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. 2018 Mar 27;12(5):574–578. doi: 10.1049/iet-nbt.2017.0265

Green synthesis of silver nanoparticles using Glaucium corniculatum (L.) Curtis extract and evaluation of its antibacterial activity

Ali Reza Allafchian 1,✉, Seyed Amir Hossein Jalali 2,3, Farzane Aghaei 1, Hamid Reza Farhang 2
PMCID: PMC8676330  PMID: 30095415

Abstract

The metal nanoparticles, due to interesting features such as electrical, optical, chemical and magnetic properties, have been investigated repeatedly. Also, the mentioned nanoparticles have specific uses in terms of their antibacterial activity. The biosynthesis method is more appropriate than the chemical method for producing the nanoparticles because it does not need any special facilities; it is also economically affordable. In the current study, the silver nanoparticles (AgNPs) were obtained by using a very simple and low‐cost method via Glaucium corniculatum (L.) Curtis plant extract. The characteristics of the AgNPs were investigated using techniques including: X‐ray diffraction, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy. The SEM and TEM images showed that the nanoparticles had a spherical shape, and the mean diameter of them was 53.7 and 45 nm, respectively. The results of the disc diffusion test used for measuring the anti‐bacterial activity of the synthesised nanoparticles indicated that the formed nanoparticles possessed a suitable anti‐bacterial activity.

Inspec keywords: silver, nanoparticles, antibacterial activity, nanomedicine, nanofabrication, X‐ray diffraction, transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectra

Other keywords: green synthesis, silver nanoparticles, Glaucium corniculatum Curtis extract, antibacterial activity, metal nanoparticles, biosynthesis method, X‐ray diffraction, transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, SEM, TEM, spherical shape, disc diffusion test, Ag

1 Introduction

In the recent decades, the nanotechnology science, coupled with its hallmark features, has created a new frontier of knowledge, such that its scope has been extended from human body to various industrial fields; it is, in fact, developing continuously. Consequently, the nature and its extraordinary features have been one of the fundamental issues for biology researchers. It is worth mentioning that different communities of microorganisms can have a significant effect in further developing the nano science [1, 2]. One of the novel areas is nanobiotechnology; it has developed a wide range of specialised biology studies; also, it is the fusion of biotechnology and nanotechnology. In other words, nanobiotechnology has provided the tools and other related technologies to ensure the development of stimulation models for bio accumulated components using biology. On the other hand, the use of the knowledge and the prevalent techniques in biology has been common to manoeuvre in genetic, molecular and cellular processes; this has been focused on building products and services in the nano scale in various fields ranging from agriculture to medicine [3, 4, 5].

The nanoparticles, due to specific properties such as shape, size, distribution pattern and morphology, have had a significant impact in all aspects of human life [6, 7, 8, 9, 10]. Among them, the metal nanoparticles such as silver, platinum, gold and palladium have been considered due to many applications in various scientific fields such as pharmaceuticals, medicine, biomedical engineering and health [10, 11, 12]. AgNPs, among all nanoparticles with metal origin, are more recognised than the other nanoparticles because their numerous applications have been proved in different scientific areas [13, 14]. Since the old times, silver, as one of the antibacterial agent, has been utilised for treating the illnesses and keeping foods and waters. That is why it can be said that the greatest application of AgNPs is in the medical industry [15]. Given the current developments of nanotechnology, AgNPs have been used in various fields of medical sciences, such that the mentioned nanoparticles have played a substantial role as one of the therapeutic agents in different medical fields considering antibacterial, antifungal, anti‐viral, anti‐inflammatory and anti‐cancerous properties; also, they are consolidating their position in the mentioned areas [16, 17]. In the synthesis of silver nanoparticles (AgNPs), the size of the particles has been prioritised; following that, the synthesised nanoparticles with characteristics such as small size and lack of the accumulation of particles have been pursued in all of the conducted studies [13, 18]. It is worth mentioning that some of the properties of AgNPs depend on their size and shape; for example, the spherical shape and tiny nanoparticles, in comparison with the rest of the forms of AgNPs, can have a better and more efficient function [12]. The synthesis of AgNPs is done by different ways or methods; they are usually carried out by physical and chemical methods; some of the most important ones are the use of gamma rays, ultraviolet radiation and electro‐chemical regeneration, and the application of synthesising chemical materials such as hydrazine, sodium borohydride, glycerol, glucose, ethylene glycol, formaldehyde, and the sodium in ammonia liquid [13]. Unfortunately, most of the above listed methods are very risky and hazardous for nature and environment due to the utilisation of some toxic and dangerous chemical matters and also, high the energy consumption required for their synthesis.

The biosynthesis or green synthesis of AgNPs using bio microorganisms and the extraction of various organs of plants (aerial parts and underground organs) have been more compatible with the environment (eco‐friendly); it has been recently introduced as an alternative method instead of using physical and chemical synthesis methods [10, 12, 19, 20, 21, 22, 23, 24]. Among the newly introduced methods, the use of the plants for the synthesis of nanoparticles, due to their numerous advantages such as wide distribution, accessibility and affordability, has been considered more than the other cases [25, 26, 27]. Previously, in other studies, numerous cases of the synthesis of AgNPs have been reported using plant extracts, thereby confirming the mentioned issue. Some of the studies carried out in this case are as follows: cycas leaf [28], alfalfa sprouts [29], Dorema ammoniacum D. [30], Azadirachta indica (Neem) leaves [19], Geranium leaf [31], Parthenium leaf [32], Cinnamomum zeylanicum bark [33], Lepidium draba [34], Gundelia tournefortii L. [35], Mangifera indica leaf [36] and phlomis leaf [37].

The present study was aimed to rapidly green synthesise AgNPs using the aqueous aerial parts extract of G. corniculatum (L.) Curtis to investigate the biomolecules responsible for the evaluation of its antibacterial activity. The green synthesis of AgNPs was conducted using G. corniculatum (L.) Curtis aerial parts extract in the mentioned research has not been reported yet. In order to evaluate and characterise the synthesised AgNPs, usual techniques such as X‐ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR) were used. Finally, the investigation of antibacterial activities of AgNPs was done and the results were compared with their respective silver nitrate.

2 Experimental

2.1 Materials

Silver nitrate was purchased from Merck Chemical Company for this study. Also, the whole glassware was washed with diluted HNO3 and distilled water and dried in the oven before use. G. corniculatum (L.) Curtis is a plant species of the Glaucium genus that belongs to Papaveraceae family. It is an annual or, sometimes, biennial flowering plant grown extensively in the West and North West of Iran between the months of July and August. Some of the most common names are as follows: blackspot hornpoppy, red horned‐Poppy, and bristly horned‐Poppy. Samples of G. corniculatum (L.) Curtis aerial parts consist of leaves, stems and flower buds collected in July, 2016 (from one of its habitats located in the western regions of Iran) during the flowering period and the vegetative phase. After that, the taxonomic identity of the plant was confirmed by comparing the collected voucher specimen with that of a known identity available in the herbarium of the Department of Natural Resources, Isfahan University of Technology, Iran.

2.2 Apparatus

The FT‐IR spectrum of AgNPs was recorded by (JASCO FT–IR/680plus, Japan) spectrometer with KBr pellets around the 400–4000 per centimetre. The pseudo crystal structure (transparent) of the AgNPs was investigated using the X‐ray diffraction analysis. The phase investigation of the synthesised nanoparticles was examined by X‐ray diffraction analysis, using X'Pert PRO MPD XRD (Philips). It was also achieved in the range of 2θ, from 20 to 80°, with the size of each step being 0.02°, 40 kV, 30 mA, and with a Cu Kα radiation (λ  = 1.5406 Å). Morphological characterisation of AgNPs was carried out with the aim of investigating the shape, size and cross section of the formed AgNPs using transmission electron microscopy (TEM) (cm30‐Philphs) and SEM (HITACHI S‐4160, Japan).

2.3 Microorganisms and growth conditions

The used bacteria, along with their specified scientific names and identification code in this research, were as follows: Escherichia coli (ATCC 35218) and Salmonella typhimurium (ATCC 14028), as Gram‐negative bacteria, and Staphylococcus aureus (ATCC 29213) and Bacillus cereus (ATCC 14579), as Gram‐positive bacteria. They were tested to perform the antibacterial activity. Also, all these bacteria strains were cultured at 37°C on the Luria Bertani agar.

2.4 Preparation of the plant extract and synthesis of AgNPs

Initially, 80 gr of the air‐dried aerial part of G. corniculatum (L.) Curtis was boiled in one litre of distilled water at 86°C for 2 h; then, it was filtered through the Whatman filter paper No. 1. The made aqueous extract was kept in the standard condition for further examinations. In order to prepare AgNPs, 0.034 gr of the solid nitrate silver was weighted by the precision scale and shed into a volumetric Balloon; also, it was brought to the volume of 200 ml. Therefore, 200 ml of the solution of silver nitrate 0.01 M was made. Following that, 25 ml of the mentioned extract was mixed with 200 ml of the solution of silver nitrate separately and the variation of solution colour was followed in the time range of 1 h at 60°C. The colour of the solution was dark after an hour. After the formation of nanoparticles, it was kept at the ambient temperature until it was completely dried.

2.5 Antimicrobial assay

The test of bacterial sensitivity was done by the disk diffusion method, using bacterial strains of B. cereus, E. coli, S. aureus and S. typhimurium [38]. At first, 30 µL of each bacterial suspension (105 bacteria/mL), with a concentration of 100 mg/L of the dispersed AgNPs in the salt phosphate buffer, and 30 µL of the solution of AgNPs, with a concentration of 100 mg/L and also, 30 µL of the plant extract were transmitted on the standard disks with the help of the micropipette. The bacterial strains were prepared at a concentration of 5.0 McFarland and sterile swabs were cultured on the Mueller–Hinton agar; subsequently, the discs were placed with Anas sterile on the culture medium. Finally, in order to evaluate the antibacterial activity, the plates were incubated at 37°C for 16 h.

3 Results and discussion

3.1 TEM and SEM analysis

The morphological structure of the surface of AgNPs was investigated by TEM and SEM. The TEM and SEM images showed that the formed nanoparticles were spherical in terms of shape; also, two other features of them, namely mean diameter and standard deviation related to the formation of the AgNPs, were 53.7 and 45 nm, respectively (Figs. 1 and 2). It is worth mentioning that the optical and electronic properties of metal nanoparticles are usually altered considerably [39]. On the other hand, the biosynthesis of AgNPs using the aerial parts extract of different plants formed a product with various dimensions; due to variable values, they are completely different from each other. In the previous studies, it has been confirmed that the formation of the small particles of AgNPs could lead to increasing nucleation; it is owing to enhancement throughout the reduction process [40].

Fig. 1.

Fig. 1

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TEM images of bio‐synthesised AgNPs by using the G. corniculatum (L.) Curtis aerial parts extract

Fig. 2.

Fig. 2

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SEM images of bio‐synthesised AgNPs by using the G. corniculatum (L.) Curtis aerial parts extract

3.2 UV–‐vis analysis

The AgNPs were characterised by UV–vis spectroscopy. As shown in UV–vis spectra, strong surface plasmon vibrations in AgNPs were excited at ∼440 nm (Fig. 3). Observation of this broad and strong surface plasmon peak has been well recognised for various metal NPs, with sizes ranging all the way from 2 to 100 nm [41].

Fig. 3.

Fig. 3

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UV–vis spectra recorded after the addition of the aerial part of G. corniculatum (L.) Curtis (25 mL) to the 0.01 M silver nitrate solution (200 mL)

3.3 XRD analysis

The sample was washed several times to remove NaCl salt and impurities. This sample was used for further experiments. The XRD patterns for the synthesised AgNPs using G. corniculatum (L.) Curtis aerial parts are shown in Fig. 4. As shown in the diagram, the peak characteristic of AgNPs was located in the range of 38.2°, 44.4°, 64.3°, 77.6°, and 81.6°. The values of the (hkl) for these angles were (111), (200), (220) and (311), respectively. Following that, it turned out that the AgNPs possessed a cubic structure along with a full sides centre. Also, the position of the mentioned peaks was matched with (JCPDS) file no. 04‐0783. In the recent study, by considering the conducted studies by other researchers, some additional peaks were observed in Fig. 4, showing, via the star sign, that the mentioned peaks were related to the crystal materials of bioorganic materials. This pattern indicated that the average size of AgNPs was calculated by Scherrer equation

D=Kλβcos⁡θ (1)

Fig. 4.

Fig. 4

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XRD spectra of AgNPs by using the G. corniculatum (L.) Curtis aerial parts extract

In the above equation, ‘D’ is the particle diameter size, ‘k’ is the Scherrer coefficient with the value from 0.9 to 1, ‘λ’ is the wavelength of X‐ray source (1.5418 Å), ‘β ½’ is the peak width of the XRD at the maximum point, and ‘θ’ is the Bragg angle. This equation showed that the average size of AgNPs was 52 nm.

3.4 FTIR analysis

The FTIR spectroscopy was used to identify biomolecules and the functional groups participating in the effective reduction and stabilisation of the newly synthesised nanoparticles. One to 2% of the sample was mixed with KBr and press powdered to take a transparent pellet. The pellet was placed in the spectrometer, obtaining a spectrum in 400– 4000 cm − 1. The FTIR spectrum of the stabilised AgNPs is shown in Fig. 5. The peak one was almost related to the absorption band of O–H or the stretching band of NH3 group at the wavelength of 3409 cm − 1. The peak two was related to the asymmetrical tensile of the C–H group at the wavelength of 2926 cm − 1 and the symmetrical tensile of the C–H group in the alkane wavelength of 2900 cm − 1. The peak three was related to the tensile of the C–H in aldehyde at the wavelength of 2854 cm − 1. The peak four was related to the asymmetric aldehyde tensile of the C=O group and saturated aliphatic at the wavelength of 1746 cm − 1. The peaks five and six are shown at the wavelength of 1384 and 1597 cm − 1 and they could be matched with the bending C–H and the tensile vibration of the aromatic rings of C–C, respectively. In addition, the peak 7 at the wavelength of 1236 cm − 1 and the peak 9 at the wavelength of 1078 cm − 1 were compatible with the tensile C–N; this could be attributed due to the presence of the amine group [42, 43, 44]. Based on the above results, it could be seen that the functional groups such as hydroxyl (O–H), carboxyl (C=O) and amine (C–N) had played a remarkable role; also, they were involved in the synthesis of AgNPs using the G. corniculatum (L.) Curtis extract. This agreed with a previous study of this plant. Different components including alkaloid were found to be the major compound and flavonoid and phenolic contents in the G. corniculatum (L.) Curtis extract [45, 46, 47].

Fig. 5.

Fig. 5

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FTIR spectra of bio‐synthesised AgNPs by using the G. corniculatum (L.) Curtis aerial parts extract

3.5 Antibacterial activity

The use of silver, as one of the most famous antibacterial substance and also, its usage in the numerous biomedical applications have been remarkable in recent decades [48, 49]. The results of the antibacterial activity test showed that in the same concentration of silver nitrate and AgNPs, the diameter of the growth inhibitory zone for the E. coli bacterium was 8.6 ± 0.4 and 9.6 ± 0.3 mm, respectively (Fig. 6, part A). Following that, the diameter of the growth inhibitory zone of the S. typhimurium bacterium in the face of the silver nitrate was 7.6 ± 0.3 mm; it was also 8.6 ± 0.2 mm for the AgNPs (Fig. 6, part B). The diameter of the growth inhibitory zone of the B. cereus bacterium in the face of the silver nitrate was 6.9 ± 0.1 mm; it was also 7.6 ± 0.2 for the AgNPs (Fig. 6, part C). Finally, the diameter of the growth inhibitory zone of the S. aureus bacterium in the face of the silver nitrate was 6.7 ± 0.2 mm; it was also 8.3 ± 0.4 mm for the AgNPs (Fig. 6, part D). All of the above results are listed in Table 1. It is worth mentioning that the mechanism of the AgNPs action against bacteria strains has not been specified yet completely. For instance, it is said that some cellular features, especially respiration and permeability, might be affected by the power of operation of cells; items such as addition, binding or aggregation of AgNPs to the cell surface membrane are some examples making these variations. The impact of AgNPs is not limited only to the cell surface membrane and it may also be in the intracellular space occurring specifically in bacteria and fungi; it causes the changes and damages in the function and interaction of the intracellular components. An obvious example of it is visible and traceable in DNA.

Fig. 6.

Fig. 6

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Antibacterial activity test using four strains bacteria: (A) E. coli, (B) S. typhimurium, (C) B. cereus and (D) S. aureus. The written numbers on petri dishes are as follows: (1) AgNPs, (2) AgNO3, (3) G. corniculatum (L.) Curtis aerial parts extract, and (4) distilled water

Table 1.

Inhibition zone (mm) of the AgNO3, AgNPs, G. corniculatum (L.) Curtis aerial parts extract and deionised water against the tested bacteria

Bacterial species Zone of inhibition, mm
AgNO3 AgNPs G. corniculatum (L.) Curtis aerial parts extract Deionised water
E. coli 8.6 ± 0.4 9.6 ± 0.3 6.4 6.4
S. typhimurium 7.6 ± 0.3 8.6 ± 0.2 6.4 6.4
S. aureus 7.7 ± 0.2 8.3 ± 0.4 6.4 6.4
B. cereus 7.1 ± 0.1 7.6 ± 0.2 6.4 6.4
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The disk's diameter was 6.4 mm.

4 Conclusion

In the recent study, the stable biosynthesis and the nearly spherical shape of the AgNPs were demonstrated using the aerial parts extract of G. corniculatum (L.) Curtis as a reducing agent of silver ions. The synthesised nanoparticles showed a strong antibacterial activity in contrast to Gram‐positive bacteria (S. aureus and B. cereus) and Gram‐negative bacteria (S. typhimurium and E. coli). Also, the AgNPs were shaped using the G. corniculatum (L.) Curtis, and it was confirmed through the measurement and characterisation techniques of the nanoparticles, such as X‐ray diffraction, transmission electron microscopy, scanning electron microscopy, and FTIR spectroscopy. The examination of the XRD spectrum pattern indicated that the synthesised nanoparticles had a crystalline structure and the particle size of the mentioned nanoparticles was 52 nm. Also, the SEM and TEM images showed that the formed nanoparticles had spherical shapes and the mean diameter of them was 53.7 and 45 nm, respectively. Consequently, the biological synthesis of AgNPs using plant materials could be regarded as one of the most appropriate and well‐matched methods with environmental conditions (eco‐friendly), in comparison to the chemical and physical synthesis.

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