Antibacterial Activity of Silver Nanoparticles Synthesized from Plant Latex

Nanoparticles produced by plants are preferred in the medical field for its safe and unpolluted product; it is also accepted as an ecofriendly, non-expensive, and non-toxic nanomaterial. In this study, silver nitrate was successfully used to produce silver nanoparticles (AgNPs) by the use extractsof 4 different latex-producing plants which belong to 2 families (Moraceae and Euphorbiaceae). The synthesis was proved by Atomic Force Microscopy (AFM).The sizes of the AgNP grains were estimated by Granularity Cumulating Distribution (GCD). The results revealed the production of AgNPs in different sizes of 103 and 82 nm using the Moraceae family and 77 and74nm using the Euphorbiaceae family.Antibacterial activity was also detected against both Gram positive and Gram negative pathogenic bacteria using the well diffusion assay. In conclusion, this source of nanoparticles can be a very useful industrial project in a goal to find new safe and economic alternatives to antibiotics.


Introduction
The word ''nano'', meaning ''dwarf '', was derived from a Greek word that refers to objects of one -billionth in size. The nanomaterials have been gathering a great attention due to their wide

ISSN: 0067-2904
Saleh and Najim Iraqi Journal of Science, 2020, Vol. 61, No. 7, pp: 1579-1588 1580 applications, including ''Nano medicine'' as well as their properties in different fields that lead to unique characteristics depending upon their overall size, shape, composition and distribution. Nan oparticles have shown significant change sdue to a wide range of applications in bio-medical, antimicrobials, sensors, electronics, catalysts, agricultural, optical fibers, bio-labeling and others [1].In spite of many applications of nanoparticles, there are certain concerns of toxicities which should be taken in mind [2], mainly including environmental applications of nanotechnology such as environmentally benign products(e.g. green chemistry or pollution prevention) and remediation of substances contaminated with hazardous materials [3].
Chemical and physical techniques have been used to synthesize nanoparticles, including heat evaporation [4], photochemical reduction [5] and many others [6,7]. These techniques have many disadvantages and efforts have been made to find sources with less damage. There is a need to explore and develop environmental friendly techniques of nanoparticle synthesis that do not include toxic materials. Biological processes for nanoparticle synthesis using bacteria, fungi, algae enzymes, and plants or plant extracts have been studied as ecofriendly alternatives tochemical and physical methods [8,9].The use of plants for nanoparticle synth esiscan be much better than other biological processes. Different types of nanoparticles have been used with different mechanisms for fighting microbial resistance, where the synthesized silver nanoparticles showed activity against many clinical strains of bacteria [10]. The first gold and silver nanoparticles were produced by living plants [11], then many plants and plant products were used to produce nanomaterial such as green tea [12], Aloe vera plant extract [13], starch [14],and lemon grass leaves extract [15]. Thus, this study was interested in investigating the possibility of using the plant latex of two latex plant families to facilitate the formation of silver nanoparticles (AgNPs) and then their use as anantibacterial material.

Materials and methods Sample collection
Plants were selected from gardens in Baghdad city Figure-1 and transferred to the laboratory where they were identified by a botanist (Table-1

Ficus carica L (common fig) Moraceae
This plant is from Asian and North America, the common name is figs and considered a good fruit to eat.

Ficus elastic (rubber bush) Moraceae
Native to tropical Asia, India, and has been known to West Indies. The plants is elliptic to oblong leaves, acuminate at apex, rounded at base, glabrous, smooth, leathery, and gray to brown when dry.

Euphorbia milii (crown of thorns) Euphorbiaceae
Native to Madagascar,this plant is straight, slender spines, and the sap is moderately poisonous.

Euphorbiaceace
Native to deserts of southern Africa and Madagascar, the plant shares the features of having a poisonous, milky, white, latexlike sap, and unique floral structures.

Synthesis of Silver nanoparticles
A concentration of 1Mof  [16]. Synthesized AgNPs were centrifuged, dried, and a final stock concentration of 500µg/ml was prepared.

Description of synthesized AgNPs
The synthesized AgNPs were analyzed by Atomic Force Microscope (AFM) pictures for characterization of morphology and size of AgNPs, which enable microscopic information plot topographies showing the structure of the surface and surface alleviation [17]. Scanning Probe Microscopy (SPM) was used to image samples over a wide area to observe dimensions at better resolution.

Detection of antibacterial activity of plant latex
The antibacterial activity of synthesized AgNPs was detected using the well diffusion assay. Dilutions of AgNPs(250, 125 and 62.5 µg /ml) were prepared from the stock concentration(500µg/ml). Indicator bacteria were activated in nutrient broth for 18hr. at and the concentration of the culture was subjected to1.5*10 8 cell/ml that confronts McFarl and turbidity tube of 0.5. Sterilized nutrient agar plates were cultured with pathogenic bacteria using cotton swab. After 5-10 minutes, wells were cut out usin nd of a r d pa r p p o on fro a on n ra on wa add d n w and n a da for 24hr. Inhibition zones were measured in mm [18].

Statistical analysis
The data were analyzed using SPSS IBM version 20 IBM. Results of study groups and assays were expressed as Mean± Standard Error, while One-sample T-test was used to calculate the significance where P-values ≤ 0.05were considered statistically significant.

Results and Discussion Characteristic of the synthesized AgNps
The latex extract of the plants showed change in color and consistency, where the color changedfrom whit e(emulsion) to deep brown with the precipitation of the particles during the process of synthesizing AgNPs.
The technique of AFM deals with images that permit quantitative measurements of the material surface as average roughness (Ra), root mean square roughness (Rq) and analysis of different angels containing 3D simulation [19]. The 2D and 3D AFM images of AgNPs synthesized by plant latex are illustrated in Figure- (Figure-5)which was estimated by the granularity cumulating distribution chart. The largest size of AgNPs was produced by Ficus caria compared to the other plant latex extracts used in the study, where this size was also higher than those produced by algae extracts (25-55 nm) and other bacteria as Pseudomonas aeruginosa (35-46 nm) [20,21]. Many plant parts and extracts showed the ability to synthesize nanoparticles. The size of AgNPs synthesized by the leaves extract of Alternantherta dentale was 50-100nm [22],by the rhizomes of Acorus calamus synthesized AgNPs was 83nm [23], by the seeds of Psoralea corylifoliawas 100-110nm [24], andby the fruits of Vitis viniferawas 30-40nm [25]. Natural latex extracted from Aevea brasilensis was used to synthesize colloidal AgNPs by easy green method with thermal treatment, with a resulted size of 10-20nm [26]. The high levels of flavonoids, carbohydrates, sapogenins and steroids act as reducing and capping agents that give stability to the synthesized nanoparticles [27].These studies show that plants could be a perfect source of nanoparticles which can be utilized in different kinds of applications.

Antibacterial activity of AgNps
Results of antibacterial activity of the AgNPs showed effects against all bacteria with variation in diameters of inhibition zones. The largest inhibition zone was detected against E.coli(20.5mm)while the smallestwas9.5mm was against Staphylococcus aureusat 500µg/ml of synthesized AgNPs ( Figure-6 and Table-2).

Mean±SE (represent triplicate experiments),con:concentration
These results agreed with related studies of synthesizing nanoparticles using plant extracts [28][29][30]. Some specific plant parts or whole plant are used for the successful and sufficientsyn thesis of nanoparticle [31]. The variation in the diameters of inhibition zones may be due to the size or shapes of the synthesized nanoparticles that may affect the growth of bacteria and cause their inhibition,which was confirmed by analyzing enzymes and cell leakage [32]. These preparations can be used for various biotechnology and medical applications for controlling pathogenic bacteria with better dispersion and, consequently, bettere fficiency in aqueous environment. The bactericidal effect of metal nanoparticles was related to their small size and high surface to volume ratio, which let the minteract closely with microbial membranes and is not only due to the release of metal ions in solution [33]. The ionized form of silver with its positive charge provide the antimicrobial property of AgNPs. These ions form complexes with DNA, especially with nucleosides, and not with the phosphate group of the nucleic acid [34]. As a bactericidal agent, some studies showed that electrostatic attraction is presented between positively charged NPs and negatively charged cells of bacteria. These NPs accumulate within the membranes of bacteria and penetrate inside the cells causing the damage [35]. Other studies showed that silver atoms bind to (-SH) group of bacterial enzymes, resulting in a S-Ag stable bonds which cause deactivation of the enzyme. Others proposed that Ag ions which enter the cell disrupt the pyrimidine and purine base pairs, finally disrupting the hydrogen bond between DNA parallel strands and causing denaturation. This interaction with macromolecules of the bacterial cell involves the electron release mechanism as well as free radical formation [36]. The inhibition of protein and cell wall synthesis induced by NPs has been explained by the accumulation of precursor envelope proteins or outer membrane destabilization, which finally leads to energy leakage [37]. According to the results of this study, Gram negative bacterial was more susceptible to the AgNPs than the Gram positive bacteria. This can be explained as the cell wall of Gram positive bacteria is thicker and built of peptidoglycan molecules, as well as its negative charge that causes the positive charge of Ag ions to be trapped outside the cell, as compared to Gram negative bacteria with a very thin cell membrane [38].