Green synthesis and stabilization of silver nanoparticles using Lysimachia foenum-graecum Hance extract and their antibacterial activity

Abstract The Lysimachia foenumgraecum Hance extract (LHE) was used for silver nanoparticles (AgNPs) synthesis. In this study, the herbal plant of Lysimachia foenumgraecum Hance (LH) was extracted with deionized water and we are the first to successfully use LHE as reducing and stabilizing agents for the green synthesis of AgNPs. The concentration of LHE used in this study was in the range of 0.003 to 1.0 wt%. Aqueous colloidal solutions of AgNPs reduced and stabilized by LHE show long-term stability due to the steric stabilization effect. This can be confirmed by zeta potential measurements which afforded values approximately of 0 mV, indicating the steric stability of AgNPs colloidal solutions synthesized by LHE. Furthermore, the obtained AgNPs colloidal solutions show superior antibacterial effect to gram-positive bacteria (Staphylococcus aureus) comparing to Chloramphenicol (positive control). AgNPs with LHE 0.003 wt% affords the highest antibacterial effect to S. aureus showing an inhibition zone diameter of 19.08 ± 0.67 mm; which is superior to Chloramphenicol.


Introduction
Let F denote a eld and let V denote a vector space over F with nite pos pair A, A * of diagonalizable F-linear maps on V, each of which acts on an ei irreducible tridiagonal fashion. Such a pair is called a Leonard pair (see [13, D A, A * is said to be self-dual whenever there exists an automorphism of the e swaps A and A * . In this case such an automorphism is unique, and called th The literature contains many examples of self-dual Leonard pairs. For inst ated with an irreducible module for the Terwilliger algebra of the hypercube (s Leonard pair of Krawtchouk type (see [10, De nition 6.1]); (iii) the Leonard pa module for the Terwilliger algebra of a distance-regular graph that has a spin bra (see [1,Theorem], [3,Theorems 4.1,5.5]); (iv) an appropriately normalize (see [11,Lemma 14.8]); (v) the Leonard pair consisting of any two of a modula De nition 1.4]); (vi) the Leonard pair consisting of a pair of opposite genera bra, acting on an evaluation module (see [5,Proposition 9.2]). The example (i examples (iii), (iv) are special cases of (v).
Let A, A * denote a Leonard pair on V. We can determine whether A, A * i By [13,Lemma 1.3] each eigenspace of A, A * has dimension one. Let {θ i } d i= d values of A. For ≤ i ≤ d let v i denote a θ i -eigenvector for A. The orderin whenever A * acts on the basis {v i } d i= in an irreducible tridiagonal fashion. If then the ordering {θ d−i } d i= is also standard, and no further ordering is stand A * . Let {θ i } d i= denote a standard ordering of the eigenvalues of A. Then A, A * is a standard ordering of the eigenvalues of A * (see [7,Proposition 8.7]). and analysis of materials at the nanometer scale [1]. The control of atoms or molecules in the structure of materials at the nanometer scale allows us to adjust the properties of materials for more specific applications [2]. That is the reason why nanotechnology has received much attention from many researchers and scientists worldwide and has become more popular in both academic research and industry [3,4]. Nowadays, nanotechnology involving green synthesis of nanoparticles has become an eye-catching idea and has gained much importance and significance in recent years due to its great facility, clean processing, non-toxic chemicals used, cost-effectiveness, and being environmentally and ecofriendly. There have been many research articles focusing on the use of many kinds of plant extracts as a reducing agent for synthesis of nanoparticles; especially silver nanoparticles (AgNPs). For example, Anandalakshmi K. and co-workers used the Pedalium murex leaf extract for the synthesis of AgNPs and antibacterial activity has been studied. The results suggested that synthesised AgNPs had the highest antibacterial activity against Escherichia coli (E. coli) and B. subtilis, respectively [5]. Umadevi M. and co-workers also used the extract of D. carota extract for the synthesis of AgNPs [6]. Rajesh WR. and co-workers used the dried leaves extract of Pongamia pinnata (L) pierre for an extracellular synthesis of AgNPs [7]. Ghaedi M. and coworkers used the extract of Rosmarinus officinalis leaf and investigated its antimicrobial properties showing effective antibacterial and antifungal activities [8]. Jyoti K. and co-workers synthesized silver nanoparticles using Urtica dioica Linn. leaves and investigated their synergistic effects with antibiotics. The synthesized AgNPs exhibited higher activity than AgNO 3 solution and leaves extract [9]. Puišo J. and co-workers used lingonberry and cranberry juices in a biosynthesis of silver nanoparticles and studied their antimicrobial activity. It was found that the biosynthesized silver nanoparticles showed a broad spectrum of antimicrobial activity and were most active against Staphylococcus aureus (S. aureus), B. subtilis and B. cereus reference cultures [10]. Mražíková A. and co-workers reported the use of P. kessleri on the biosynthesis of AgNPs which showed a long-term stability at the higher  pH values and indicated similar inhibitory antimicrobial activity as gelatin/sodium citrate-AgNPs against biofilm formation [11].
However, in this research we aimed to study the synthesis of AgNPs using a new kind of Chinese herbal plant extract called Lysimachia foenum-graecum Hance (LH) as a reducing and stabilizing agent to observe the stability of synthesized AgNPs. Furthermore, antibacterial property has been studied to observe the antibacterial activity of these stabilized AgNPs.
LH is a plant in Lysimachia genus of the Primulaceae family ( Figure 1). It is about 50 cm tall and has a scented smell, and is mostly found in the Guangxi and Yunnan provinces of China. Commonly, it has been used to make perfume, pest repellent, alleviate pain and Chinese medical treatment for common colds and headaches [12]. However, the chemical constituent found in LH is triterpenoid saponins, flavonoids and glycosides [12,13]. Surprisingly, saponins found in LHE is the main ingredient which plays an important role in the stabilization of AgNPs in water. According to our experiment, when Lysimachia foenum-graecum Hance extract (LHE) was dissolved in deionized water, it produces a little foam on the solution surface. Foaming is a common and very visible effect that can occur when surfactants are present in water. Because saponins are natural surfactants showing the unique properties of foaming and emulsifying agents, they can act as a stabilizer in our experiments for AgNPs synthesis.
Surfactants or surface-active agents are large selfassembled molecular substances that possess a hydrophilic part and hydrophobic part (lipophilic part) [14,15]. Due to the amphiphilic nature of triterpenoid saponins found in LHE, they can lower surface tension (interfacial tension) and can also act as a stabilizing agent to create a high stability for metallic AgNPs in water; thus affording superior and long-term steric stabilization [16] of metallic AgNPs in colloidal solutions as can be seen in Figure 2.
Therefore, the first objective of the present study is the synthesis and characterization of AgNPs using LHE as a reducing and stabilizing agent. The investigation for the optimum concentration of LHE for the preparation of sterically stabilized colloidal solutions of AgNPs has been performed. The AgNPs were characterized by UV-Visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), Dynamic light scattering technique (DLS), X-ray diffraction (XRD) and transmission electron microscopy (TEM) measurements to observe the formation, stabilization, size and shape of AgNPs. The second objective is to evaluate the effect of green synthesized AgNPs on gram-negative and gram-positive bacteria, namely, E. coli and S. aureus, respectively using the disc diffusion method.

Chemicals and materials
AR-grade silver nitrate (AgNO 3 ) was purchased from Labscan (RCL Labscan Limited, Bangkok, Thailand). on the nutrient agar using sterile cotton buds. Whatman filter paper (no:1) discs of 75 mm diameter were dipped in AgNO 3 0.025 wt%, LHE 1.0 wt% and different dilutions of biosynthesized AgNPs varying from 0.003 wt%, 0.01 wt%, 0.05 wt%, 0.1 wt%, 0.5 wt% and 1.0 wt%. Each Petri plate containing nutrient agar was loaded with four antibacterial testing samples. The Petri plates were incubated at 37°C for 12 h and then examined for the appearance of a clear area around the disc by measuring the diameter of inhibition zones using a ruler which was recorded and expressed in millimeters.

Sample characterization
UV-Visible (UV-Vis) absorption measurements were performed using a UV-Vis spectrometer (Jusco, V530, Jasco International Co., Ltd., Tokyo, Japan) in dual beam mode. Fourier-transform infrared (FTIR) data were collected using an FTIR spectrometer (Nicolet 6700, Thermo Fisher Scientific). A laser particle sizer (Malvern Zetasizer Nano ZS, Malvern Instruments limited, Worcestershire, UK) equipped with a He-Ne laser at 633 nm, 4 mW was used to determine particle size and zeta potential at 25°C by dynamic light scattering (DLS) in back scattering mode. An X-ray diffractometer (Rigaku Miniflex 600 X-ray diffractometer using Cu Kα x-ray radiation, Rigaku Corporation, Tokyo, Japan) was used to obtain the diffraction patterns of the AgNPs on a glass slide. A transmission electron microscope (TEM, Philips Tecnai 12, FEI Company, OR, USA) was used to investigate the size and morphology of AgNPs.

Spectroscopic measurements
UV-Vis spectroscopy was employed to investigate the formation and stabilization of AgNPs in aqueous solution containing LHE. The AgNPs solutions were subjected to test on the first day after heat treatment. It can be observed that, the absorbance intensity of AgNPs gradually reduced with respect to the concentration of LHE used in the reduction process from 0.003 wt%, 0.01 wt%, 0.05 wt%, 0.1 wt%, 0.5 wt% and 1.0 wt% ( Figure 3). The absorbance peaks appeared around λ max = 410-430 nm for the whole concentration range of 0.003-1.0 wt% of LHE resulting from the excitation of the surface plasmon vibration in the AgNPs; indicating the formation of AgNPs in this

Preparation of Lysimachia foenumgraecum -Hance extract
100 g of LH dried leaves were cut into small pieces in a Moulinex blender and were boiled for 3 h at 80°C with deionized water. The ratio of LH dried leaves to deionized water was 1 g : 10 mL after boiling for 3 h. The solution was then filtered and lyophilized to obtain brown extracted powder. The LHE was used as a reducing agent and stabilizing agent.

Silver nanoparticles synthesis
The green synthesis of AgNPs with LHE as a reducing and stabilizing agent was performed as follows: Solution of silver nitrate 25 mL, 0.025 wt% of Ag was added into 25 mL of LHE solution. The concentration of LHE in the mixture was prepared as follows: 0.003 wt%, 0.01 wt%, 0.05 wt%, 0.1 wt%, 0.5 wt% and 1.0 wt%. The solution mixture was then stirred until it became homogeneous. The reaction mixture was stirred overnight (12 h) at room temperature and the temperature was increased to 90°C for 1 h in the final reaction process. The AgNPs formation was observed by the appropriate color change and had been kept in the dark condition at room temperature prior to use.

Antimicrobial activity
The synthesized AgNPs were tested for antimicrobial activity using the disc diffusion method following standards and guidelines from the Clinical Laboratory Standards Institute (CLSI  [17,18] depends on some factors such as size and shape of AgNPs. The peak position and peak shape of AgNPs in Figure 3 can be used roughly to predict the size and shape of AgNPs indicating that they are quasispherical in shape and AgNPs size should be approximately around 10 to 20 nm [19][20][21]. It can also be seen that when the LHE concentration increased, the absorbance intensity also increased. This can be interpreted that the functional groups in LHE help to promote the reduction of silver metal ions in aqueous solution. Figure 4 shows color intensity of AgNO 3 aqueous solution with LHE before the reduction process takes place (Figure 4a), AgNPs colloidal solution after overnight reduction ( Figure 4b) and AgNPs colloidal solution after overnight reduction followed by heating at 90°C for 1 h (Figure 4c). To make a clearer observation, the dilution series of colloidal solution in each figure (Figures 4a-c) which were labeled as A1 to A10, B1 to B10 and C1 to C10 represent the concentration of LHE in the concentration range from 0.003 wt%, 0.006 wt%, 0.01 wt%, 0.025 wt%, 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.3 wt%, 0.5 wt% and 1.0 wt%. It can be seen that the solution color in Figures 4b and 4c changed from light yellow to pale orange, orange, dark orange, brown, and dark brown and the colors are more intense than the solution in Figure 4a; indicating the formation of AgNPs colloidal solution and plasmon resonance absorption. This result corresponds to the UV-Vis spectra in Figure 3 that the color intensity and absorbance intensity of AgNPs colloidal solutions increased with the reduction process taking place. Furthermore, the reduction process of AgNPs by LHE can proceed faster and completely achieved by heating. Figure 5 shows the UV-Vis spectra of colloidal solution of AgNPs reduced by LHE to observe the stability of the series of colloidal solutions ranging from 0.003 to 1.0 wt%.
The observations were made on the 2nd, 3rd, 4th and 10th week and the AgNPs solution has been kept in the dark condition at room temperature. It can be noticed that good UV-Vis spectra were observed until the 10th week which confirms the stability of AgNPs colloidal solution reduced by LHE. Furthermore, after the AgNPs colloidal solutions had been kept for more than 6 months, they still showed good stability of AgNPs colloidal dispersion, thus indicating the superior dispersion of this AgNPs colloidal system. These results are superior to the work published by Velgosova O. and co-workers [22], where the biologically synthesized AgNPs using algae Parachlorella kessleri stored in the dark at room temperature showed better long-term stability but weak particle agglomeration was observed at approximately the 100th day. Furthermore, the stability of our biologically synthesized AgNPs using LHE can be stored without chemical stabilizer such as polyvinyl alcohol (PVA) [23]. Figure 6 shows FTIR spectra of pure LHE and dried powder obtained from AgNPs colloidal solutions at the different LHE concentrations. FTIR spectra of LHE show a broad peak at around 3300 cm -1 , which is attributed to the -OH functional group (H-bond stretching). Some other peaks at 2927 cm -1 and 1386 cm -1 can also be observed, which can be attributed C-H stretching vibration and C-H bending vibration respectively. The remaining two others at 1590 cm -1 and 1022 cm -1 also observed can be attributed to C=C stretching vibrations in the aromatic ring and oligosaccharide linkage absorptions to sapogenins C-O-C, respectively. As we mentioned earlier, saponins found in LHE is the main ingredient which plays an important role in the stabilization of AgNPs in water. These results agree with the FTIR spectra of saponins in crude extract of soapnuts reported by Meshari et al. [24]. It can also be observed that the high similarity between LHE and AgNPs reduced by LHE indicated the same compounds existed in all samples. The FTIR spectra of saponin in LHE and AgNPs reduced by LHE demonstrated the characteristic of triterpenoid saponin absorptions of -OH, C-H, and C=C, while the absorptions of C-O-C indicated the glycoside linkages to the sapogenins. It should be noted that the extract consists of differences in saponin content and composition. In general, saponins consist of a hydrophobic aglycone structure with hydrophilic sugar residues linkage. The steric stabilization mechanism of the AgNPs depends on the extensive hydration of the hydrophilic sugar residues in the adsorbed saponin layer [25,26]. Furthermore, it can be observed that as the concentration of LHE was increased, clear peaks in the spectra were revealed. The marked shifts at 3300 cm −1 , 1386 cm −1 and 1022 cm −1 also observed are related to the adsorption of LHE constituents on the AgNPs surface which are aiding the reduction and stabilization of AgNPs.

X-ray diffraction
The powder of AgNPs obtained from AgNPs colloidal solution by the reduction of LHE (after heat treatment) at the different concentrations of 0.003, 0.01, 0.05, 0.1, 0.5 and 1.0 wt% were subjected to analysis of their crystalline nature through X-ray diffraction. A comparison of our XRD spectrum with the standard confirmed that AgNPs in our experiment were in the form of nanocrystals, as evidenced by the XRD patterns showing four diffraction peaks at 2θ values of around 38°, 44°, 64° and 78° which are indexed as the (111), (200), (220) and (311) Bragg reflections, respectively. These diffraction peaks are well consistent with the standard data file (JCPDS No. 04-0783) and can be indexed as the face-centered cubic structure of silver (Figures 7a-f). XRD patterns of the synthesized AgNPs show a strong diffraction peak corresponding to the (200) facet at the first few synthesis conditions (0.003 and 0.01 wt% of LHE) while the others show a strong diffraction peak corresponding to the (111) facet; especially at the high LHE concentration range (0.1 to 1.0 wt%). These results suggest the predominance of (111) plane orientation of AgNPs in the synthesis condition of 0.01 to 1.0 wt% of LHE.

Zeta potential and size distribution
To observe the charge on the particle surface and particle size of AgNPs, Dynamic Light Scattering (DLS) technique was used to determine the zeta potential and size of AgNPs. Surprisingly, AgNPs colloidal solutions obtained from LHE reduction at the concentration of 0.003 to 1.0 wt% show zeta potential values of approximately zero in all samples as shown in Table 1. This result indicates that the stability of AgNPs colloidal solution obtained from LHE reduction is not due to electrostatic stabilization but is due to steric stabilization afforded  by the natural surfactants in LHE as we had described earlier and also shown in Figure 2. In terms of particle size, it can be observed that the AgNPs colloidal solution with LHE concentration of 0.003 wt% shows a peak of mean particle size (175 ± 71 nm); while other AgNPs with LHE concentrations of 0.01, 0.05, 0.1, 0.5 and 1.0 show three different mean particle sizes as shown in Table 1. It can be observed that, the first and second peak represent the small and large size of AgNPs obtained from the reduction while the third peak might be the size  Table 1: Zeta potentials and particles sizes of AgNPs colloidal solutions obtained from LHE reduction (after heat treatment) at the concentration range between 0.003 to 1.0 wt%.

LHE concentration (wt%)
Mean zeta potential (mV) Mean particle size (1st peak-nm) Mean particle size (2nd peak-nm) of the self-assembled surfactant aggregates or surfactant micelle that existed at high concentrations of LHE (0.01 to 1.0 wt%). However, particle sizes obtained using Dynamic Light Scattering (DLS) and TEM are different. DLS gives the hydrodynamic size (the size of the nanoparticle plus the liquid layer around the particle) while TEM gives the actual size of the nanoparticle [27,28]. This is the reason to explain why size measured by DLS is different from TEM. Thus, the mean particle size at the 2nd peak of LHE 0.03, 0.01, 0.05, 0.1, 0.5 and 1.0 wt% (175 ± 71 nm, 108 ± 61 nm, 95 ± 50 nm, 106 ± 65 nm, 140 ± 72 nm and 158 ± 76 nm, respectively) should be the hydrodynamic diameters of AgNPs coated with phytochemicals (hydrodynamic radius) because phytochemicals coatings on AgNPs are resulting in substantial changes in the hydrodynamic radius. This result suggests that the phytochemicals (triterpenoid saponins, flavonoids and glycosides) found in LHE are capped on AgNPs. Furthermore, only one mean particle size at the 2nd peak of LHE 0.003 wt% was observed at this condition representing the optimized concentration for AgNPs synthesis resulting in good antibacterial activity to S. aureus which will be discussed in the last section. However, the mean particle size at the 1st peak of LHE 0.003 wt% was not observed, which may be due to the concentration of AgNPs in solution being too low.

TEM morphology
TEM images of AgNPs prepared by the green reduction using LHE of concentration range from 0.003 to 1.0 wt% are shown in Figure 8. It can be observed that at the lowest LHE concentration of 0.003 wt%, the AgNPs size observed by TEM is approximately 20 nm in size (Figure 8a). When the LHE concentration is increased (0.01 to 1.0 wt%), the combination of AgNPs size range started to appear as shown in Figures 8b-f. This result corresponds to the result observed by DLS that multiple peaks of mean particle size were observed at LHE concentration range from 0.01 to 1.0 wt%. Furthermore, it should be noted that there is no big difference in the particle size of AgNPs which were observed by TEM in all synthesis conditions which is in keeping with the DLS technique which also showed no big difference in particle size observed. However, as we mentioned in the previous section, the particle size of AgNPs determined by DLS and TEM are different due to the enveloped state of particles in media. DLS gives a hydrodynamic diameter/ radius, while TEM gives an actual particle size in the dry state. Thus, the size obtained by these two characterization techniques are different. Furthermore, it should be noted that surfactants can influence or possess ability to control crystal growth [29,30]. This is the reason to explain why the size of nanoparticles do not vary outstandingly with the concentration of plant extract in this experiment. This is because nanoparticles are capped by surfactants found in phytochemicals in LHE.

Antimicrobial activity
The antibacterial activity of synthesized AgNPs were subjected to test with gram-negative bacteria E. coli and gram-positive S. aureus. AgNPs in all synthesis conditions were tested for antibacterial activity comparing with pure DI water (negative control), Chloramphenicol (positive control), pure LHE and AgNO 3 (equivalent to 0.025 wt% of Ag). Figure 9 shows the position measurement of inhibition zone. The negative control and positive control were measured in each condition for comparison. The inhibition zone (average value ± standard deviation) of pure LHE, AgNO 3 and AgNPs at the different concentrations of LHE were measured 6 times in each experiment (labelled with numbers 1 to 6). The inhibition zone diameters of all samples are recorded in Table 2 , which is considered to be more effective than larger particles, resulting in a superior effective binding to the bacterial membrane or ease to reach cellular proximity [31][32][33]. The superior antibacterial activity of AgNPs at this condition (LHE 0.003 wt%) suggests this condition to be optimal for the green synthesis of AgNPs in this system. However, a major difference in the inhibition zone between AgNO 3 and green-synthesized AgNPs with LHE 0.003 wt% to S. aureus might be explained by previous findings that silver ions produced in an electrolytic way are better antibacterial agents than dissolved silver compounds [34,35]. While in the case of E. coli, a small difference in the inhibition zone can be observed due to the negatively charged lipopolysaccharide which makes Gram-negative bacteria more resistant than the Gram-positive bacteria [36,37].

Conclusions
In this research, we are the first to successfully synthesize AgNPs by green synthesis using Lysimachia foenum-graecum Hance extract (LHE) as reducing and stabilizing agent. Synthesized AgNPs with different LHE concentrations show quasi-spherical shape with long term stability in water. In this experiment, it can be seen that LHE can be used as a reducing and superior long-term stabilizing agent for AgNPs. Furthermore, AgNPs aqueous colloidal solutions show advantage in antimicrobial activity. AgNPs with LHE 0.003 wt% (smallest LHE concentration) affords the highest antibacterial effect to S. aureus showing an inhibition zone diameter of 19.08 ± 0.67 mm; which is superior to Chloramphenicol. The results suggest that synthesizing AgNPs by using LHE is a facile pathway, cost-effective, safe to the environment and living organisms and also shows a superior stability with outstanding antimicrobial activity to S. aureus.