Nanotechnology is the 21st century's most promising emerging technology, widely used in information, biological, pharmaceutical, chemical, aerospace, energy, defense and other fields, which has huge market potential. Nanoparticles are particles in the particle diameter between 1~100nm, also referred to as ultrafine particles. Excellent performance of nanomaterials depends on its unique microstructure. Nanoparticles having a small size effect, surface effect, quantum size effect and macroscopic quantum tunneling effect, which shows a material different from conventional thermal, optical, electrical, magnetic, catalytic and sensitive properties. Nano-silver material has a very stable chemical and physical properties that has a very excellent performance in many aspects of electrical, optical and catalytic, now widely used in many fields of ceramic materials, environmentally friendly materials and coatings. When silver particles are embedded in the different substrates, the original material exhibits different electrical and optical properties. Nowadays, study on silver nanoparticles is still a hot spot with a wide application.
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At present, nano-silver antibacterial nonwoven fabric as raw material of a variety of medical, health, household and industrial use antibacterial products have been listed. On the domestic market, the successful launch of nano-silver wound paste, paste nanosilver burns and has dual protective effect of nano silver antimicrobial masks. In recent years, a seriou of products have developed in market, such as a nano-silver antibacterial sanitary napkins, sanitary pads nano-silver, nano-silver antibacterial diapers etc. The newly developed nano-silver antibacterial deodorant antibacterial socks and various types of air filter media, antibacterial wipes, antibacterial mop, antibacterial wipes and the like. Today because of people's increasing emphasis on health care, nanosilver has excellent antimicrobial efficacy of its products and more widely.
Nano silver can also be applied to antibacterial interior wall paint. Antibacterial interior wall paint can effectively inhibit and kill harmful bacteria in the environment, reduce environmental microbiological hazards on the human body, thus achieving the purpose of the clean environment, the protection of human health, improve the living environment of great significance. Inorganic antibacterial agent is present advantages in safety, durability, broad-spectrum antimicrobial resistance, heat resistance, etc., to overcome the disadvantages of conventional paint mildew fungicides greater toxicity, a soluble, heat, poor durability, and increasingly draw people's attention.
Currently there are natural antibacterial materials antibacterial materials, organic materials and inorganic synthetic antibacterial antibacterial materials. In general, natural antibacterial material has the advantage of high security, but it generally short-lived, poor heat resistance, easy re-processing; synthetic organic antibacterial materials having a wide range antibacterial, sterilization speed, etc., but in general it side effects is relatively large, easy hydrolysis, life is short; nano-silver is an inorganic antibacterial material, from the current situation of people in research and development of nano-silver watch, which has a high safety, good heat resistance, antibacterial wide range continuous sterilization of long shelf life and other advantages. It is precisely because of the nano silver antimicrobial material having the above characteristics and quantum effects of nanomaterials itself and a small effect size and great specific surface features, it attracts so many researchers to do researches and development.
The visual color change from pale yellow to dark brown in response to time can be seen as evidence of silver ion reduction to AgNP. The change in color of biosynthesized AgNP is due to the excitation of surface plasmon resonance (SPR). Several studies done on the synthesis of AgNP via medicinal plant suggest the absorption peak around 412–470 nm with the duration of synthesis from 4 h till 24 h, these include medicinal plants, such as Abutilon indicum, Aegle marmelos, Azadirachta indica, Calliandra haematocephala, Calotropis procera, Carica papaya, Helicteres isora, Lawsonia inermi, Leptadenia reticulate, Rheum palmatum, Tecomella undulata, Tagetes erecta, Urtica dioica. The rate of color change from light yellow to dark brown varied in these studies, the earliest color change began within 1 h till 4 h4,20,23,24,30,31,32,33,34,35,36,37,38. Alternatively, different studies utilizing non-medicinal plants for the AgNP synthesis, such as Allium cepa, Chenopodiastrum murale, Cyperus rotundus, Eleusin indica, Euphorbia hirta, Melastoma malabathricum, Musa acuminate, Pachyrhizus erosus, Rubus glaucus exhibited absorption peak from 401–780 nm and was synthesized for 72 h till 14 days. The color change of AgNP synthesized via C. murale turned to brown color after incubating overnight39,40,41,42,43. The difference in color change rate might be due to the different properties of the plant, specifically, the medicinal plant contains a wide range of phytochemicals, such as flavonoids, polyphenols, terpenoids, etc.44 that assist in the formation of silver nanoparticles. Iravani et al.5 reported in their studies that flavonoids, polyphenols, terpenoids, alkaloids and proteins are the main constituents responsible for the reduction and stabilization of silver nanoparticles. Figure 1 shows the result of color change of the synthesized silver nanoparticle with different organs of Carduus crispus, such as stem, flower and the whole plant. It can be seen that different plant organs affected differently on silver nanoparticle synthesis, and particularly whole plant extract facilitated better silver nanoparticle formation compared to the stem and flower extract. The synthesis of silver nanoparticles with whole plant extract exhibited a darker color change. The variation in color change might be due to the different phytochemical content in the plant organs. Following the visual color change study, the formation and stability of silver nanoparticles synthesized with flower, stem, and whole plant of Carduus crispus were characterized using a UV–Vis spectrophotometer (Fig. 2). The results revealed that silver nanoparticles synthesized with whole plant (AgNP-W) exhibited higher absorption compared to silver nanoparticles synthesized using plant organs such as flower (AgNP-F) and stem (AgNP-S). The higher absorption is directly proportional to the higher yield of silver nanoparticles in colloidal solution45. Additionally, the size of the synthesized silver nanoparticle was studied by observing the shift of the absorption peak towards a longer or shorter wavelength8,46. In Fig. 2 b-d, silver nanoparticles were measured at various times, and according to our results, the AgNP-W exhibited blueshift in contrast to AgNP-F and AgNP-S, which can be interpreted as the formation of smaller-sized silver nanoparticles.
Figure 1Color changes in biosynthesized silver nanoparticle with different parts of Carduus crispus. S-stem, F-flower and W-whole plant.
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Figure 2UV–Vis spectra for the reaction mixture containing of silver nanoparticles synthesized from Carduus crispus flower (AgNP-F), stem (AgNP-S) and whole plant (AgNP-W). Shown are the UV–Vis absorption spectra from 370 to 700 nm of all plant organs and synthesized (A) AgNPs, (B) AgNP-W, (C) AgNP-S, and (D) AgNP-F.
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Zeta potential explains the stability, dispersion and surface charge of the nanoparticles. The zeta potential greater than + 30 mV or less than − 30 mV indicates high stability of nanoparticles in dry powder form31. The high negative value produces repulsion between similarly charged particles in suspension, therefore resisting aggregation47. Several studies were done on silver nanoparticle synthesis with a medicinal plant such as Potentilla fulgens, Alpinia calcarata, Pestalotiopsis micospora, Urtica dioica, Jatropha curcas which resulted inzeta potential of − 18 mV, − 19.4 mV, − 35.7 mV, − 24.1 mV, and − 23.4 mV respectively4,6,12,47,48. Our results showed that zeta potential of the synthesized AgNP-W, AgNP-S, AgNP-F had an average zeta potential of − 46.0 2 ± 4.17 (AgNP-W), − 54.29 ± 4.96 (AgNP-S) and − 42.64 ± 3.762 (AgNP-F) (Table 1). The zeta potential of AgNP-S exhibited a higher average value compared to the AgNP-W and AgNP-F, this may be due to the presence of different phytochemicals in each sample that reduces and cap silver nanoparticles. The results of the zeta potential analysis suggest that silver nanoparticles synthesized with Carduus crispus exhibit high stability and resist agglomeration. Figure 3 showed that zeta potential values of AgNP-W, AgNP-S, and AgNP-F fall within the normal distribution curve, which indicates that synthesized silver nanoparticles are fairly monodisperse.
Table 1 Average zeta potential and mobility of AgNP-W, AgNP-S and AgNP-F.Full size table
Figure 3Zeta potential analysis of (A) AgNP-W, (B) AgNP-F and (C) AgNP-S.
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The presence of the functional groups capping AgNP synthesized using Carduus crispus is analyzed by FTIR and shown in Fig. 4. The presence of various organic compounds in the plant reveals multiple peaks compared to the chemical method where only a few and strong peaks are displayed49,50. The results of our FTIR analysis showed the presence of several functional groups in AgNP-W, AgNP-S, AgNP-F. Additionally, the functional groups in AgNP-F and AgNP-S were present in AgNP-W samples as well, this may be attributed to the various phytochemicals capping the silver nanoparticles that are found both in flower and stem of Carduus crispus. The strong characteristic bands at ~ 3418 cm−1 to 3429 cm−1 and 2361 cm−1 in all samples AgNP-S, AgNP-F, AgNP-W are assigned to the O–H stretching/N–H stretching of amides and 2361 cm−1 to the C≡C stretching. Additionally, the weak band at ~ 1017 cm−1 to 1022 cm−1 and ~ 828 cm−1 assigned to carbohydrates and –C = O bending were found in all samples AgNP-S, AgNP-F, and AgNP-W. C–O stretching is present in AgNP-F which was observed from the very strong band at 1353 cm−1. The weak bands at 2922 cm−1 and 2857 cm−1 of CH3 stretch of alkane/carboxylic acids present in AgNP-F and were absent in AgNP-S. The band detected at ~ 3418 cm−1 to 3429 cm−1 and 1618.35 cm−1 correspond to the presence of phenolic compounds and flavonoids, and the band found on 1021.35 cm−1 indicates carboxylic acid, ester, and ether groups of proteins and metabolites that may be involved in the synthesis of nanoparticles33. Our result show that the strong band detected at 1611 cm−1 and 1017 cm−1 from AgNP-F correspond to the presence of flavonoids and proteins. On the other hand, weak bands detected at ~ 1696 cm−1 to 1371 cm−1 correspond to alcohol, carboxylic acids, alkyl halides/carboxylic acids/ester, alkenes/alkyl halides/aromatics, alkynes/alkyl halides stretch that peaks found from AgNP-S. According to Baumberger27 the major compounds detected in Carduus crispus are flavonoids and coumarins, in addition, alkaloids, saccharides, essential oil, rubber and lipids contained in small quantities which is in line with the presence of flavonoids and phenolic compounds in our synthesized AgNP. The AgNP-F and AgNP-S contained different functional groups that correspond to various compounds, and AgNP-F revealed that it has a strong correlation with flavonoids from Carduus crispus. The results of FTIR and UV–Vis spectra analysis confirm that these functional groups are the capping and reducing agents responsible for the synthesis of AgNPs.
Figure 4Fourier transform infrared spectra of (a) AgNP-W, (b) AgNP-S, and (c) AgNP-F.
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The crystalline nature of the synthesized AgNP was confirmed by X-ray crystallography. The XRD pattern of the nanoparticles was analyzed with an XRD instrument and shown in Fig. 5. Bragg reflection of the 2θ peaks was observed at 32.25˚ to 81.62˚ and corresponded to (111), (200), (220), (311), (222) plane lattice which can be indexed to the face-centered cubic crystal nature of the silver. The average crystallite size was calculated using the Scherrer equation. The average crystallite sizes were 13 nm (AgNP-F), 14 nm (AgNP-W) and 36 nm (AgNP-S). The results of our study are in line with other published literature, the crystal nature of silver nanoparticles synthesized with Tagetes erecta 31, Urtica dioica4, Aegle marmelos was face-centered cubic with diffraction peaks of (111), (200), (220), (311) respectively 34. PCCS is a technique based on the Brownian motion that measures the average nanoparticle size (grain size). In Fig. 6, the average particle size of AgNP-W, AgNP-F and AgNP-S was 99.6 nm, 22.5 nm and 145.1 nm respectively. The difference between PCCS and XRD analysis lies in the measurement method of the particle. Application of the Scherrer equation on XRD data gives the average crystallite size, specifically the size of a single crystal inside the particle or grain. The morphological and elemental analysis was done on Scanning Electron Microscope (SEM) and Energy Dispersive X-Ray Spectroscopy (EDX). The elemental composition of the synthesized silver nanoparticle was assessed using EDX spectroscopy (Table 2). The results in Fig. 7 showed that AgNP-W, AgNP-S, and AgNP-F contained silver and potassium elements together with several other elements that differed in AgNP-F and AgNP-S samples, i.e. AgNP-F included phosphorus 2.8%, potassium 15.2%, and AgNP-S had calcium 7.5%, pottassium 15.5% elements. In contrast, AgNP-W contained all the elements including the elements that differed in AgNP-F and AgNP-S. Interestingly, the silver element in AgNP-F had the highest content of 82% compared to AgNP-W and AgNP-S which had a silver content of 79% and 77% respectively. Another observation on EDX analysis revealed that AgNP-W, AgNP-F, AgNP-S did not show the presence of nitrogen peak, this indicates that trace ions from AgNO3 are absent in the samples. The size of biosynthesized AgNP-W, AgNP-F and AgNP-S was determined with Atomic Force Microscopy (AFM). Figure 8 show that the size of nanoparticles differed, for instance, AgNP-W had a size of 70 nm, AgNP-F with size 33 nm and AgNP-S with size 131 nm. Figure 8 (A-C, E–G and I-K) represents the two dimensional images of AgNP-W, AgNP-F and AgNP-S. Figure 8 (D, H and L) shows the three dimensional image of AgNP-W, AgNP-F and AgNP-S respectively. The different composition of plant organs, such as stem, flower and whole plant could be the reason for the observed variability in, color change, UV–Vis absorption, EDX, FTIR. In addition, the results of AFM data and XRD show that the synthesis of AgNP can be manipulated with different plant organs.
Figure 5XRD spectra of (a) AgNP-W, (b) AgNP-F, (c) AgNP-S. Peaks are appeared at 111, 200, 220, 311 and 222.
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Figure 6PCCS analysis: particle number distribution of synthesized AgNP-W (A), AgNP-F (B) and AgNP-S (C).
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Table 2 Elemental composition of the synthesized silver nanoparticles by Carduus cripus.Full size table
Figure 7EDX spectra for (A) AgNP-F, (B) AgNP-S and (C) AgNP-W along with SEM image area (inset).
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Figure 8Atomic force microscopy images (2D and 3D) of silver nanoparticles on siliconized cover slide; AgNP-W (A–D), AgNP-F (E–H) and AgNP-S (I–L).
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The antibacterial activity of silver nanoparticles was studied against pathogenic bacterial strains of gram-negative E.coli and gram-positive M.luteus using the well diffusion method (Fig. 9). Standard antibiotics such as Penicillin G and Chloramphenicol, plant extracts, AgNO3 and distilled water were chosen as the control group. The results of the antibacterial activity showed that all synthesized silver nanoparticles had efficient antibacterial activity against both gram-negative E.coli and gram-positive M.luteus bacterial strains. The inhibition zone of AgNP-F, AgNP-W and AgNP-S against E.coli and M.luteus were 6.5 ± 0.3, 6 ± 0.2, 5.5 ± 0.2 and 7.5 ± 0.3, 7 ± 0.2, 7.7 ± 0.4 mm respectively. The plant extract and AgNO3 did not reveal any antibacterial activity against both E.coli and M.luteus, which can be interpreted that AgNP-W, AgNP-F, and AgNP-S are solely responsible for the antibacterial activity. The mode of action of AgNPs against bacteria is not completely understood yet. However, several hypotheses are explaining the antibacterial activity of silver nanoparticle: (1) generation of reactive oxygen species; (2) release of Ag + ions from AgNPs denaturize proteins by bonding with sulfhydryl groups; (3) attachment of AgNPs on bacteria and subsequent damage to bacteria4,11,24. The multiple published reports on the antibacterial activity of silver nanoparticles against gram-negative and gram-positive bacteria showed that silver nanoparticles had a slight antibacterial activity on gram-positive bacteria6,22,31,36. Interestingly, AgNP synthesized by Carduus crispus exhibited effective inhibition on both gram-positive and gram-negative bacteria which can be interpreted that the antibacterial activity of silver nanoparticles (AgNP-W, AgNP-F and AgNP-S) is not affected by the difference in the bacterial wall.
Figure 9Petri dishes showing the zone of inhibition of synthesized AgNP-W on (A) M. luteus and (B) E. coli, and AgNP-F on (C) M. luteus and (D) E. coli, AgNP-S on (E) M. luteus and (F) E. coli (AgNP: silver nanoparticle, AgNO3: silver nitrate, DW: distilled water, PE: plant extract).
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Cytotoxicity is considered as an important indicator for cell viability, therefore in this study we employed crystal violet assay to investigate the effect of different concentration of AgNP-W, AgNP-F and AgNP-S on the adherent human hepatoma cell line HepG2 (Fig. 10). The liver is an important organ with detoxifying effect, additionally, it is considered as an accumulation site for AgNPs51. In this study, the untreated HepG2 cell lines revealed significant adherence to the well plate. On the other hand, the treated cells with nanoparticles exhibited small decrease in cell viability after 24 h incubation at 3 to 17 µg/ml. The cell viability of these treated groups with AgNP-W, AgNP-F and AgNP-S were 87.93 ± 4.87%, 92.24 ± 1.21% and 86.20 ± 2.43% at 17 µg/ml. The toxicity of AgNPs to bacteria and human cells is widely known, however, the result of our study suggests that AgNPs synthesized by medicinal plant Carduus crispus with concentration of 3 to 17 µg/ml have low toxicity on HepG2 cell line (Fig. 10A,B). In addition, biosynthesized silver nanoparticles possessed efficient antibacterial activity against Gram-negative and Gram-positive bacteria (Fig. 9). The antibacterial activity of the synthesized AgNPs and their low toxicity to human cells may enable further application in biomedical field. The low toxicity of biosynthesized AgNPs to adherent human cells are similar to other published reports52.
Figure 10A microscopic pictures of HepG2 cells treated with AgNPs for 24 h in cell culture: control (A), AgNP-W (B), AgNP-F (C) and AgNP-S (D). After 24 h, the cell toxicity effect was examined with Crystal Violet (E).
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