biosynthesis processes, gold and silver metal nanoparticles were first investigated in plant extract-assisted synthesis. Various plants [including aloe vera ( Aloe barbadensis Miller), Oat ( Avena sativa ), alfalfa ( Medicago sativa ), Tulsi ( Osimum sanctum ), Lemon ( Citrus limon ), Neem ( Azadirachta indica ), Coriander ( Coriandrum sativum ), Mustard ( Brassica juncea ) and lemon grass ( Cymbopogon flexuosus )] have been utilized to synthesize silver nanoparticles and gold nanoparticles, as listed in Table 2 . The major part of this type of research has explored the ex vivo synthesis of nanoparticles, while metallic nanoparticles can be formed in living plants (in vivo) by reducing metal salt ions absorbed as soluble salts. The in vivo synthesis of nanoparticles like zinc, nickel, cobalt, and copper was also observed in mustard ( Brassica juncea ), alfalfa ( Medicago sativa ), and sunflower ( Helianthus annuus ) [ 27 ]. Also, ZnO nanoparticles have been prepared with a great variety of plant leaf extracts such as coriander ( Coriandrum sativum ) [ 28 ], crown flower ( Calotropis gigantean ) [ 29 ], copper leaf ( Acalypha indica ) [ 30 ], China rose ( Hibiscus rosa - sinensis ) [ 31 ], Green Tea ( Camellia sinensis ) [ 32 ], and aloe leaf broth extract ( Aloe barbadensis Miller) [ 33 ]. Readers can refer to the work of Iravani [ 34 ] for a comprehensive overview of plant materials utilized for the biosynthesis of nanoparticles.
Solvent system-based “green” synthesis
Solvent systems are a fundamental component in the synthesis process, whether it is “green” synthesis or not. Water is always considered an ideal and suitable solvent system for synthesis processes. According to Sheldon, “the best solvent is no solvent, and if a solvent is desirable then water is ideal” [ 35 ]. Water is the cheapest and most commonly accessible solvent on earth. Since the advent of nanoscience and nanotechnology, the use of water as a solvent for the synthesis of various nanoparticles has been carried out. For instance, synthesized Au and Ag nanoparticles at room temperature using gallic acid, a bifunctional molecule, in an aqueous medium [ 36 ]. Gold nanoparticles were produced via a laser ablation technique in an aqueous solution. The oxygen present in the water leads to partial oxidation of the synthesized gold nanoparticles, which finally enhanced its chemical reactivity and had a great impact on its growth [ 37 ].
In the literature, “green” synthesis consists of two major routes:
Wherein water is used as a solvent system.
Wherein a natural source/extract is utilized as the main component.
Both of these routes have been covered in the coming section according to the present literature. Hopefully, our efforts will help researchers gain a better knowledge of ‘green’ synthesis methods, the role of toxic/non-toxic solvents (or components), and renewable resources derived from natural sources. Ionic and supercritical liquids are one of the best examples in this emerging area. Ionic liquids (ILs) are composed of ions that have melting points below 100 °C. Ionic liquids are also acknowledged as “room temperature ionic liquids.” Several metal nanoparticles (e.g., Au, Ag, Al, Te, Ru, Ir, and Pt) have been synthesized in ionic liquids [ 38 , 39 , 40 , 41 ]. The process of nanoparticle synthesis is simplified since the ionic liquid can serve as both a reductant and a protective agent.
ILs can be hydrophilic or hydrophobic depending on the nature of the cations and anions. For example, 1-butyl-3-methyl imidazolium (Bmim) hexafluorophosphate (PF6) is hydrophobic, whereas its tetrafluoroborate (BF4) analogue is hydrophilic. Since both species are ionic in nature, they can act as catalysts [ 40 , 42 , 43 , 44 , 45 ]. Bussamara et al. have performed a comparative study by controlling the synthesis of manganese oxide (Mn 3 O 4 ) nanoparticles using imidazolium ionic liquids and oleylamine (a conventional solvent). They found that smaller sized nanoparticles (9.9 ± 1.8 nm) were formed with better dispersity in ionic liquids than in the oleylamine solvent (12.1 ± 3.0 nm) [ 46 ]. Lazarus et al. synthesized silver nanoparticles in an ionic liquid (BmimBF4). The synthesized nanoparticles were in both smaller isotropic spherical and large-sized anisotropic hexagonal shaped forms [ 47 ]. An electrochemical method was employed for this purpose [ 48 ]. Ionic liquid was used in the electrolytic reaction as a substitute for water without mechanical stirring. For the first time, Kim et al. developed a one-phase preparation technique for gold (Au) and platinum (Pt) nanoparticles by means of thiol-functionalized ionic liquids (TFILs). TFILs acted as a stabilizing agent to produce crystalline structures with small sizes [ 49 ]. Dupont et al. used 1-n-butyl-3-methylimidazolium hexafluorophosphate (which is room temperature ionic liquid) for synthesizing Ir(0) nanoparticles by Ir(I) reduction. The average size of synthesized nanoparticles was ~ 2 nm. Interestingly, the ionic liquid medium is impeccable for the production of recyclable biphasic catalytic systems for hydrogenation reactions [ 50 ].
The benefits of using ionic liquids instead of other solvents include the following. (a) Many metal catalysts, polar organic compounds, and gases are easily dissolved in ILs to support biocatalysts. (b) ILs have constructive thermal stabilities to operate in a broad temperature range. Most of these melt below room temperature and begin to decompose above 300 or 400 °C. As such, they allow a broader synthesis temperature range (e.g., three to four times) than that of water. (c) The solubility properties of IL can be modulated by modifying the cations and anions associated with them. (d) Unlike other polar solvents or alcohols, ILs are non-coordinating. However, they have polarities comparable to alcohol. (e) ILs do not evaporate into the environment like volatile solvents because they have no vapor pressure. (f) ILs have dual functionality because they have both cations and anions. The problems associated with the biodegradability of ionic liquids make them not acceptable for synthesis of metallic nanoparticles. To diminish these non-biodegradability issues, many new potentially benign ionic liquids are being developed with maximum biodegradation efficiency [ 51 , 52 , 53 , 54 ]. The innumerable ILs are used to synthesize various metallic nanoparticles as listed in Table 3 .
Likewise, ordinary solvents can be converted into super critical fluids at temperatures and pressures above critical point. In the supercritical state, solvent properties such as density, thermal conductivity, and viscosity are significantly altered. Carbon dioxide is the most feasible super critical, non-hazardous, and inert fluid [ 55 , 56 ]. Also, supercritical water can serve as a good solvent system for several reactions. As, water has critical temperature of 646 K and pressure of 22.1 MPa [ 57 ]. Silver and copper NPs can be synthesized in supercritical carbon dioxide [ 58 ]. Sue et al. suggested that decreasing the solubility of metal oxides around the critical point can lead to super saturation and the ultimate formation of nanoparticles [ 59 ]. Kim et al. synthesized tungsten oxide (WO 3 ) and tungsten blue oxide nanoparticles by using sub- and supercritical water and methanol [ 60 ].
Stability and toxicity of the nanoparticles
The environmental distribution and transport of released nanoparticles depend on their ability to make metastable aqueous suspensions or aerosols in environmental fluids. The stability of the nanoparticles in the environment can therefore be evaluated by estimating their propensity to aggregate or interact with the surrounding media. Aggregation is a time-dependent phenomena associated with the rate of particle collision while the stability of the suspension is largely determined by the size of the particles and affinity toward other environmental constituents. The “green” synthesis of AgNPs from tea leaf extraction was found to be stable after entering the aquatic environment [ 61 ]. Likewise, the stability of AgNPs (in aqueous medium) manufactured using plant extracts and plant metabolites was confirmed from the resulting material [ 62 ]. Surface complexation is also reported to affect the intrinsic stability of nanoparticles by regulating its colloidal stability. The nature and stability of nanoparticles were theoretically predicted through a mechanistic understanding of the surface complexation processes [ 63 ]. The colloidal stability (or rate of dissolution) of nanoparticles can be regulated by controlling the particle size and surface capping or through functionalization techniques [ 64 , 65 ]).
Transformation of nanoparticles is an essential property to consider when assessing their environmental impact or toxicity. For instance, sulfurization of AgNPs greatly reduced their toxicity due to the lower solubility of silver sulfide [ 66 ]. For similar reasons, the use of biocompatible stabilizing agents (e.g., biodegradable polymers and copolymers) have opened up a “greener” avenue of nanomaterial surface engineering. Such techniques can impart remarkable stability, e.g., in situ synthesis of AuNPs capped with Korean red ginseng root [ 67 ]. Apart from surface chemistry, other key structural features determining the nanomaterial toxicity are the size, shape, and composition of the nanomaterials [ 68 ]. Toxicity analysis of AgNP synthesized using plant leaf extracts showed enhanced seed germination rates in the AgNP chemical treatment for activation than the corresponding control treatments [ 69 ]. However, the mechanism of such rate enhancement effects was not reported.
Mechanism of “green” synthesis for metals and their oxide nanoparticles
Microorganism-based mechanism.
There are different mechanisms for the formation of nanoparticles using different microorganisms. First, metallic ions are captured on the surface or inside the microbial cells, and then these arrested metal ions are reduced into metal nanoparticles by the action of enzymes. Sneha et al. [ 70 ] described the mechanism of microorganism-assisted silver and gold nanoparticles formed via Verticillium sp. or algal biomass based on the following hypothesis. (a) First, the silver or gold ions were captured on the surface of fungal cells via electrostatic interactions between ions and negatively charged cell wall enzymes. (b) Then, silver or gold ions were bioreduced into silver or gold nuclei, which subsequently grew. The two key aspects in the biosynthesis of nanoparticles are NADH (nicotinamide adenine dinucleotide) and NADH-dependent nitrate reductase. Kalishwaralal et al. [ 71 ] demonstrated that the nitrate reductase was responsible for the production of bioreduced silver nanoparticles by B. licheniformis . Nonetheless, the bioreduction mechanisms associated with the production of metal salt ions and the resulting metallic nanoparticles formed by microorganisms remain unexplored.
Plant leaf extract-based mechanism
For nanoparticle synthesis mediated by plant leaf extract, the extract is mixed with metal precursor solutions at different reaction conditions [ 72 ]. The parameters determining the conditions of the plant leaf extract (such as types of phytochemicals, phytochemical concentration, metal salt concentration, pH, and temperature) are admitted to control the rate of nanoparticle formation as well as their yield and stability [ 73 ]. The phytochemicals present in plant leaf extracts have uncanny potential to reduce metal ions in a much shorter time as compared to fungi and bacteria, which demands the longer incubation time [ 74 ]. Therefore, plant leaf extracts are considered to be an excellent and benign source for metal as well as metal oxide nanoparticle synthesis. Additionally, plant leaf extract play a dual role by acting as both reducing and stabilizing agents in nanoparticles synthesis process to facilitate nanoparticles synthesis [ 75 ]. The composition of the plant leaf extract is also an important factor in nanoparticle synthesis, for example different plants comprise varying concentration levels of phytochemicals [ 76 , 77 ]. The main phytochemicals present in plants are flavones, terpenoids, sugars, ketones, aldehydes, carboxylic acids, and amides, which are responsible for bioreduction of nanoparticles [ 78 ].
Flavonoids contain various functional groups, which have an enhanced ability to reduce metal ions. The reactive hydrogen atom is released due to tautomeric transformations in flavonoids by which enol-form is converted into the keto-form. This process is realized by the reduction of metal ions into metal nanoparticles. In sweet basil ( Ocimum basilicum ) extracts, enol- to keto-transformation is the key factor in the synthesis of biogenic silver nanoparticles [ 79 ]. Sugars such as glucose and fructose exist in plant extracts can also be responsible for the formation of metallic nanoparticles. Note that glucose was capable of participating in the formation of metallic nanoparticles with different size and shapes, whereas fructose-mediated gold and silver nanoparticles are monodisperse in nature [ 80 ].
An FTIR analysis of green synthesized nanoparticles via plant extracts confirmed that nascent nanoparticles were repeatedly found to be associated with proteins [ 81 ]. Also, amino acids have different ways of reducing the metal ions. Gruen et al. [ 82 ] observed that amino acids (viz cysteine, arginine, lysine, and methionine are proficient in binding with silver ions. Tan et al. [ 83 ] tested all of the 20 natural α-amino acids to establish their efficient potential behavior towards the reduction of Au 0 metal ions.
Plant extracts are made up of carbohydrates and proteins biomolecules, which act as a reducing agent to promote the formation of metallic nanoparticles [ 34 ]. Also, the proteins with functionalized amino groups (–NH 2 ) available in plant extracts can actively participate in the reduction of metal ions [ 84 ]. The functional groups (such as –C–O–C–, –C–O–, –C=C–, and –C=O–) present in phytochemicals such as flavones, alkaloids, phenols, and anthracenes can help to generate metallic nanoparticles. According to Huang et al. [ 85 ], the absorption peaks of FTIR spectra at (1) 1042 and 1077, (2) 1606 and 1622, and (3) 1700–1800 cm −1 imply the stretching of (1) –C–O–C– or –C–O–, (2) –C=C– and (3) –C=O, respectively. Based on FTIR analysis, they confirmed that functional groups like –C–O–C–, –C–O–, –C=C–, and –C=O, are the capping ligands of the nanoparticles [ 86 ]. The main role of the capping ligands is to stabilize the nanoparticles to prevent further growth and agglomeration. Kesharwani et al. [ 87 ] covered photographic films using an emulsion of silver bromide. When light hit the film, the silver bromide was sensitized; this exposed film was placed into a solution of hydroquinone, which was further oxidized to quinone by the action of sensitized silver ion. The silver ion was reduced to silver metal, which remained in the emulsion.
Based on the chemistry of photography, we assumed that hydroquinone or plastohydroquinone or quinol (alcoholic compound) serve as a main reducing agent for the reduction of silver ions to silver nanoparticles through non-cyclic photophosphorylation [ 87 ]. Thus, this experiment proves that the biomolecules and heterocyclic compounds exist in plant extract were accountable for the extracellular synthesis of metallic nanoparticles by plants. It has already been well established that numerous plant phytochemicals including alkaloids, terpenoids, phenolic acids, sugars, polyphenols, and proteins play a significant role in the bioreduction of metal salt into metallic nanoparticles. For instance, Shanakr et al. [ 88 ] confirmed that the terpenoids present in geranium leaf extract actively take part in the conversion of silver ions into nanoparticles. Eugenol is a main terpenoid component of Cinnamomum zeylanisum (cinnamon) extracts, and it plays a crucial role for the bioreduction of HAuCl 4 and AgNO 3 metal salts into their respective metal nanoparticles. FTIR data showed that –OH groups originating from eugenol disappear during the formation of Au and Ag nanoparticles. After the formation of Au nanoparticles, carbonyl, alkenes, and chloride functional groups appeared. Several other groups [e.g., R–CH and –OH (aqueous)] were also found both before and after the production of Au nanoparticles [ 89 ]. Thus, they proposed the possible chemical mechanism shown in Fig. 3 . Nonetheless, the exact fundamental mechanism for metal oxide nanoparticle preparation via plant extracts is still not fully tacit. In general, there are three phases of metallic nanoparticle synthesis from plant extracts: (1) the activation phase (bioreduction of metal ions/salts and nucleation process of the reduced metal ions), (2) the growth phase (spontaneous combination of tiny particles with greater ones) via a process acknowledged as Ostwald ripening, and (3) the last one is termination phase (defining the final shape of the nanoparticles) [ 90 , 91 ]. The process of nanoparticle formation by plant extract is depicted in Fig. 4 [ 92 ].
Schematic for the reduction of Au and Ag ions [ 89 ]
Mechanism of nanoparticle formation by plant leaf extract [ 228 ]
Environmental remediation applications
Antimicrobial activity.
Various studies have been carried out to ameliorate antimicrobial functions because of the growing microbial resistance towards common antiseptic and antibiotics. According to in vitro antimicrobial studies, the metallic nanoparticles effectively obstruct the several microbial species [ 93 ]. The antimicrobial effectiveness of the metallic nanoparticles depends upon two important parameters: (a) material employed for the synthesis of the nanoparticles and (b) their particle size. Over the time, microbial resistance to antimicrobial drugs has become gradually raised and is therefore a considerable threat to public health. For instance, antimicrobial drug resistant bacteria contain methicillin-resistant, sulfonamide-resistant, penicillin-resistant, and vancomycin-resistant properties [ 94 ]. Antibiotics face many current challenges such as combatting multidrug-resistant mutants and biofilms. The effectiveness of antibiotic is likely to decrease rapidly because of the drug resistance capabilities of microbes. Hence, even when bacteria are treated with large doses of antibiotics, diseases will persist in living beings. Biofilms are also an important way of providing multidrug resistance against heavy doses of antibiotics. Drug resistance occurs mainly in infectious diseases such as lung infection and gingivitis [ 95 ]. The most promising approach for abating or avoiding microbial drug resistance is the utilization of nanoparticles. Due to various mechanisms, metallic nanoparticles can preclude or overwhelm the multidrug-resistance and biofilm formation, as described in Figs. 5 and 6 .
Schematic for the multiple antimicrobial mechanisms in different metal nanoparticles against microbial cells [ 96 ]
Various mechanisms of antimicrobial activity of metal nanoparticles [ 93 ]
Various nanoparticles employ multiple mechanisms concurrently to fight microbes [e.g., metal-containing nanoparticles, NO-releasing nanoparticles (NO NPs), and chitosan-containing nanoparticles (chitosan NPs)]. Nanoparticles can fight drug resistance because they operate using multiple mechanisms. Therefore, microbes must simultaneously have multiple gene mutations in their cell to overcome the nanoparticle mechanisms. However, simultaneous multiple biological gene mutations in the same cell are unlikely [ 96 ].
Multiple mechanisms observed in nanoparticles are discussed in Table 4 . Silver nanoparticles are the most admired inorganic nanoparticles, and they are utilized as efficient antimicrobial, antifungal, antiviral, and anti-inflammatory agents [ 97 ]. According to a literature survey, the antimicrobial potential of silver nanoparticles can be described in the following ways: (1) denaturation of the bacterial outer membrane [ 98 ], (2) generation of pits/gaps in the bacterial cell membrane leading to fragmentation of the cell membrane [ 99 , 100 ], and (3) interactions between Ag NPs and disulfide or sulfhydryl groups of enzymes disrupt metabolic processes; this step leads to cell death [ 101 ]. The shape-dependent antimicrobial activity was also examined. According to Pal et al. [ 102 ], truncated triangular nanoparticles are highly reactive in nature because their high-atom-density surfaces have enhanced antimicrobial activity.
The synthesis of Au nanoparticles is highly useful in the advancement of effective antibacterial agents because of their non-toxic nature, queer ability to be functionalized, polyvalent effects, and photo-thermal activity [ 103 , 104 , 105 ]. However, the antimicrobial action of gold nanoparticles is not associated with the production of any reactive oxygen species-related process [ 106 ]. To investigate the antibacterial potential of the Au nanoparticles, researchers attempted to attach nanoparticles to the bacterial membrane followed by modifying the membrane potential, which lowered the ATP level. This attachment also inhibited tRNA binding with the ribosome [ 106 ]. Azam et al. [ 107 ] examined the antimicrobial potential of zinc oxide (ZnO), copper oxide (CuO), and iron oxide (Fe 2 O 3 ) nanoparticles toward gram-negative bacteria ( Escherichia coli , Pseudomonas aeruginosa ) and gram-positive bacteria ( Staphylococcus Aureus and Bacillus subtilis ). Accordingly, the most intense antibacterial activity was reported for the ZnO nanoparticles. In contrast, Fe 2 O 3 nanoparticles exhibited the weakest antibacterial effects. The order of antibacterial activities of nanoparticles was found to be as ZnO (19.89 ± 1.43 nm), CuO (29.11 ± 1.61 nm), and Fe 2 O 3 (35.16 ± 1.47 nm). These results clearly depicts that the size of the nanoparticles also play a momentous role in the antibacterial potential of each sample [ 107 ]. The anticipated mechanism of antimicrobial action of ZnO nanoparticles is: (1) ROS generation, (2) zinc ion release on the surface, (3) membrane dysfunction, and (4) entry into the cell. Also, the antimicrobial potential of ZnO nanoparticles is concentration and surface area dependent [ 108 ]. Mahapatra et al. [ 109 ] determined the antimicrobial action of copper oxide nanoparticles towards several bacterial species such as Klebsiella pneumoniae , P. aeruginosa , Shigella Salmonella paratyphi s. They found that CuO nanoparticles exhibited suitable antibacterial activity against those bacteria. It was assumed that nanoparticles should cross the bacterial cell membrane to damage the crucial enzymes of bacteria, which further induce cell death. For instance, green synthesized nanoparticles show enhanced antimicrobial activity compared to chemically synthesized or commercial nanoparticles. This is because the plants [such as Ocimum sanctum (Tulsi) and Azadirachta indica (neem)] employed for synthesis of nanoparticles have medicinal properties [ 110 , 111 ]. For example, green synthesized silver nanoparticles showed an efficient and large zone of clearance against various bacterial strains compared to commercial silver nanoparticles (Fig. 7 ) [ 112 ].
Schematic for the antimicrobial activity for the five bacterial strains: a Staphylococcus aureus , b Klebsiella pneumonia , c Pseudomonas aeruginosa , d Vibrio cholera , and e Proteus vulgaris . Numbers of 1 through 6 inside each strain denote: (1) nickel chloride, (2) control ciprofloxacin, (3) Desmodium gangeticum root extract, (4) negative control, (5) nickel NPs prepared by a green method, and (6) nickel NPs prepared by a chemical method [ 229 ]
Catalytic activity
4-Nitrophenol and its derivatives are used to manufacture herbicides, insecticides, and synthetic dyestuffs, and they can significantly damage the ecosystem as common organic pollutants of wastewater. Due to its toxic and inhibitory nature, 4-nitrophenol is a great environmental concern. Therefore, the reduction of these pollutants is crucial. The 4-nitrophenol reduction product, 4-aminophenol, has been applied in diverse fields as an intermediate for paracetamol, sulfur dyes, rubber antioxidants, preparation of black/white film developers, corrosion inhibitors, and precursors in antipyretic and analgesic drugs [ 113 , 114 ]. The simplest and most effective way to reduce 4-nitrophenol is to introduce NaBH 4 as a reductant and a metal catalyst such as Au NPs [ 115 ], Ag NPs [ 116 ], CuO NPs [ 117 ], and Pd NPs [ 118 ]. Metal NPs exhibit admirable catalytic potential because of the high rate of surface adsorption ability and high surface area to volume ratio. Nevertheless, the viability of the reaction declines as a consequence of the substantial potential difference between donor (H 3 BO 3 /NaBH 4 ) and acceptor molecules (nitrophenolate ion), which accounts for the higher activation energy barrier.
Metallic NPs can promote the rate of reaction by increasing the adsorption of reactants on their surface, thereby diminishing activation energy barriers [ 119 , 120 ] (Fig. 8 ). The UV–visible spectrum of 4-nitrophenol was characterized by a sharp band at 400 nm as a nitrophenolate ion was produced in the presence of NaOH. The addition of Ag NPs (synthesized by Chenopodium aristatum L. stem extract) to the reaction medium led to a fast decay in the absorption intensity at 400 nm, which was concurrently accompanied by the appearance of a comparatively wide band at 313 nm, demonstrating the formation of 4-aminophenol [ 121 ] (Fig. 9 ).
Schematic of the metallic NP-mediated catalytic reduction of 4-nitrophenol to 4-aminophenol [ 120 ]
UV-visible spectra illustrating Chenopodium aristatum L. stem extract synthesized Ag NP-mediated catalytic reduction of 4-NP to 4-AP at three different temperatures a 30 °C, b 50 °C, and c 70 °C. Reduction in the absorption intensity of the characteristic nitrophenolate band at 400 nm accompanied by concomitant appearance of a wider absorption band at 313 nm indicates the formation of 4-AP [ 121 ]
Removal of pollutant dyes
Cationic and anionic dyes are a main class of organic pollutants used in various applications [ 122 ]. Organic dyes play a very imperative role due to their gigantic demand in paper mills, textiles, plastic, leather, food, printing, and pharmaceuticals industries. In textile industries, about 60% of dyes are consumed in the manufacturing process of pigmentation for many fabrics [ 123 ]. After the fabric process, nearly 15% of dyes are wasted and are discharged into the hydrosphere, and they represent a significant source of pollution due to their recalcitrance nature [ 124 ]. The pollutants from these manufacturing units are the most important sources of ecological pollution. They produce undesirable turbidity in the water, which will reduce sunlight penetration, and this leads to the resistance of photochemical synthesis and biological attacks to aquatic and marine life [ 125 , 126 , 127 ]. Therefore, the management of effluents containing dyes is one of the daunting challenge in the field of environmental chemistry [ 128 ].
The need for hygienic and safe drinking water is increasing day by day. Considering this fact, the use of metal and metal oxide semiconductor nanomaterials for oxidizing toxic pollutants has become of great interest in recent material research fields [ 129 , 130 , 131 ]. In the nano regime, semiconductor nanomaterials have superior photocatalytic activity relative to the bulk materials. Metal oxide semiconductor nanoparticles (like ZnO, TiO 2 , SnO 2 , WO 3 , and CuO) have been applied preferentially for the photocatalytic activity of synthetic dyes [ 31 , 132 , 133 , 134 ]. The merits of these nanophotocatalysts (e.g., ZnO and TiO 2 nanoparticles) are ascribable to their high surface area to mass ratio to enhance the adsorption of organic pollutants. The surface energy of the nanoparticles increases due to the large number of surface reactive sites available on the nanoparticle surfaces. This leads to an increase in rate of contaminant removal at low concentrations. Consequently, a lower quantity of nanocatalyst will be required to treat polluted water relative to the bulk material [ 135 , 136 , 137 , 138 ]. Like metal oxide nanoparticles, metal nanoparticles also show enhanced photocatalytic degradation of various pollutant dyes; for example, silver nanoparticles synthesized from Z. armatum leaf extract were utilized for the degradation of various pollutant dyes [ 127 ] (Fig. 10 ).
Schematic for the reduction of a safranine O, b methyl red, c methyl orange, and d methylene blue dyes using silver NPs synthesized from Z. armatum leaf extract by metallic nanoparticles [ 136 ]
Heavy metal ion sensing
Heavy metals (like Ni, Cu, Fe, Cr, Zn, Co, Cd, Pb, Cr, Hg, and Mn) are well-known for being pollutants in air, soil, and water. There are innumerable sources of heavy metal pollution such as mining waste, vehicle emissions, natural gas, paper, plastic, coal, and dye industries [ 139 ]. Some metals (like lead, copper, cadmium, and mercury ions) shows enhanced toxicity potential even at trace ppm levels [ 140 , 141 ]. Therefore, the identification of toxic metals in the biological and aquatic environment has become a vital need for proper remedial processes [ 142 , 143 , 144 ]. Conventional techniques based on instrumental systems generally offer excellent sensitivity in multi-element analysis. However, experimental set ups to perform such analysis are highly expensive, time-consuming, skill-dependent, and non-portable.
Due to the tunable size and distance-dependent optical properties of metallic nanoparticles, they have been preferably employed for the detection of heavy metal ions in polluted water systems [ 145 , 146 ]. The advantages of using metal NPs as colorimetric sensors for heavy metal ions in environmental systems/samples include simplicity, cost effectiveness, and high sensitivity at sub ppm levels. Karthiga et al. [ 147 ] synthesized AgNPs using various plant extracts used as colorimetric sensors for heavy metal ions like cadmium, chromium, mercury, calcium, and zinc (Cd 2+ , Cr 3+ , Hg 2+ , Ca 2+ , and Zn 2+ ) in water. Their as-synthesized Ag nanoparticles showed colorimetric sensing of zinc and mercury ions (Zn 2+ and Hg 2+ ). Likewise, AgNPs synthesized using mango fresh leaves and dried leaves (fresh, MF-AgNPs and sun-dried, MD-AgNPs) exhibited selective sensing for mercury and lead ions (Hg 2+ and Pb 2+ ). Also, AgNPs prepared from pepper seed extract and green tea extract (GT-AgNPs) showed selective sensing properties for Hg 2+ , Pb 2+ , and Zn 2+ ions [ 147 ] (Fig. 11 ).
Schematic of metal removal using metal oxides prepared by green synthesis. Left— a digital images and b absorption spectra of neem bark extract-mediated silver NPs (NB-AgNPs) with different metal ions and concentration-dependent studies of c Hg 2+ and d Zn 2+ . Right— a digital images and b absorption spectra of fresh mango leaf extract-mediated silver NPs (MF-AgNPs) with different metal ions and c concentration-dependent studies of Pb 2+ removal [ 147 ]
Conclusion and future prospects
‘Green’ synthesis of metal and metal oxide nanoparticles has been a highly attractive research area over the last decade. Numerous kinds of natural extracts (i.e., biocomponents like plant, bacteria, fungi, yeast, and plant extract) have been employed as efficient resources for the synthesis and/or fabrication of materials. Among them, plant extract has been proven to possess high efficiency as stabilizing and reducing agents for the synthesis of controlled materials (i.e., controlled shapes, sizes, structures, and other specific features). This review article was organized to encompass the ‘state of the art’ research on the ‘green’ synthesis of metal/metal oxide nanoparticles and their use in environmental remediation applications. Detailed synthesis mechanisms and an updated literature study on the role of solvents in synthesis have been reviewed thoroughly based on the literature available to help encounter the existing problems in ‘green’ synthesis. In summary, future research and development of prospective ‘green’ materials/nanoparticle synthesis should be directed toward extending laboratory-based work to an industrial scale by considering traditional/present issues, especially health and environmental effects. Nevertheless, ‘green’ material/nanoparticle synthesis based on biocomponent-derived materials/nanoparticles is likely to be applied extensively both in the field of environmental remediation and in other important areas like pharmaceutical, food, and cosmetic industries. Biosynthesis of metals and their oxide materials/nanoparticles using marine algae and marine plants is an area that remains largely unexplored. Accordingly, ample possibilities remain for the exploration of new green preparatory strategies based on biogenic synthesis.
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Authors’ contributions
JS, KHK and PK made substantial contributions to interpretation of literature; drafted the article and revised it critically. All made substantial contributions to draft the article and revised it critically for important intellectual content and gave approval to the submitted manuscript. All authors read and approved the final manuscript.
Acknowledgements
The corresponding author (KHK) acknowledges a supporting Grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, & Future Planning (No. 2016R1E1A1A01940995). Dr. Pawan Kumar would like to thank SERB and UGC, New Delhi, for the ‘Empowerment and Equity Opportunities for Excellence in Science’ video file No. EEQ/2016/00484 and the UGC-BSR Start Up-Research Project.
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The corresponding author (KHK and PK) acknowledges a supporting grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, & Future Planning (No. 2016R1E1A1A01940995) and ‘Empowerment and Equity Opportunities for Excellence in Science’ video file No. EEQ/2016/00484 and the UGC-BSR Start Up-Research Project.
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Department of Nanotechnology, Sri Guru Granth Sahib World University, Fatehgarh Sahib, Punjab, 140406, India
Jagpreet Singh & Mohit Rawat
Department of Chemical, Biological & Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata, 700 098, India
Tanushree Dutta
Department of Civil & Environmental Engineering, Hanyang University, Seoul, 04763, South Korea
Ki-Hyun Kim & Pallabi Samddar
Department of Nano Science and Materials, Central University of Jammu, Jammu, J & K, 180011, India
Pawan Kumar
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Singh, J., Dutta, T., Kim, KH. et al. ‘Green’ synthesis of metals and their oxide nanoparticles: applications for environmental remediation. J Nanobiotechnol 16 , 84 (2018). https://doi.org/10.1186/s12951-018-0408-4
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Green synthesis of nanoparticles: current developments and limitations.
Recent studies of green synthesis of nanoparticles had been summarized.
The limitations of green synthesis had been evaluated.
the expectation for the future green synthesis had been suggested.
Nanoscale metals are widely used in many fields such as environment, medicine, and engineering that synthesis of nanoscale metals is a timely topic. At present, nanoscale metals are mainly synthesized by chemical methods that have unintended effects such as environmental pollution, large energy consumption, and potential health problems. In response to these challenges, green synthesis, which uses plant extracts instead of industrial chemical agents to reduce metal ions, has been developed. Green synthesis is more beneficial than traditional chemical synthesis because it costs less, decreases pollution, and improves environmental and human health safety. In this review, current developments in the green synthesis of nanoparticles of gold (Au NPs), silver (Ag NPs), palladium (Pd NPs), copper (Cu NPs), and iron and its oxide (Fe NPs) were evaluated. Major findings reveal the complexity in geographical and seasonal distributions of plants and their compositions that green synthesis is limited by time and place of production as well as issues with low purity and poor yield. However, considering current environmental problems and pollution associated with chemical synthesis, green synthesis offers alternative development prospects and potential applications.
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Special Issue "Plant-based green synthesis of nanoparticles: production, characterization and applications"
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Nanotechnology is an interdisciplinary area of research taking advantage of core techniques used in various disciplines like chemistry, engineering, physics and biological sciences, and leading to the development of novel strategies to manipulate minute particles resulting in the production of nanoparticles (NPs). These NPs may be defined as particles with at least one dimension ranging from 1–100 nm. Nanotechnology deals with the synthesis, development and applications of a variety of NPs. These NPs are generally produced via laborious and hazard-prone physical and chemical methods. According to the safety-by-design principle, during the last decade a large array of safe, facile, cost effective, reproducible and scalable green synthesis approaches of NPs have been developed. Among these green biological methods, plant-based biosynthesis of NPs is considered a gold technique due to easy availability and the diverse nature of plants. The potential of plant extracts to produce NPs that have definite size and shape, as well as composition, is of great importance. Moreover, the great diversity of phytochemicals readily available in plant extracts can be utilized in this green approach as the natural stabilizing and reducing agents for the biosynthesis of NPs. Plant-derived NPs are also prone to present less harmful side effects to the human population as compared to chemically synthesized NPs, and exhibit high biological potential with applications in various domains such as in agriculture (e.g. in precision farming with controlled release of agrochemicals, target-specific delivery of biomolecules, more efficient nutrients absorption, detection and control of plant diseases, etc.), in food science and technology (e.g. in processing, storage and packaging processes), in bioengineering (biocatalysts, photocatalysts, biosensors, etc.), in cosmetic (e.g. sunscreen, anti-aging, hair growth, bioactive compounds delivery, nanoemulsion, etc.) or in nanomedicine and human health protection (e.g. antimicrobial, antiparasitic, antiproliferative, pro-apoptotic, pro- or anti-oxidative depending on the context, anti-inflammatory activities, etc.). Recently, NPs have also emerged as a novel effective elicitor in plant in vitro systems with the ability to enhance the synthesis of bioactive secondary metabolites, thus further increasing the potential applications spectra of plant-based NPs.
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Green synthesis of silver nanoparticles from Tectona grandis seeds extract: characterization and mechanism of antimicrobial action on different microorganisms
- Akhil Rautela 1 ,
- Jyoti Rani 1 &
- Mira Debnath (Das) 1
Journal of Analytical Science and Technology volume 10 , Article number: 5 ( 2019 ) Cite this article
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Green synthesis of silver nanoparticles makes use of plant constituents, like carbohydrates, fats, enzymes, flavonoids, terpenoids, polyphenols, and alkaloids, as reducing agents to synthesize silver nanoparticles. The present study for the first time utilized seed extract of Tectona grandis (teak) for reduction of 1 mM silver nitrate solution to silver nanoparticles. The method proved to be very simple, cost-efficient, and convenient. Synthesis of nanoparticles was confirmed by visual detection in which the colorless solution gets changed to a brown-colored solution. Further characterization was done by UV-visible spectroscopy, XRD, FTIR analysis, SEM/EDS, FESEM, and TEM. Size of silver nanoparticles was found to be 10–30 nm approximately as determined by transmission electron microscopy (TEM). Energy-dispersive spectra (EDS) revealed that nanoparticles contain silver in its pure form. Well diffusion method showed the antimicrobial effect of AgNPs on different microorganisms with the zone of inhibition of 16 mm for Staphylococcus aureus , 12 mm for Bacillus cereus , and 17 mm for E. coli when 50 μg of AgNPs was used. Minimum inhibitory concentration was found to be 5.2, 2.6, and 2.0 μg/ml for Bacillus cereus, Staphylococcus aureus, and E. coli respectively. Mode of action of antimicrobial activity of nanoparticles was investigated by determining leakage of reducing sugars and proteins, suggesting that AgNPs were able to destroy membrane permeability.
Introduction
Nanotechnology is the process of synthesizing particles which are in the nano range, ranging from approximately 1 to 100 nm. They have large surface area to volume ratio due to which they possess optical properties as they are small enough to confine their electrons and produce quantum effects by which their detection becomes easy. Intensive research is being done on silver nanoparticles (AgNPs) owing to their wide range of applications in medical devices (He et al. 2013 ), pharmaceuticals (Kumar et al. 2011 ), clothing (Vigneshwaran et al. 2007a ), and water purification (Lin et al. 2013 ) due to their antimicrobial properties and also in adsorption of metals and pesticides (Asthana et al. 2016 ; Das et al. 2012 ), sensing of food adulterants (Ping et al. 2012 ), detection of DNA (Thompson et al. 2008 ), etc.
Chemical reduction which is the most commonly used method for synthesis of nanoparticles uses an organic solvent, like ethylene glycol (Wiley et al. 2004 ), and reducing agents, like hydrazine (Guzmán et al. 2009 ), sodium borohydride (Song et al. 2009 ), trisodium citrate (Rashid et al. 2013 ), and ascorbate (Qin et al. 2010 ). There was an earnest need for the development of cleaner and safer methods as chemical reduction gives low yield, requires complicated purification and high energy. This gave rise to green synthesis which utilizes microorganisms (bacteria (Natarajan et al. 2010 ), fungi (Vigneshwaran et al. 2007b ), yeast (Kowshik et al. 2002 ), actinomycetes (Sastry et al. 2003 )) and plant extracts for the reduction of silver to silver nanoparticles. Synthesis of nanoparticles from microbes can be extracellular (Kowshik et al. 2002 ) or intracellular (Mukherjee et al. 2001 ). The major disadvantage of the use of microbial source is the maintenance of aseptic conditions, high cost of isolation, and their maintenance in culture media due to which plants promise to be excellent sources for reducing agents for the synthesis of nanoparticles. Parts of plants used for synthesis of AgNPs are leaf (Prakash et al. 2013 ), bark (Sathishkumar et al. 2009 ), seeds (Bar et al. 2009 ), roots (Suman et al. 2013 ), etc. There are numerous examples of AgNPs synthesis from diverse plant sources, like Chrysanthemum morifolium ( He et al. 2013 ) , Cassia auriculata ( Kumar et al. 2011 ) , Mimusops elengi ( Prakash et al. 2013 ) , Cinnamon zeylanicum ( Sathishkumar et al. 2009 ) , Jatropha curcas ( Bar et al. 2009 ) , and Morinda citrifolia ( Suman et al. 2013 ) . Constituents of plants, like carbohydrates, fats, enzymes, flavonoids, terpenoids, polyphenols, and alkaloids, are capable of reducing silver to nanoparticles.
Antimicrobial activity of silver nanoparticles has been reported in many research papers. The mechanism of antibacterial action of silver nanoparticles is a topic of debate and is not well understood. But many assumptions and theories are there. In one of the study on E. coli and Staphylococcus aureus, it was seen that silver ions get released by nanoparticles and accumulate around the cell wall or inside the cell and affect DNA replication and interact with thiol groups of protein, inducing protein inactivation (Feng et al. 2000 ). This also leads to the formation of reactive oxygen species (Matsumura et al. 2003 ).
Tectona grandis , commonly called as timber, teak, Sagun, has potential medicinal value. It is widely used for making furniture, cabinets, and musical instruments (Indira and Mohanadas 2002 ). Its bark has an antibacterial compound 5-hydroxy-1,4-naphthalenedione (Juglone) which shows antibacterial activity against Listeria monocytogenes and methicillin-resistant Staphylococcus aureus (MRSA) (Neamatallah et al. 2005 ). It is also used for the treatment of anemia and shows anti-hemolytic anemia activity (Diallo et al. 2008 ). Its seeds are acclaimed to be hair tonic, which is reported to increase the number of hair follicles in the anagenic phase (Jaybhaye et al. 2010 ). Its leaves, bark, and wood show antioxidant properties, with wood showing the highest (98.6%) inhibition against DPPH (Krishna and Jayakumaran 2010 ).
In the present study, Tectona grandis seed extract was utilized for the reduction of silver nitrate to silver nanoparticles, and characterization of the synthesized nanoparticles was carried out by UV-visible spectroscopy, scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDX), field emission scanning electron microscope (FESEM), transmission electron microscope (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR) analysis. Antimicrobial activity of the synthesized nanoparticles was investigated against representative human pathogenic microorganisms ( Bacillus cereus, Staphylococcus aureus , and Escherichia coli ). Minimum inhibitory concentration of silver nanoparticles against these microorganisms was determined. Besides, the mechanism of action of antimicrobial activity was understood by detecting leakage of reducing sugars and proteins through DNS and Bradford’s method, which indicated that killing of microorganisms was through the destruction of membranous structure and permeability.
Materials and methods
Tectona grandis seeds were collected from BHU campus. Before washing, the outer covering of the harvested seeds was removed. Seeds were dried at room temperature for 3–4 days so that moisture gets removed completely. Dried seeds were crushed to fine powder and stored in dry and airtight container for further use. Silver nitrate (AgNO 3 ) was purchased from SRL, India. All other reagents were of analytical grade and used as received. All the solutions were freshly prepared with double distilled water for the experimental procedure and were kept in the dark to avoid any photochemical reaction.
Microorganisms
Antimicrobial activity of silver nanoparticles was investigated against Gram-positive bacteria ( Bacillus cereus ; MTCC 9817 and Staphylococcus aureus ; MTCC 7443) and Gram-negative bacteria ( Escherichia coli ; MTCC 443). Nutrient agar containing peptone, beef extract, and NaCl was used to maintain bacterial strains. Prior to the experiment, bacterial strains were inoculated in nutrient broth to encourage the growth of microorganisms in the exponential phase.
Tectona grandis seed extract preparation
Five grams of seed powder was carefully weighed and added in an Erlenmeyer flask of 250 ml containing 50 ml of double distilled water. The mixture in the flask was heated in a water bath for 15–20 min at 80 °C. After boiling, the extract was filtered using a muslin cloth to remove any coarse material. Volume was made up to 100 ml using double distilled water. The prepared extract was stored at 4 °C and used within 1 week.
Synthesis of silver nanoparticles using seed extract
1 mM silver nitrate solution in double distilled water was the source of silver. Silver nitrate and seed extract were mixed together in a ratio of 1:9. The reaction mixture was heated below the boiling point and continuously stirred at 800 rpm using magnetic stirrer. The mixture turned reddish brown in color within 1 h. The whole reaction was carried out in the dark. The obtained suspension of Ag/ T. grandis was centrifuged at 15,000 rpm for 45 min. The pellet containing silver nanoparticles was washed 3–4 times with deionized water to remove silver ions and seed extract residue. The precipitated nanoparticles were lyophilized. Lyophilized nanoparticles were stored in a cool, dry, and dark place and further their characterization was carried out.
Characterization of synthesized nanoparticles
UV-1800 Shimadzu spectrophotometer was used to obtain UV-visible spectra of AgNPs at different time intervals using seed extract diluted with water (1:9) as a blank. Further shape and size of formed nanoparticles were determined by SEM, FESEM equipped with EDS, and TEM. A lyophilized sample of AgNPs was subjected to Zeiss EVO-18 scanning electron microscope at 20 kV to study the morphological features of silver nanoparticles. Further, the shape, morphology, and elemental mapping of AgNPs were studied using field emission scanning electron microscopy (NOVA NanoSEM 450). For this purpose, the lyophilized sample was sonicated for a sufficient amount of time, the smear was made on a platinum grid, and allowed to dry overnight under vacuum. The grid was then coated with a thin film of palladium and finally subjected to FESEM. Transmission electron microscopy was done to exactly determine the size of nanoparticles. Sonicated sample was loaded on a carbon-coated copper grid and was allowed to dry overnight in a vacuum and subjected to transmission electron microscopy (FEI-TECNAI G 2 20 TWIN). The crystalline nature of AgNPs was confirmed by XRD pattern obtained from Rigaku-MiniFlex 600 X-ray diffractometer at 2 θ range from 0 to 100°. The sample for XRD measurement was prepared by casting the powder of silver nanoparticles on a glass slide and subsequently air-drying it under ambient conditions. The pattern was recorded by CuKα radiation with λ of 1.5406 Å at a voltage of 40 kV and current of 15 mA with a scan rate of 10°/min. Presence of functional groups present in AgNPs was determined by using FTIR (ALPHA BRUKER Eco-ATR). Four per centimeter resolution was taken in all spectra with 500–4000 cm −1 IR range.
Antibacterial assay
Well diffusion method is commonly used to check the antimicrobial activity of the nanoparticles. The antimicrobial activity of synthesized AgNPs was checked using this method. Three microorganisms, namely E. coli , Bacillus cereus, and Staphylococcus aureus were used for this purpose. 1 mg/ml solution of AgNPs was made in Milli Q water and was sonicated properly. Overnight grown culture in Luria-Bertani broth of the mentioned microorganisms was taken and diluted to an optical density of 1. Diluted bacterial suspension (500 μl) was spread uniformly on three different Luria-Bertani agar plates for three different microorganisms. After spreading, two wells were cut on each plate using well borer of approximately 10 mm diameter. One well was filled with 50 μg and the other with 100 μg of AgNPs solution in all the three plates. The plates were incubated at 37 °C for 24 h, and the zone of inhibition was measured.
Determination of minimum inhibitory concentration (MIC)
Minimum inhibitory concentration is defined as the minimum concentration of the material that inhibits the growth of the particular microorganisms. The method is based on growing microorganisms at varying concentrations of AgNPs in suspension. Sterile test tubes, containing 5 ml of Luria-Bertani broth were taken. In the test tubes, varying concentrations of AgNPs were added and one test tube was taken as control. Then, the test tubes including the control were inoculated with an equal volume (200 μl) of freshly prepared bacterial suspension diluted to an optical density of 0.5. The inoculated test tubes were incubated in a shaker incubator at 250 rpm and 37 °C for 24 h. The next day, absorbance was taken using UV-visible spectrophotometer at 600 nm, and the graph was plotted against optical density and the concentration of AgNPs. The concentration giving the least optical density corresponds to MIC of silver nanoparticles for that particular microorganism. The above method is repeated with all the three microorganisms, namely E. coli , Bacillus cereus , and Staphylococcus aureus .
Determination of the effect of AgNPs on leakage of membrane
In this leakage of reducing sugars and proteins through the membrane was determined. For this, overnight grown cultures of E. coli , Bacillus cereus , and Staphylococcus aureus were taken, and 2 ml sample was withdrawn from each culture and was marked as 0 h sample. One milliliter of AgNPs solution (1 mg/ml) was added to each culture and was incubated at 200 rpm and 37 °C. Now, the sample was withdrawn after 2 h, 4 h, and 6 h from each culture. All the samples were centrifuged at 10,000 rpm for 5 min. Pellet was discarded and the supernatant was preserved at − 30 °C immediately, and the concentration of reducing sugar and proteins were determined by DNS and Bradford’s method, respectively, as soon as possible.
Results and discussion
Nanoparticles synthesis initiates once the T. grandis seeds extract was introduced into 1 mM AgNO 3 solution. The gradual color change of AgNO 3 / T. grandis solution from colorless to yellow and finally to reddish brown indicates the formation of silver nanoparticles as shown in Fig. 1 . This color change is due to the surface plasmon vibration, an optical property which is unique to the noble metals (Ibrahim 2015 ). The formation of AgNPs was further confirmed by using UV-visible spectroscopy, scanning electron microscopy, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy.
Visual detection [( a ) 0 min. ( b ) 30 min. ( c ) 1 h]
Formation of the nanoparticles in the aqueous solution was further confirmed by the UV-visible spectroscopy. The wavelength scale was fixed between 300 and 600 nm, and the solution was scanned in this range. Maximum absorbance at 440 nm was observed, which is characteristic of silver nanoparticles (Bahuguna et al. 2016 ). The curve (Fig. 2 ) shows an increase in absorbance with the increase in incubation time (30 min, 45 min, and 1 h) of silver nitrate and seed extract.
Absorption spectra of silver nanoparticles obtained at different time intervals
Scanning electron microscopy images of the lyophilized silver nanoparticles showed mostly spherical particles of a size below 100 nm as shown in Fig. 3 . The image was a blurred one as the SEM present at the institute was not able to take images of particles below 100 nm. Therefore, FESEM of the nanoparticles was done.
SEM image of AgNPs
FESEM (Fig. 4 ) clearly shows the presence of synthesized nanoparticles. The nanoparticles were oval, spherical in shape. Most of the nanoparticles were aggregated, and few individual particles were also observed (Suman et al. 2013 ).
FESEM image of AgNPs
Elemental mapping of AgNPs by FESEM-EDX shows the presence of 94% of Ag and 6% of oxides as shown in Fig. 5 . Elemental analysis of AgNPs was confirmed by EDX as shown in Fig. 6 . A strong signal of the peak was observed at 3 KeV which is typical for absorption of metallic silver nanoparticles. The absence of other elements confirms the purity of prepared nanoparticles.
FESEM mapping
EDX analysis
Further, an insight into the morphology and size details of AgNPs was provided by transmission electron microscopy. TEM image as shown in Fig. 7 clearly demonstrates that the AgNPs were spherical in shape. The image shows agglomerates of small grains and some dispersed nanoparticles, confirming the results obtained by SEM and FESEM (Guzmán et al. 2009 ). The synthesized AgNPs were in the range of 10–30 nm. The selected area diffraction pattern (Fig. 7 , inset) confirms the face-centered cubic (fcc) crystalline structure of metallic silver.
TEM image of AgNPs (inset shows the SAED pattern of nanocrystalline silver)
Figure 8 shows the XRD pattern of silver nanoparticles which confirmed the crystalline nature of AgNPs. The four distinct diffraction peaks at 2 θ values of 38.05 θ , 44.23 θ , 64.41 θ , and 76.66 θ can be indexed to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) reflection planes of face-centered cubic structure of silver. In addition to the Bragg peaks representative of silver nanocrystals, additional peaks were also observed at 27.77 θ , 32.18 θ , 46.17 θ , and 54.752 θ . Presence of these peaks was due to seed extract which contains organic compounds and is responsible for the reduction of silver ions and stabilization of resultant nanoparticles (Ibrahim 2015 ).
X-ray diffractogram of AgNPs
FTIR measurements were carried out to identify the major functional groups in the seed extract and their possible involvement in the synthesis and stabilization of silver nanoparticles. The spectrum of seed powder and synthesized AgNPs is represented in Fig. 9 . Seed extract showed several peaks indicating the complex nature of the biological material. The bands appearing at 1745, 1643, 1508, and 1038 cm −1 were assigned to stretching vibration of C=O bond of carboxylic acid or ester, N–C=O amide bond of proteins, nitro compounds, C–N amine bond, respectively. There was a shift in the peaks in synthesized silver nanoparticles which suggests that functional groups of seed extract participate in the formation of AgNPs.
FTIR spectra of AgNPs
The synthesized AgNPs were investigated for antimicrobial activity by using well diffusion method. Growth inhibition was observed after 24 h on plates loaded with 50 and 100 μg of AgNPs (Fig. 10 ). Bacterial growth suppression around the well was due to the release of diffusible inhibitory compounds, i.e., silver nanoparticles.
Zone of inhibition of AgNPs against [( a ) B. cereus , ( b ) S. aureus , and ( c ) E. coli ]
From Table 1 , it is evident that Gram-negative bacteria ( E. coli ) show a higher zone of inhibition, in comparison to Gram-positive bacteria ( B. cereus and S. aureus ) at the same concentration of AgNPs. This difference could be explained by variation in the composition of the cell wall of Gram-positive and Gram-negative bacteria. In Gram-positive bacteria, the cell wall is constituted of a thick peptidoglycan layer, consisting of short peptide cross-linked linear polysaccharide chains. This leads to a more rigid structure, increasing difficulties in penetration of the silver nanoparticles. On the other hand, the Gram-negative bacteria’s cell wall is composed of a thinner peptidoglycan layer (Shrivastava et al. 2007 ).
After confirmation of antimicrobial activity of synthesized AgNPs through well diffusion assay, minimum inhibitory concentration (MIC) of AgNPs against B. cereus , S. aureus, and E. coli was determined. Broth dilution method is the most commonly used technique to determine the MIC of antimicrobial agents against different microorganisms (Fig. 11 ). After 24 h of incubation, no growth of B. cereus , S. aureus , and E. coli was seen in the test tubes supplemented with 5.2, 2.6, and 2.0 μg/ml of silver nanoparticles, and the optical density was 0.016, 0.016, and 0.02, respectively (Fig. 12 ).
Broth dilution assay to detect MIC against [( a ) B. cereus , ( b ) S. aureus , and ( c ) E. coli ]
Average bacterial growth after 24 h with varying concentrations of AgNPs. [( a ) B. cereus , ( b ) S. aureus, and ( c ) E. coli ]
MIC results are in relation to the fact that a larger zone of inhibition corresponds to smaller minimum inhibitory concentration (Mohanty et al. 2010 ).
The action of the mechanism of silver nanoparticles on microorganisms is still a topic of debate. Figures 13 and 14 reveal that AgNPs could enhance the permeability of the membrane, and thus leakage of reducing sugars and proteins. There was an increase in the release of sugar and proteins in all the microorganisms as the incubation time increases. At 0 h, in control and treated samples, amount of sugar and proteins were almost the same, as there was no time of contact between AgNPs and microorganisms. But at 2, 4, and 6 h amount of sugar and proteins were higher as compared with control in all the cases, which suggests that AgNPs may have expedited leakage of sugar and proteins from the cytoplasm of microorganisms.
Leakage of reducing sugar from ( a ) B. cereus , ( b ) S. aureus , and ( c ) E. coli cells treated with AgNPs
Leakage of proteins from ( a ) B. cereus , ( b ) S. aureus , and ( c ) E. coli cells treated with AgNPs
The maximum amount of sugars and proteins is released by E. coli (112.86 and 12.9 μg/ml, respectively) when treated with AgNPs. Whereas, the least amount is released by B. cereus .
In conclusion, a simple, safe, and one-step process is utilized for the synthesis of silver nanoparticles by using the Tectona grandis seed extract. Seed extracts act as a reducing agent for nanoparticles synthesis. No chemical reagent or surfactant template is required in the process, which consequently established the bioprocess with the advantage of being environmentally friendly. TEM analysis revealed that nanoparticles were in the range of 10–30 nm and spherical. SAED and XRD pattern confirmed the crystalline nature of AgNPs. Synthesized nanoparticles showed antimicrobial activity which was investigated by agar well diffusion method against B. cereus , S. aureus , and E. coli . Minimum inhibitory concentration against these pathogens was determined by the broth dilution method which was found to be 5.2, 2.6, and 2.0 μg/ml for B. cereus, S. aureus , and E. coli , respectively. MIC results were in accordance with the zone of inhibition, i.e., more zone of inhibition less MIC value and vice-versa. Mechanism of action of antimicrobial activity was found to be the change in permeability of membrane by detecting the release of reducing sugars and proteins through leaky membrane, which was detected by DNS and Bradford’s method, respectively.
Abbreviations
- Silver nanoparticles
Energy-dispersive spectra
Field emission scanning electron microscopy
Fourier transformation infrared
Minimum inhibitory concentration
Scanning electron microscopy
Transmission electron microscopy
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Acknowledgements
We thank Central Instrument Facility, IIT-BHU for providing us the necessary instrumentation facilities. We are grateful to the Ministry of Human Resource and Development (MHRD), India for financial aid.
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AR carried out all the experiments mentioned in the manuscript as this was his M.Tech dissertation work. JR helped to draft the manuscript. MD is the Senior Professor and was the supervisor of the dissertation work. All authors read and approved the final manuscript.
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Rautela, A., Rani, J. & Debnath (Das), M. Green synthesis of silver nanoparticles from Tectona grandis seeds extract: characterization and mechanism of antimicrobial action on different microorganisms. J Anal Sci Technol 10 , 5 (2019). https://doi.org/10.1186/s40543-018-0163-z
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Green synthesis and characterization of silver nanoparticles for reducing the damage to sperm parameters in diabetic compared to metformin
- Iman A. Mohammed Ali 1 ,
- Ali Ben Ahmed 1 &
- Hazim Ismail Al-Ahmed 2
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The present study used physics to synthesize silver nanoparticles using aqueous extract of fresh garlic as reducing and as a stabilizing agent silver nitrate solution. This method has proven to be environmentally friendly and safe for the synthesis of stable silver nanoparticles. The acquisition of silver nanoparticles was confirmed by optical detection, that is, by changing the color of the liquid to transparent orange and then blackish brown. Then, the characterization was confirmed using other assays. In this study, it was found that the absorption peak of silver nanoparticles was at a wavelength of 420 nm and the particle size ranged between [50–350] nm. The surface roughness of silver oxide/silver nanoparticles was 9.32 nm with an average square roughness of 21.19 nm, and the energy dispersive spectra showed that the absorption peak was in the region of 3 keV, indicating that the nanoparticles contained crystalline silver. In this study, the stability of the silver nanoparticles was good, as ZP reached (− 19.5). The results confirm that the conductivity increases with the increase in frequency due to the high energy of the photons, which causes the electrons to vibrate in the energy levels and thus increase the energy in the mitochondria and increase the movement of sperm in the Diabetic mice treated with doses of silver nanoparticles. The toxic effect of silver nanoparticles has been evaluated in other studies, in addition to evaluating antioxidants, antifungals, treating cancer cells, regulating cholesterol levels, the effect of these nanoparticles on sex cells in pregnant female mice, heart tension, and many other tests. In this study, the activities and efficacy of silver nanoparticles on sperms were determined in male mice with diabetes caused by STZ, and the treatment period was long (35 days) so that the evaluation period was a complete life cycle of male sex cells and within a long period of time and at an average nano size. This has not been studied in other previous studies. The results indicate that the biosynthesis of silver nanoparticles using garlic plant led to positive results on sperm treatments by contributing to an increase in the number of sperm with reactivation and a decrease in abnormalities in addition to a decrease in mortality due to diabetes. This is evidence that the synthesis of silver nanoparticles using garlic plant size (50–350 nm) can treat impotence and be used in the future in the treatment of many diseases without side effects.
Introduction
Nanotechnology is described in this study. It is a technique that has attracted a lot of interest in recent years. Nanoparticles represent all types of particles that fall within a diameter of 1–100 nm, and are made of carbon, metal, metal oxides, or organic compounds 1 . When compared with its larger counterpart (Bulk), nanoparticles have distinctive physical, chemical and biological characteristics due to their small size and large surface area, which led to improved interaction or stability during the physical and chemical process, increased mechanical strength and other factors 2 , 3 , 4 . Nanoparticles are distinguished by their different shapes and sizes 5 , in addition to their different diameters. Nanoparticles may be spherical or cylindrical in shape and can be tubular, spiral, flat or irregular in shape and size and range from 1 to 100 nanometers 6 , 7 . Nanotechnology is rapidly developing, and many intensive studies have been conducted to synthesize nanoparticles with distinctive characteristics in terms of cost, speed of completion and the distinctive characteristics of the resulting nanoparticles 8 . NPs have been used in a variety of applications including cooking utensils, renewable energies, agricultural pest control and have been used extensively in the medical field, treating a wide range of diseases, as well as transporting medicines and improving the quality of materials 9 . Silver nanoparticles are among the most important and most common nanoparticles due to the unique properties they possess, such as good conductivity and stability. They are also used in the manufacture of therapeutic alloys, in the treatment of burns and infections resulting from wounds, as anti-cancer cells, and against viruses, bacteria and free radicals 10 , and can be used in the manufacture of tools in contact with food, as a result, it can cause direct contact between silver nanoparticles and workers in this field, and this in turn can cause semi-chronic toxic effects and may interact with the health of the organism, as it was found that AgNPs have the ability to precipitate in The kidneys, testicles, lungs, heart and other organs of the body result in the generation of reactive oxygen species in living cells and can cause immunological or neurotoxicity 11 , on the other hand, many studies have shown that exposure to silver nanoparticles can treat many diseases resulting from External and chronic causes, such as diabetes, pressure, or immune diseases 12 , 13 , 14 . There are few studies that show the effect of silver nanoparticles on sex cells, as many have shown Among the studies, there is a relationship between the amount of the dose once and the approved period in which the infected body is exposed to a quantity of therapeutic nanoparticles again and between the health of the sperm 15 . There are various methods for the synthesis of nanoparticles, including chemical methods, in which highly dangerous compounds are used and toxic for the purpose of minimization and stabilization, causing environmental damage in addition to being expensive and time consuming with high energies. Biological and physical methods are among the ideal methods used for the synthesis of nanoparticles because they are simple, harmless, environmentally friendly, and effective at the same time 16 , 17 . Research and studies have increased to use the biophysical method to manufacture nanoparticles, as it is considered the best to use effective plant-derived compounds such as polyphenols, flavonoids, anthocyanins, ellagic acid and other plant materials that can improve the properties of silver nanoparticles and reduce toxicity resulting from the use of nanoparticles. Metals and salts used in the manufacture of commercial silver nanoparticles 18 .
Our current study focuses on the synthesis of silver nanocomposites derived from biological sources to reduce harmful ions and toxicity resulting from substances that are a source of stabilization and reduction 19 . An aqueous extract of garlic was used 20 , 21 , and testing the toxicity of silver nanoparticles on the sperm of healthy mature mice, and evaluating the role of therapeutic silver nanoparticles in stimulating sperm movement and reducing deformities in sperms as a result of STZ-induced diabetes, and possibly death in some of them and a decrease in the number of sex cells. Treatment with manufactured silver nanoparticles was tested with a long treatment period of up to 35 consecutive days and at a rate of one dose per day, that is, according to the entire life cycle of the sperm, which was not observed in another research. The results indicate that there is no significant toxicity resulting from the silver nanoparticles, and this is considered a success in using a quick and inexpensive method of synthesis, it can be used in the treatment of many diseases and negative effects resulting from diabetes.
Experimental section
Synthesis materials.
Silver nitrate, Streptozotocin obtained from Sigma Corporation, USA industry was used. Fresh garlic was purchased from the traditional medicine store in Baghdad. The garlic plant sample was classified by an expert at the University of Baghdad, College of Science, Department of Biology (AlliumL type/Alliaceae family) 22 . Ethanol was taken from DUKSAN Company The deionized water was used during the preparation of the aqueous extract and all the tools used were washed using distilled water and left to dry using a hot oven before use.
Prepare fresh garlic leaf extract
Fresh garlic was used after washing several times with deionized water (DIW) to remove dust particles, then the plant was left in the air to dry to remove residual moisture. Dry garlic was ground using an ordinary grinder. 15 g of finely ground garlic powder was dispersed in 500 ml DIW, which is of very high purity, using a magnetic stirrer for 30 min at 100 °C. Then, the solution was filtered through filter paper and centrifuged at 4000 rpm for 30 min to remove any impurities and obtain a clear solution. The extract is kept in the refrigerator for later use in the preparation of silver nanoparticles 23 .
Synthesis of silver nanoparticles by green method
The preparation was carried out according to 24 , 25 with some modification. 2 g of silver nitrate was dissolved in 25 ml of distilled water by a 600 RPM magnetic stirrer for 30 min. After that, we add 25 ml of garlic extract gradually with continuous magnetic stirring for an hour at 80 °C. Then a precipitate will form and the color of the solution will turn black. The solution was then left overnight and the precipitate was separated by centrifuge and washed with water and ethanol more than once. The precipitate is dried in an oven at 85 °C for 4 h.
Characterization techniques
X-ray diffraction analysis (xrd).
It is a technique used to study the arrangement of atoms inside crystals, where X-ray diffraction of silver nanoparticles was measured using XRD analysis. The examination was carried out by placing the sample in a centrifuge at 10,000 rpm for 15 min. The precipitate was collected and the resulting silver pellets dried at 50 °C in an oven. The size of silver nanoparticles is calculated using the Scherer equation, which is shown below, \(D = K.\lambda/\beta.{\text{cos}}\theta\) , knowing that the constant k (geometric factor) is equal to \(0.94\) 24 .
Field emission scanning electron microscope (FE-SEM)
The structure of silver nanoparticles and the size and shape of the nanoparticles were studied using scanning electron microscope (EDS-Mapping-Line-EBSD) made in Germany. The examination was carried out after placing the sample in a centrifuge at a rotation speed of 10,000 rpm for 15 min, after which the sample was washed with distilled water and dried at a temperature of 50 degrees Celsius. The sample was placed on a platinum mesh coated with palladium and the sample was analyzed by the radiation passing through the sample and the image was at a dispersion spectrum of (250 INCA Energy) 26 .
Atomic force microscopy (AFM)
Atomic force microscopy was used to examine the surface morphology of silver nanoparticles produced by garlic extract. The examination was carried out after the examined sample was dispersed and placed on a small glass slide under the microscope and at room temperature 27 .
Transmission electron microscopy (TEM)
It is a very powerful technique in materials science that can be described by a beam of high-energy electrons passing through very thin samples. The properties of silver nanoparticles in terms of particle shape and size were studied using TEM technology. An amount of the dried precipitate of nano silver was dissolved in ethanol alcohol, the suspension was placed in the ultrasonic bath for 15 min, then a drop is taken from the suspension and placed on a carbon-coated copper grid. We note that after the sample dries and forms a partially transparent layer, the sample is examined and the resulting image is formed from the shadow of the electron beam falling on the sample 28 .
UV–visible spectra analysis
The UV–visible spectrum of stable silver nanoparticles and aqueous extract of fresh garlic plant was recorded using a UV–Vis spectrometer (Shimadzu-Japanese uv-2450) at 1 nm resolution to ensure the reduction of silver Ag + ions to AgO using garlic extracts as a reducing agent. Samples were scanned in the 300–800 nm range, with a scanning speed of 475 nm/min, at 1 cm optical path and at room temperature. UV–vis absorption spectra were recorded 24 h after incubating the AgNO 3 solution with garlic extract. Distilled water was used as a blank reference for background yellowing from other sources 29 .
Zeta potential measurement
The surface electric charge of Ag NPs was determined by measuring the most stable particles when electrostatic repulsion occurs between the particles. Zeta potential was determined using HAS 300. Zeta sizer based on photon correlation spectroscopy 30 . The analysis time was 60 s, and the average zeta potential was determined. Dispersion was determined as such without dilution.
Dynamic light scattering (DLS)
The hydrodynamic diameter of silver nanoparticles in solution was determined by dynamic light scattering (DLS) and multiple scattering laser diffraction method. Malvern Zeta sizer from Origin/Germany was used 31 .
Biotechnological part
Animals experiment.
Thirty healthy male albino mice weighing about 30 ± 5 g used in the experiment were purchased from Biotechnology Research Center/Al-Nahrain University. All animals were kept in standard conditions of 22 ± 3 °C with a constant 12 h dark and 12 h light exposure cycle, and in a controlled environment at an equilibrium humidity of 50 ± 5%. The animals were left for a week to acclimatize to the experimental conditions while being provided with the standard healthy diet of food and water. The experiment was conducted in the animal house of Al-Nahrain University.
Stimulating experimental diabetes mellitus in albino mice
Diabetes was induced in all male experimental albino mice, except for the healthy control, by giving them a single dose of fresh streptozotocin after being dissolved in saline in an amount \(\left( {200 \;{\text{mg}}/{\text{kg}}} \right)\) of body weight. This dose stimulated diabetes mellitus in male rats, in which hyperglycemia was measured by measuring blood glucose 3–6 days after streptozotocin administration. Using a glucose meter, blood was drawn from the tail vein of mice White mice that showed a blood glucose level higher than 245 mg/dL were taken and with this methodology these animals were selected alone for the current study 32 .
Sperm analysis
The mice were divided into sex groups that were forced to observe the mice. The first group is the negative control group shown, watching these mice. The second group, control diabetic mice, took doses of streptozotocin, the third group was diabetic and treated with metformin (600 mg/kg body weight/day) for 28 days, the fourth group was diabetic and treated with Silver and the fifth was diabetic and treated silver nanoparticles (2 mg/kg body weight/day) 33 . Sperm motility, normal and damaged morphology, and sperm count were examined, and the number was found to be at least 150 sperms from each mouse. Sperm activity and motility analysis was performed by Makler Chamber and using a light microscope (Olympus Corporation, Tokyo, Japan). Where motility was expressed as fast (Grade A) and slow (Grade B) sperm, non-progressive (Grade C) and non-motile (Grade D) sperm. On the other hand, the shapes of sperms were identified by adjusting the optical microscope to magnification × 40 34 .
Ethical declaration
The authors declare that: (1) All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by ethics committee in Biotechnology research center, at University of Al-Nahrain, Baghdad 35,095 Iraq: Application number 60, Reference number 21-3, date 20 May 2022. (2) All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments. (See attached file: “Research Ethics Checklist (Animals)”).
Results and discussion
Xrd analysis.
X-ray diffraction is an important technique for determining crystal structure. It is used to determine the atomic arrangement, lattice parameters, crystal size 35 . Figure 1 shows the pattern of Ag/AgO NPs prepared by the biosynthesis method. There are (8) peaks are shown with different intensities. The diffraction angles at 27.9°, 32.2°, and 54.72° corresponding to planes (100), (111), and (220) respectively, confirm the behavior of AgO Nps and agreement with JCPDS (01–076-1489), while, the diffraction angles at 38.2° and 46.3°, 34.74°, 77.23°, and 81.82° corresponding to planes (111), (200), (220), (311), and (222) respectively. These results illustrated the AgNPs has been prepared, and agreement with the JCPDS (00-001-1167). The crystallite size of Ag/AgO NPs was calculated from the full width half-maximum, Bragg reflections by the Debye–Scherrer equation 36 :
where \(D\) is the crystallite size, \(\lambda = 1.5406\) Å is the wavelength of X-ray, \(\beta\) is the full width half maximum (FWHM) of the peak in radians, and \(\theta\) is the Bragg angle. The crystallite size of Ag/AgO NPs are shown in Table 1 .
XRD pattern of Ag/AgO NPs.
FE-SEM–EDS analysis
The field emission scanning electron microscope (FESESM) provides the ability to study the topography of the surfaces of nanomaterials and determine the possibility of their application in different fields. The morphological images of Ag/AgO NPs created using the biosynthetic process are shown in Fig. 2 a (1 μm) and Fig. 2 b (100 nm). The findings indicate the existence of spherical nanoparticles, which resemble clusters of spherical nanoparticles, with different nano-diameter.
FESEM images of AgNPs at ( a ) 1 µm and ( b ) 300 nm.
AgNPs existence and crystalline structure were study by using EDS analysis. It is widely known that surface Plasmon resonance causes Ag spherical nanoparticles to have a characteristic optical absorption peak about \(3{ }\;{\text{keV}}\) . Figure 3 displayed the absorption peak in the \(3{ }\;{\text{keV}}\) area, demonstrating that NPs were made of crystalline silver.
EDS of Ag/AgO NPs.
The presence of a high content of oxygen may be attributed to the presence of silver oxides in the prepared sample, these results are consistent with the results of XRD analysis. The present results are consistent with the results of the work 37 .
TEM analysis
TEM is one of the advanced analytical measurement tools used for imaging and distinguishing the size and shape of nanoparticles 38 . Figure 4 a,b show the TEM Image of Ag/AgO NPs prepared by the biosynthesis method. The results show that the nanoparticles have a spherical shape and with some aggregation. Figure 4 c explains the histogram size distribution on Ag/AgO NPs with particles size ranging from \(\left[ {50 - 350 } \right] \;{\text{nm}}\) .
TEM Images of ( a , b ) Ag/AgO NPs prepared by biosynthesis method with different magnification, and ( c ) the histogram size distribution.
AFM analysis
The roughness and surface morphology of AgO/AgNPs are indicated in the AFM images, and Fig. 5 . This image was carried out by Naio Nanosurf software, version 3.10.0.28 39 . The results showed that the surface roughness of AgO/AgNPs was \(9.32\;{\text{nm}}\) with an average square roughness of \(21.19\;{\text{nm}}\) , as shown in the Fig. 5 . The present results are consistent with those of the TEM analysis.
AFM images of AgO/AgNPs.
Optical properties
Uv–visible spectra analysis.
Figure 6 a shows the optical absorption spectrum of AgO/AgNPs prepared using garlic extract. The results show that there is an absorption peak at the wavelength \(420\;{\text{nm}}\) that can be attributed to the presence of the surface plasmon resonance (it is the result of the collective movement of free electrons in the silver when light falls on it), which is a characteristic of AgNPs This result is close to the source 40 . The present results are consistent with the results of the work 41 , 42 . Figure 6 b shows the absorption spectrum at a wavelength range (250–800) nm of garlic plant. The peak of the optical absorption spectrum was observed at wavelength 272 nm when using an aqueous garlic plant extract, and this is close to the studies presented in 43 .
UV–visible absorbance spectra of AgO/AgNPs ( a ) extract garlic and ( b ) garlic plant.
Optical absorption coefficient
The attenuation of light intensity as it passes through a substance is described by the absorption coefficient \(\left( \alpha \right)\) . It may be thought of as the total of a material's absorption cross-sections for an optical process per unit volume. The optical absorption coefficient \(\left( \alpha \right)\) can be calculated by the following equation 26 .
where \(A\) is absorbance, \(d\) is thickness and \(ln\left( {10} \right) = 2.30\) .
Figure 7 shows the relation between the optical absorption coefficient \(\left( \alpha \right)\) and the wavelength of AgO/AgNPs prepared by garlic plant extract. The results indicate a peak absorption coefficient at the wavelength of \(420\;{\text{nm}}\) with a high absorption edge close in the ultraviolet region. This curve represents the optical absorption coefficient (α) behavior of AgNPs 44 , 45 .
Variations of absorption coefficient with wavelength of AgO/AgNPs.
Urbach energy
Figure 8 shows the \(Ln\left( \alpha \right)\) versus \(h\nu\) plots obtained for the Ag nanoparticles thin film sample. This plot can be divided into three regions for analysis. The first region belongs to the weak absorption (WAT-Region). It represents the transitions that take place from a tail state located above the valence band to another tail state located below the conduction band, and/or from a tail state located below the conduction band to another tail state located above the valence band. In this WAT region, \(\alpha\) follows \(h\nu\) according to the following relationship 46 :
where \(\alpha_{0}\) is a constant, \(h\nu\) is the photon energy and \(E_{WAT}\) represents the weak absorption tail energy.
Variations of Ln(α) with incident photon energy of AgO/AgNPs.
The Urbach region (U region) (Fig. 8 ) represents the electronic transitions that take place from an extended valence band state to another tail state below the conduction band and/or d from a conduction band state extended to another tail state above the valence band. The \(E_{u}\) can be calculated by the following equation 46 :
where \(\alpha_{0}\) is a constant, \(h\nu\) is the photon energy and \(E_{u}\) is the Urbach energy.
The Urbach energy was calculated by the inverse of slope to the curve. In Fig. 8 we have the Urbach energy \(\left( {E_{u} = 0.25\;{\text{eV}}} \right)\) this low value indicates low density of localized states in the bandgap of Ag nanoparticles thin film. So minimal impurities in our prepared Ag/AgO film.
Optical bandgap analysis
The determination of the optical bandgap \(E_{g}\) was based on this Tauc formula:
After plotting \(\left( {\alpha h\nu } \right)^{2}\) in function of the photon energy \(\left( {h\nu } \right)\) , the bandgap value could be determined using the extrapolating of the linear portion to \(\alpha = 0\) . As can be seen in Fig. 9 , the Tauc plot obtained for the Ag nanoparticles thin film sample. It was obtained from the T region in Fig. 8 as it represents the longest transition. Figure 9 show the relation between the photon energy (eV) and \(\left( {\alpha h\nu } \right)^{2}\) of AgO/AgNPs, by drawing the tangent with the x-axis the direct optical energy gap can be calculated. From the Fig. 9 , the results confirm that the energy gap directly was \(3.39\;{\text{eV}}\) , this meaning the sample needs less energy to stimulate the electrons to move between the energy bands. The current results are in agreement with the results of work 47 .
Tauc plot of AgO/Ag NPs prepared by garlic plant extract.
Refractive index
It has been reported that, the refractive index \(n_{r}\) is a fundamental parameter of optical materials that plays a very important role in optical device designing. Thus, controlling the refractive index of optical nanomaterials makes them convenient for a wide range of applications in industrial and medical applications such as display devices, light-emitting diodes (OLEDs), optical communications, and antibacterial activities. The band gap values can be used to calculate a high frequency refractive index \(n_{r}\) according to the empirical relation applicable to different varieties of compounds:
where \(T_{s}\) is the percent transmittance and A is absorbance.
As can be seen Fig. 10 , the value of the refractive index for the Ag-NPs in the range \(0.113 - 0.116\) . This is close to the usual value of silver \(\left( {0.135} \right)\) 48 . The difference is that our compound is formed by AgO nanoparticles in addition to Ag nanoparticles.
Plot of refractive index as function energy of AgO/Ag NPs.
Optical conductivity
The optical conductivity \(\left( {\sigma_{opt} } \right)\) for this sample was calculated using the absorption coefficient \(\alpha\) , and the refractive index \(n\) data using the following relation 49 :
where \(c\) is the velocity of light in free space, \(\alpha\) is the absorption coefficient and \({ }n_{r}\) is the refractive index.
The optical conductivity of a material determines the relationship between the amplitude of the induced electric field and the density of the induced current in the material for any given frequency. This linear response function is a generalization of electrical conductivity, which is often considered in terms of static electric fields with slow or time-independent differences. It has to do with how conductive a material is, or how much electricity can flow through it. The conductivity of a particular material depends on the frequency of the electric field (that is, how fast it changes) 50 , 51 . Figure 11 shows the photoconductivity as a function of the photon energy (frequency) of silver nanoparticles.
The relation between optical conductivity and energy of AgO/Ag NPs.
The results confirm that the conductivity increases with increasing frequency due to the high energy of the photons, causing the electrons to vibrate in the energy levels.
Figure 12 shows the surface charge measurements of both AgO/AgNPs. The results confirm the dispersion of AgO/AgNPs, which have a zeta potential of − 19.5 (mV) and a mobility of − 1.532 (μmcm/Vs). The present results show a good dispersion state of NPs in liquids 52 . The negative value in this test confirms the occurrence of repulsion between the silver nanoparticles and proves that they are very stable.
Zeta potential values of (A) AgO/AgNPs.
Dynamic light scattering (DLS) analysis
Figure 13 shows the size distribution (by intensity and volume) of AgO/AgNPs prepared by the plant extract. From the figure, the results confirm that the highest peaks of AgO/AgNPs distribution were between 95 and 310 nm. The present results are in close agreement with the results of TEM analysis.
Size Distribution of Ag/AgONPs by ( A ) Intensity and ( B ) Volume. At Temperature 25 °C and Duration 60 s.
Sperms parameters
The results presented in Table 2 and Figs. 14 and 15 indicate that the effect of treatment of Metformin, silver and silver nanoparticles, respectively, on sperm treatments, where the motility and vitality increased significantly \(\left( {P \le 0.05} \right)\) for the group treated with silver nanoparticles when compared to the negative control. While the group treated with silver and Metformin, a significant \(\left( {P \le 0.05} \right)\) increase in movement was observed, but with a less effect. While the abnormalities and the number of deaths were significant as a result of diabetes, where there was a decrease in mortality and Abnormalities in the treated groups.
Sperm without tail & sperm without Head. Slide stained with Nigrosine and Eosin stain (100×).
Dead sperm which take eosin stain and abnormal tail (folded tail sperm). Slide stained with Nigrosine and Eosin stain (100×).
The results showed that the effect of silver nanoparticles on the treatments was positive and significantly increased in motility and living with a significant decrease in deformities and thus mortality in the treated groups. Silver nanoparticles prepared using the environmentally friendly garlic plant protect sperm by increasing the oxidative activity in the immune system, thus preventing reactive oxygen species from lipid peroxidation stimulation responsible for subsequent sperm damage. Our result clearly indicates the important role of silver nanoparticles in various medical applications 53 , 54 .
In this study, silver nanoparticles were successfully generated using a simple, efficient and inexpensive protocol from fresh garlic plant as reducing agent, and silver nanoparticles with a diameter of \(\left[ {50 - 350 } \right] \;{\text{nm}}\) were obtained. They were well crystallized, spherical, stable, and compatible with living cells. This study proved that silver nanoparticles have a great importance and an effective role against diabetes compared to Metformin, and silver nanoparticles succeeded in reducing the high percentage of damage in sperm transactions resulting from diabetes, and thus cause an increase in motility to reach the maximum value with reduced abnormalities.
Data availability
The datasets generated and/or analyzed during the current study are available in the department of biology at university of Baghdad-Iraq: [email protected] (See attached file: “Plant identification certificate). No DNA sequence was performed in this work.
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Ali, I.A.M., Ahmed, A.B. & Al-Ahmed, H.I. Green synthesis and characterization of silver nanoparticles for reducing the damage to sperm parameters in diabetic compared to metformin. Sci Rep 13 , 2256 (2023). https://doi.org/10.1038/s41598-023-29412-3
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DOI : https://doi.org/10.1038/s41598-023-29412-3
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