A Review on Synthesis, Optimization, Mechanism, Characterization, and Antibacterial Application of Silver Nanoparticles Synthesized from Plants

MOE Key Laboratory of Cluster Science, Beijing Key Laboratory of Photoelectronic Electrophotonic Conversion Materials, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 102488, China Department of Pure and Applied Chemistry, Ladoke Akintola University of Technology, Ogbomoso, Nigeria Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana, USA


Introduction
Bacterial infections had remained a major threat facing medical industry since the occurrence of antibiotic-resistant bacterial strains [1]. e overdosage of antibiotics in treating infectious diseases coupled with other side effects (like strain, diagnosis, and treatment, hypersensitivity, immunesuppression, and allergic effects) has been linked to the causes of drug resistance. Reports on the usage of AgNPs as a therapeutic agent in curing bacterial infection or as an alternative to antibiotic drugs showed good effect on multidrug resistant bacteria [2]. e presence of protein caps on AgNPs has been observed to be a great advantage in promoting the binding and stabilization on bacterial cell surface which promote the binding and absorption of drug on human cells [3].
Nanotechnology is the scientific approach of synthesizing particles of nanoscale in the range of 1 to 100 nm [4]. e large surface area to volume ratio exhibited by nanoparticles has been reported to enhance their optical properties to confine their electrons and yield quantum effects that are easily detectable. Diverse application of AgNPs in pesticide [5,6], food sensing [7], textile [8], medical [9], DNA detection [10], water [11] and pharmaceutical industries has been reported to increase its demand.
Limitation of the chemical method of synthesizing AgNPs has been traced down to the health and environmental implications associated with the organic solvents such as ascorbate, trisodium citrate, hydrazine, sodium borohydride, and sodium borohydride used as reducing and stabilizing agent in chemical synthesis of AgNPs [12]. In addition, literatures had also made it clear that chemical reduction method also produces low yield, high purity, require high energy, and expensive [13,14].
Quest to eradicate this problem as demand for AgNPs increases had led to the synthesis of AgNPs from biological substances (microorganisms and plants). Intracellular and extracellular synthesis of AgNPs from microbes such as Penicillium oxalicum GRS-1 [15], Aspergillus fumigatus BTCB10 [16], and Trichoderma longibrachiatum [17] have been documented. However, the following set back was recorded; expensive cost of isolation, difficulties in maintaining aseptic conditions, culture media, and toxicity of some microorganism [18].
It is worth knowing that the phytosynthesis of AgNPs puffer solution to the limitations associated with both the chemical and microbial methods of synthesis because the reduction process and mechanism of phytosynthesis of AgNPs are based on the biomolecules found in the plant extract; therefore, they are said to be cheap, environmental friendly, easy, and cost-effective [19]. e following biomolecules: terpenoids, carbohydrates, tannins, fats, enzymes, flavonoids, and phenols have been reported proficient for the reduction of silver to nanoparticles [20].

Synthesis of AgNPs from Plants
In synthesis of AgNPs from plant encourage large-scale production of AgNPs, which is a remedy to the high demand of AgNPs, plant leaf is more preferable than the whole plant in the synthesis of extracellular AgNPS [21]. Plant extract possesses diverse active biomolecules such as phenolic acids, sugars, terpenoids, alkaloids, polyphenol, and proteins that play vital role in this biological reduction and stabilization of silver ions [22]. e structure of some active biomolecules that are capable of reducing and stabilizing silver ions are shown in Table 1. e size and shape of AgNPs depend on the following parameters: volume of plant extract used, concentration of silver, temperature, reaction time, and pH value of the reaction [23]. e procedure for the synthesis of AgNPs entails collection and identification of plants part (seeds, fruits, leaves, rhizomes, roots, barks, pulps, stem, pod, and latex), purification of plant parts by sorting and washing, drying of plants parts at room temperature to prevent evaporation of volatile biomolecules, pulverization of dried plant parts to aid rapid extraction, extraction with water or other solvent at minimum temperature if heat is required, preparation of silver salt usually in concentration of 1-5 mM, and combination of plants extract with prepared silver salt in ratio of 1 : 5. Application of minimal heat to the mixture with continuous stirring until a color change has been observed; finally, the solution will be filtered and incubated [24]. Report has shown that the biosynthesis of AgNPs could be completed in the silver salt solution within minutes at temperature of 25°C; however, the types of secondary metabolites present in plant extract have a great significance effect. Also, the concentration of the extract, concentration of silver salt, temperature, pH, and contact time matter [25]. e chemical and physical methods are equally employed in the synthesis of AgNPs. e chemical synthesis entails the use of some hazardous chemicals and reagents which can cause some health and environment implications for the reduction of the silver ions and stabilization of formed nanoparticles, while the physical method makes use of electric current in generation of electron needed for AgNPs synthesis. Electrospraying, laser pyrolysis, laser ablation, and evaporation-condensation rampantly used physical methods of AgNPs synthesis [26][27][28]. e flow chart illustrating the synthesis of AgNPs is given in Figure 1.

Optimization Conditions for the Synthesis of
AgNPs from Plants Extract e roles of reaction parameters are very imperative in optimizing the size, stability, and yield of biosynthesized AgNPs. Alteration of concentration of the plant extract, pH, time of incubation, concentration of silver salt, and temperature has been reported as good optimizing condition for the synthesis of AgNPs [29]. Properties of AgNPs greatly depend on optimization conditions. Many researchers had reported pH values in the range of 2 to 14 for AgNPs synthesis [13]. Nevertheless, a pH value of 7 has been reported as an optimal condition for monitoring the complete reduction of Ag + to Ag 0 during the synthesis of AgNPs using Pinus eldarica bark extract [30]. Also maximum nanoparticles synthesis occurred between pH of 7 and 9 during the synthesis of AgNPs using Fusarium oxysporum [31]. e synthesis of AgNPs can be attained at different temperatures but 25°C (room temperature) has been documented as optimal for the synthesis of small size and spherical AgNPs [32]. Maximum yield of biosynthesized AgNPs was recorded at room temperature using aqueous Aloe barbadensis leaf extract [3]. Similar result was obtained from the synthesis of AgNPs using Annona squamosa peel extract [33]. Temperature has been found to be a good parameter in determining the nature of peaks of synthesized AgNPs, and low temperature corresponds to sharp peaks, while higher temperature is associated with broad peaks. e synthesis of AgNPs using the Aspergillus fumigatus revealed maximum yield and desired particle size of 322.8 nm with sharp peaks at 25°C. With an increase in temperature, AgNPs with increased size and broad peaks were produced [16]. Concentration of silver salt played vital role in estimating the level of agglomeration in the synthesis of AgNPs and concentration above 10 mM resulted in increase in the surface plasmon resonance (SPR) band, agglomeration buildup of silver, and blurred surfaces. A period of 10 minutes has been regarded as the optimal incubation time for the AgNPs [34].

Mechanism of AgNPs Synthesis
e biosynthesis of AgNPs is a simple and facile method achieved by mixing silver nitrate (AgNO 3 ) with biological substances usually microorganism (fungi, bacterial and algae) or extract from plant's part serving as the capping and stabilizing agents [35]. e reduction and stabilization of silver ions from Ag + to Ag 0 has been traced to the presence of OH functional groups in plants biomolecules such as proteins, amino acids, enzymes, steroids, alkaloids, polyphenols, quinones, tannins, saponins, carbohydrates, flavonoids, and vitamins in plants extracts or microorganism [35,36], though the right mechanism involved in the reduction and stabilization of silver ions has not been clearly understood because biomolecules varies from plant to plants, hence, the complexity of the mechanism because 4000 phytochemicals had been known in plants [36]. erefore, the synthesis of AgNPs from plants needs more detailed research as to divulge the appropriate biomolecules serving as the capping and stabilizing agent. Nevertheless, numerous plants are described to support the synthesis AgNPs. Some of such plants are documented in Table 2. e illustration of the possible mechanism involved in the synthesis of AgNPs from plant extract is given in Figure 2. e conversion of the hydroxyl groups from a secondary metabolite quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one) usually found in plant extract to the 3,5,7-trihydroxy-2-(4-hydroxy-3-oxocyclohexa-1,5-dien-1yl)-4H-chromen-4-one generates reducing equivalents that are used for the conversion of silver metal ions (Ag + ) into elemental (Ag 0 ) nanostructures [36]. is usually accomplished a color change during nanoparticles synthesis.

Characterization of AgNPs Synthesized from Plants
Characterization is required for determination of the structural and morphological properties of AgNPs. e major techniques used for the characterization of AgNPs are X-ray diffraction (XRD) for identification of the crystalline or amorphous natures of AgNPs, scanning, and transmission electron microscopy (SEM) for evaluation of size and morphology of synthesized AgNPs; Fourier transform infrared (FTIR) is used for identification of functional groups used in reduction of silver salt. For each of the characterization techniques, a different method of sample preparation is required [64].

XRD Analysis
XRD analysis determines the crystallite size of AgNPs. is is achieved with Philips diffractometer of 'X' pert company and a nanochromatic Cu K α radiation of (λ �1.54060 A) radiation. e width of XRD peaks is used for the size determination following Scherrer's formula, D � 0.86λ/β cos θ.
e three diffraction peaks of (111), (200), and (220) represent the facecentered cubic silver. e sharpness of these peaks illustrates formation of particles the nanosize [64]. Another study also reported the indexing peak of AgNPs displayed strong Braggs reflection which correspond to the reflections of face centered cubic of silver as shown in Figure 3.

Scanning Electron Microscope (SEM)
is technique is used to study the size and shape of the nanoparticles. In SEM technique, electrons are used for the formation of an output image instead of light [66]. e synthesized AgNps is placed on a stub made for SEM analysis. e stub is usually made up of small cylinder copper of 1 mm diameter. One layer of the stub contains carbon. When the synthesized AgNps is placed on the carbon material, the stub is attached to a holder. e holder can take up to seven samples [63]. A spherical, hexagonal, round, oval, cuboidal, and triangular shape of AgNPs has been detected using the SEM techniques as shown in Table 2. An example of SEM microgram of synthesized AgNPs is shown in Figure 4. Aside from the morphological assessment of AgNPs, SEM analysis can also be useful for the estimation of stability of AgNPs; SEM analysis of AgNPs synthesized from Carica papaya leaf extract showed that increase in concentration of plant extract led to agglomeration and destabilization of AgNPs [67].

Fourier-Transform Infrared (FTIR) Spectroscopic Technique
FTIR technique is useful for the determination of the functional groups in the plants biomolecules that are responsible for the reduction and stabilization of the silver ion. For FTIR analysis, synthesized AgNPs and plant extract could be in the solid or liquid state. e dry method of analysis synthesized AgNPs and plant extract was carried out by mixing 1% (w/w) AgNPs and plant extract with 100 mg of potassium bromide powder separately. e mixture was properly pressed into a sheer slice and was scanned by on Journal of Chemistry FTIR spectrometer (FTIR Nicolet 5700, ermo Corp. USA) at a resolution of 2 cm −1 [65]. e FTIR analysis of synthesized AgNPs and plant extract had revealed that functional groups such amides and amino carboxyl are responsible for the stabilization of synthesized AgNPs [68][69][70][71]. e FTIR spectrum illustrating that plant extract contained some biomolecules is shown in Figure 5.

Transmission Electron Spectroscopy (TEM)
Determination of size distribution of AgNPs is very vital because they possess different physical and chemical features depending on their shape and size [73]. In the preparation of AgNPs for TEM analysis, small amount of AgNPs is placed on carbon coated copper grids. A ray of photons is transmitted through an ultrathin dried grid containing the AgNPs and interacting with it as it passes through [22]. e electrons are transmitted through the AgNPs to produce an interaction that results in the formation of an image. e image is magnified and recorded by an imaging device [74]. e use of medicinal plants for the synthesis of AgNPs is not only for the enhancement of their antimicrobial properties but also to regulate the size and shape of AgNPs [37]. Numerous plant extracts have been used for the synthesis of AgNPs and different morphologies have been obtained in Table 2. SEM microgram showing a spherical shape synthesized nanoparticles is shown in Figure 6.

UV-Vis Spectroscopy
e UV-Vis spectroscopy is used to measure the plasmon absorbance responsible for characteristic colors observed during the synthesis of AgNPs.
e absorption of the electrons on the surface of AgNPs and electromagnetic radiation by incident light produces oscillations. e absorption maximum for AgNPs has been reported to be in the range 0.5 and 0.7. e plasmon resonance produces a peak near 400 nm which confirmed the synthesis of AgNPs [71].  Journal of Chemistry e UV-Vis spectroscopic analysis of AgNPs synthesized from plant and their corresponding plasmon resonance peaks are shown in Figure 7 and Table 1.

Energy-Dispersive X-Ray Spectroscopy
e elemental composition of synthesized AgNPs is determined with the energy-dispersive X-ray technique [76]. e working principle of an energy-dispersive X-ray is based on the collision of electrons with the nanoparticles to produce X-rays, the uniqueness of the atomic structure of each element in making a distinct set of peaks on its X-ray spectrum results to the characterization of the elements. e distinct peak at 2.8-3.2 keV on the EDX is associated with the presence of silver [77]. EDX analysis of AgNPs shown in Figure 8 revealed an optical absorption peak at 3 keV [75].

Antibacterial Activity of Silver Nanoparticles
Recently, the increase in antibiotic-resistant microbes is becoming alarming and worrisome, and certain pathogens associated with some infections which increase the mortality rate in human and method of treatment with available antibiotic are proving abortive cost. erefore, the need to    Journal of Chemistry develop novel antibacterial agents with good potency against MDR strains is of great importance [78]. AgNPs possess some features that can inhibit the growth of the MDR organism. e concentration, shape, colloidal state, and size are the major parameter that determine the antibacterial efficacy of AgNPs [79]. It has been established by researchers that the lower the size of AgNPs, the higher its stability and biocompatibility. e effectiveness of AgNPs against bacteria, fungi, and viruses has been established. e antibacterial activity of AgNPs has been recorded to be greater than that of silver nitrate [80]. AgNPs with smaller particle size are found more lethal because of their larger surface area and easy adsorption. Hence, AgNPs toxicity depends largely on size [80,81]. e ability of AgNPs to form an attachment on the bacterial cell wall and membrane, its capability in destruction of the structures within the cell, production of reactive oxygen species, and free radicals that stimulate cellular toxicity and oxidative stress and regulation of the signal transduction pathway are the distinct antibacterial actions of AgNPs [82]. When microorganisms are treated with AgNPs, AgNPs binds on the cell wall of the pathogen and causes the shrinkage of the cytoplasm and busting of the Journal of Chemistry cell wall [83]. e interaction of AgNPs with sulfur-containing enzyme such as O-acetylserine sulfhydrylase (CysK) changes destroys the cell wall morphology of the pathogen [84]. AgNPs has been recommended for the treatment of infections caused by Gram-negative bacteria because AgNPs display higher toxicity against the Gram-negative bacteria than Gram-positive bacteria because of the thinner cell wall and small amount of peptidoglycan they have [85]. e antibacterial activities of AgNPs synthesized from some plants extracts are shown in Table 3.

Conclusion
Over the years, many efforts have been invested into the development of safer method of synthesizing AgNPs. e morphological evaluation of AgNPs synthesized from plants using various characterization techniques revealed variations in size, crystallinity, and shape distribution which is a huge challenge and a major limitation. is suggests that the regulation of silver salt concentration, volume of plant extract, pH value of the solution, reaction temperature, and time have crucial roles in procurement of Ag NPs with uniform size and shape distribution. All the same, the production of AgNPs with uniform morphological properties is of importance. Furthermore, the identification of the exact functional group in the biomolecules of plant accountable for the reduction and stabilization of silver ion required more investigation. Aside from the investigation of the antibacterial potency of AgNPs, the in vivo examination should also be investigated to assess their biocompatibility and toxicity level so that novel and potential therapeutic agent for bacterial can be produced.