Antibacterial activity and characteristics of silver nanoparticles biosynthesized from Carduus crispus

In recent years’ synthesis of metal nanoparticle using plants has been extensively studied and recognized as a non-toxic and efficient method applicable in biomedical field. The aim of this study is to investigate the role of different parts of medical plant Carduus crispus on synthesizing silver nanoparticles and characterize the produced nanoparticle. Our study showed that silver nanoparticles (AgNP) synthesized via whole plant extract exhibited a blue shift in absorption spectra with increased optical density, which correlates to a high yield and small size. Also, the results of zeta potential, X-ray diffraction, photon cross-correlation spectroscopy analysis showed the surface charge of − 54.29 ± 4.96 mV (AgNP-S), − 42.64 ± 3.762 mV (AgNP-F), − 46.02 ± 4.17 mV (AgNP-W), the crystallite size of 36 nm (AgNP-S), 13 nm (AgNP-F), 14 nm (AgNP-W) with face-centered cubic structure and average grain sizes of 145.1 nm, 22.5 nm and 99.6 nm. Another important characteristic, such as elemental composition and constituent capping agent has been determined by energy-dispersive X-ray spectroscopy and Fourier transform infrared. The silver nanoparticles were composed of ~ 80% Ag, ~ 15% K, and ~ 7.5% Ca (or ~ 2.8% P) elements. Moreover, the results of the FTIR measurement suggested that the distinct functional groups present in both AgNP-S and AgNP-F were found in AgNP-W. The atomic force microscopy analysis revealed that AgNP-S, AgNP-F and AgNP-W had sizes of 131 nm, 33 nm and 70 nm respectively. In addition, the biosynthesized silver nanoparticles were evaluated for their cytotoxicity and antibacterial activity. At 17 µg/ml concentration, AgNP-S, AgNP-F and AgNP-W showed very low toxicity on HepG2 cell line but also high antibacterial activity. The silver nanoparticles showed antibacterial activity on both gram-negative bacterium Escherichia coli (5.5 ± 0.2 mm to 6.5 ± 0.3 mm) and gram-positive bacterium Micrococcus luteus (7 ± 0.4 mm to 7.7 ± 0.5 mm). Our study is meaningful as a first observation indicating the possibility of using special plant organs to control the characteristics of nanoparticles.


Scientific Reports
| (2021) 11:21047 | https://doi.org/10.1038/s41598-021-00520-2 www.nature.com/scientificreports/ cles 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 solution 45 . Additionally, the size of the synthesized silver nanoparticle was studied by observing the shift of the absorption peak towards a longer or shorter wavelength 8,46 . 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.
Zeta potential analysis. 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 form 31 . The high negative value produces repulsion between similarly charged particles in suspension, therefore resisting aggregation 47 . 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 respectively 4,6,12,47,48 . 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.   www.nature.com/scientificreports/ band at 1353 cm −1 . The weak bands at 2922 cm −1 and 2857 cm −1 of CH 3 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 nanoparticles 33 . 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 Baumberger 27 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.

XRD, PCCS, SEM/EDX and AFM analysis.
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   www.nature.com/scientificreports/ 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 AgNO 3 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.    Antibacterial activity. 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).  www.nature.com/scientificreports/ In-vitro cytotoxicity assay. 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 AgNPs 51 . 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 reports 52 .

Conclusion
The synthesis of silver nanoparticles via biological method, specifically plant extracts provides a natural, ecofriendly, cost-effective, rapid synthesis of silver nanoparticles. The present study reports the synthesis of silver nanoparticles with medicinal plant Carduus crispus in reducing silver ions and stabilizing the silver nanoparticles. It has been reported that medicinal plants are a rich source of phenolic compounds such as flavonoids and phenolic acids, etc. Additionally, plant organs contain different contents of phenolic compounds, therefore flower, stem, and whole plant of Carduus crispus were chosen for this study. Afterwards, the synthesized silver nanoparticles were characterized using visual color change, UV-Vis spectroscopy, zeta potential, FTIR, XRD, PCCS, SEM-EDX and AFM. The characterization of AgNP-W, AgNP-F, and AgNP-S revealed that AgNP-W had a higher yield, synthesis rate, and smaller-sized silver nanoparticles. The zeta potential conveys the stability and the result of all the synthesized silver nanoparticles showed the zeta potential value of − 46.0 2 ± 4.17 (AgNP-W), www.nature.com/scientificreports/ − 54.29 ± 4.96 (AgNP-S), and − 42.64 ± 3.762 (AgNP-F) which indicates highly stable silver nanoparticles. The variation in zeta potential may be due to the different phytochemical properties of the plant. Then FTIR analysis was utilized to study the role of phytochemical properties in plants for the synthesis of silver nanoparticles, the results showed that different functional groups in AgNP-F and AgNP-S were also present in AgNP-W samples as well. And based on the UV-Vis spectra analysis, AgNP-W and AgNP-F had the highest absorbance compared to AgNP-S, therefore we can conclude that the functional groups present and coincided in both AgNP-F and AgNP-W may play a contributing role in capping and synthesis of silver nanoparticles, these include functional groups with bands at 2922. 28    www.nature.com/scientificreports/ flavonoids and phenolic compounds. The EDX analysis detected the following elements, such as silver, potassium, phosphorus in AgNP-F; silver, potassium, calcium, chloride, and phosphorus in AgNP-W; finally, silver, potassium, calcium in AgNP-S samples. The synthesized silver nanoparticles had an average crystallite sizes of 14 nm (AgNP-W), 13 nm (AgNP-F) and 36 nm (AgNP-S) with face-centered crystal structure and average grain sizes of 99.6 nm (AgNP-W), 22.5 nm (AgNP-F) and 145.1 nm (AgNP-S). The sizes detected in AFM was 70 nm (AgNP-W), 33 nm (AgNP-F) and 131 nm (AgNP-S). Although the method of synthesis varied in AgNP-F, AgNP-W, and AgNP-S, their antibacterial activity showed efficient inhibition on both gram-negative and gram-positive bacteria. Based on these results, we can conclude that silver nanoparticles synthesized by whole plant of Carduus crispus have a faster rate of synthesis, higher yield with a smaller size, and high antibacterial activity against both gram-negative and gram-positive bacteria. The overall results show that the effectiveness of the synthesis of the flower for AgNP appears similar to using whole plant. Additionally, we have shown that the process of synthesizing nanoparticles can be manipulated with specific organs of plant, for example, particle size and synthesis duration, biological effect, etc. Our study is meaningful as a first observation indicating the possibility of using special plant organs to control the characteristics of nanoparticles. Moreover, further studies are required in this area.

Methods
Chemicals and plant. Preparation of plant extract. The whole plant was washed with tap water in order to remove the adhering dust and soil particles, followed by washing with distilled water. 100 ml of distilled water was added to 5 g of Carduus crispus and boiled for 15 min, then cooled at ambient temperature. Afterward, it was filtered by Whatman filter paper and centrifuged twice at 10,000 rpm to obtain a plant extract. Finally, the extract was ready for the synthesis of AgNP.
Synthesis of silver nanoparticle. The aqueous plant extract of Carduus crispus and AgNO 3 (1 mM) were mixed with the ratio of 1:16, then the solution was exposed to the daylight and the reaction took place at the various time at room temperature. In order to obtain silver nanoparticles in powdered form, the solution was vaporized on a vacuum evaporator, and the final product of AgNP was kept inside the oven at a temperature of 300 °C for 4 h.

Characterization of AgNP synthesized by Carduus crispus. AgNP was successfully synthesized by
using Carduus crispus. A color change from pale yellow to colloidal dark brown indicated the formation of silver nanoparticles. UV-Vis spectra analysis offers an insight into the synthesis and stability of the AgNP. Formation of the biosynthesized AgNP was determined by the UV-Vis spectrophotometer (Shimadzu UV-2500PC Series) at 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 12 h, 24 h and was carried out at 350-700 nm range. FTIR spectrum was recorded in the range of 500 to 4000 cm −1 through the potassium bromide powder method using FTIR spectrophotometer (Prestege-21, Shimadzu, Japan) for understanding the constituent capping and reducing agents of silver nanoparticles. Also, elemental composition of the synthesized silver nanoparticles was analyzed with an energy dispersive X-ray spectroscope instrument (TM-10000 with EDX). To identify the structural phase present in the AgNP, XRD was performed by XRD instrument (Shimadzu, Maxima-X-7000) operating at 40 kV with a current of 30 mA and Co-Ka radiation. And crystalline size was determined by Scherrer equation. In order to understand the size distribution and surface charge, the zeta potential (ZetaCompact, CAD Instruments, France) and Photon Cross-correlation Spectroscopy (PCCS) (NANOPHOX 1 nm to 10,000 nm, Sympatec GmbH, Germany) methods was used for dispersed nanoparticles of silver.
Atomic force microscope (AFM) measurement. The size of the synthesized AgNP was analyzed with Atomic Force Spectroscopy (SPA 300, Seiko Inc., Japan). First, the siliconized glass cover slides selected for the AFM measurement were immersed in ultra-pure water and sonicated for 10 min with ultra sonicator, afterwards the siliconized glass cover slides were rinsed with ethanol solution and air-dried in laminar box at RT, then the samples for AFM analysis were prepared by drying the AgNP suspension on prepared siliconized glass cover slide at RT. Finally, Atomic Force Microscope was used to analyze the morphology and size of the samples via golden silicon probe (GSG11) with tip curvature radius of 10 nm.
Determination of anti-bacterial activity using well diffusion method. The agar well diffusion method was used to study the antibacterial activity of the synthesized silver nanoparticle. Broth medium was used to subculture bacteria and was incubated at 37 °C for 24 h, afterwards, overnight cultures were taken and spread on the agar plates to cultivate a uniform microbial growth plate. The bacterial strains were gram-negative Escherichia coli and gram-positive Micrococcus luteus. And silver nitrate, plant extract, antibiotics (Penicillin G against Micrococcus luteus and Chloramphenicol against Escherichia coli) were chosen as the control group for