Synthesis of Silver Nanoparticles Using L.Rosa Flowers Extracts: Thermodynamic and Kinetic Studies on the Inhibitoty Effects of Nanoparticles on Creatine Kinase Activity

The present work investigates the synthesis of silver nanoparticles (AgNPs) by a biological method using L.Rosa flower extract and silver nitrate as precursors. Optimum conditions of synthesis were studies, such as pH, temperature, concentration of extract, concentration of silver nitrate, and stability with time. Characterization of AgNPs was carried out using UV-visible Spectroscopy, Scanning Electron Microscopy, X-Ray Diffraction, Fourier Transform Infrared and Transmission Electron Microscopy. The biosynthesized AgNPs exhibited inhibitory effects on creatine kinase activity in the sera of patients with myocardial infarction, compared with control subjects. Thermodynamic and kinetic studies of creatine kinase were performed. Further studies on other biological activities were performed to exploit AgNPs full potential. In conclusions, the present study utilize a simple, cheap and environmentally green method to synthesize silver nanoparticles. This single step procedure is more suitable for large scale production as it is rapid and eliminates the elaborate processes employed in the other bio-based protocols (e.g. by using fungi and bacteria).


Introduction
with heating for one hour at 70 C o . After that, its maximum absorbance was measured using UV-Visible spectrophotometer. The reduction of Ag + to Ag 0 nanoparticles was indicated by the alteration in color of the solution from yellow to brownish yellow and finally deep brown. This process was affected by several parameters, such as flower extract concentration, AgNO 3 concentration, temperature, pH value, contact time, and stability with time. The sample was washed with distilled water twice then dried to obtain the synthesized silver nanoparticles for characterization. Optimization conditions of the synthesis of silver nanoparticles To optimize different conditions of the plant extract-mediated AgNPs synthesis method, the maximum absorbance was recorded at each optimization experiment using UV-visible spectrophotometry.

pH optimization
The optimum pH of the reaction was maintained over different ranges and adjusted by using 0.1 N HCl and 0.1 N NaOH.

Time optimization
The contact time of the reaction was optimized by using different time periods. The reaction time was monitored at 0, 15, 30, 45, 60, 75, 90, 105 and 120 min with stirring and heating at 70 ℃ for 1, 2, 3, 4, 5, 6 and 7 days. The mixture was then stored in dark at room temperature [15].

Concentration of flowers extract
Similarly, the concentration of flowers extract was optimized by increasing the volume (0.25, 0.50, 0.75, 1.0, 1.25 and 1.50 ml) of 5% flower extract with two milliliters of the constant concentration of AgNO 3 (2× 10 -2 ), then the volume was completed to 20 ml with deionized water [17] Stability study After the optimization of the various conditions for AgNPs-plant reaction, the solution was kept in the dark at room temperature and the stability of the synthesized particles was monitored for up to 60 days.

Characterizations of silver nanoparticles
Ultraviolet-visible absorption spectra were recorded at 37℃ using a Shimadzo UV-1800 spectrophotometer. A drop of the solution containing the nanoparticles was deposited on a Cu grid covered with amorphous carbon. An aliquot of plant extract filtrate containing silver nanoparticles was tested for scanning electron microscopy (SEM) using SEM S-4160. X-ray diffraction (XRD) pattern was obtained using a Shimadzu XRD-6000 diffractometer with Cu K,α (λ= 1.54056 A˚) to confirm the green synthesis of AgNPs. Fourier transform infrared (FTIR) spectra were recorded at 37℃ on a Shimadzo FTIR 84005 spectrophotometer. To prepare a sample for FT-IR, the plant extract with AgNPs was dried at 60 ℃ for one hour, then mixed with suitable amount of KBr. Morphology of silver nanoparticles was examined using Atomic Force Microscope (AFM, Model AA300 Angstrom advanced). Transmission electronic microscopy (TEM) study was performed using a Carl Zeiss EM 900.

Anti-bacterial activity
Testing the activity of AgNPs as anti-bacterial agent was adjusted using the well diffusion method [18] for Staphyloccocus aureus and Escherichia. coli. Brain heart infusion (BHI) broth was used to subculture the bacteria which was incubated at 37˚C for 24 h. The pathogenic bacterial strains were incubated in Mueller-Hinton agar plates. Sterile paper disks of 5mm diameter saturated with plant extract, as control, and different concentrations of the synthesized silver nanoparticles were placed in each plate. Plates were then incubated for 24 hours at 37°C. The inhibition zones were measured and tabulated.

Effects of AgNPs on CK activity
Effects of AgNPs on the activity of creatine kinase was examined. The study was conducted during the period of May 2018 to June 2018 on blood samples from 20 patients with myocardial disease (8-50 years old) admitted to the medical city hospital, Baghdad, where they were diagnosed with myocardial infarction. Twenty healthy individual (20-45 years old) were included as a control group, with the exclusion of any patientwith myocardial disease. After one-hour of diagnosis with myocardial diseases, venous blood was collected and allowed to clot at room temperature for 30 minutes. Then, the samples were centrifuged for 15 minute to separate the serum. Serum was pipetted and stored at 4 until the determination of creatine kinase activity. An assay kit purchased from biolabo-France was utilized for the spectrophotometric measurement of the level of CK. One unit of creatine kinase was defined as the conversion of one micromole of creatine to creatine phosphate per minute at 37 and PH 6.8 under specified conditions. 1.5 mL of biosynthesized AgNPs (5%) was added to the sera of each group (patient with myocardial infarction and healthy subjects), then CK activity was determined [19]. Enzyme kinetic and thermodynamic studies Parameters of creatine kinase in the absence and present of AgNPs were assessed. The reaction mixture was prepared and treated as mentioned in the CK kit assay protocol. CK activity was determined at various constant reactions of creatine phosphate (as a substrate) for the kinetic study. The data of these experiments were used to generate a linear relationship by plotting 1/v values against 1/[S] values for the control and patient's groups, according to Linweaver-Burk equation [20]. For the thermodynamic study, values of the natural logarithm of the constant of the reaction equilibrium ( ln K eq ) at different temperatures (20, 30, 37 and 45 ) were plotted against the reciprocal of absolute temperature (1/T), according to Van't Hoff equation [21]. The value of ΔHᵒ was calculated from the slope of the resulting straight line.

Results and discussion
In this study, we demonstrated the preparation of silver nanoparticles from L.Rosa flowers extract as a reducing, capping, and stabilizing agent. Synthesis of AgNPs was monitored by UV-vis spectra and the reaction was completed within 60 minute with stirring and heating at 70 . The colorless solution was turned to a brownish red color, which indicates the formation of silver nanoparticles as show in Figure-

Optimization of silver nanoparticles synthesis
Many parameters were optimized for silver nanoparticles preparation, including acidity, stability with time, and concentration variation of AgNO 3 and flowers extract. The value of the pH in the media is one of important factors playing a major role in nanoparticles preparing. As shown in Figure-2, no particle formation was recorded at acidic and alkaline pH, but the maximum peak was absorbed at neutral pH (7), when the AgNPs were synthesized. At optimum pH (7), stability of the synthesized silver nanoparticles was monitored during different time innervates (1 to 7 days). Synthesis of silver nanoparticles was started as the AgNO 3 was added into the reaction. The formation of AgNPs was noticed within 15 minute and the absorbance was increased with time up to a period of 7 days. The maximum absorption of AgNPs was measured at specified periods of time, as shown in Figure-   Furthermore, the characteristics of the in UV-vis absorption spectrum at 422 nm corresponds to the surface plasmon resonance (SPR) of silver nanoparticles. This result confirms previously reported results [22]. The phenomena of SPR occurred at 422 nm at the start of the reaction and it was stabilized in the height and shape of λmax even after the completion of the reaction. Silver nitrate with volumes of 2 ml or 3 ml supported rapid synthesis of AgNPs, as the peaks with maximum absorption were observed (Figure-4). Different concentrations of silver nitrate (AgNO 3 ) were studied for the maximum yield of the synthesized silver nanoparticles.  and pH 7, with stirring and heating at 70 C o for one hour. Thus, the optimized medium conditions(pH 7, temp. 70℃, 2 ml volume of silver nitrate and 1.25 mL of L.Rosa flowers extracts for 60 min.) supported the maximum production of silver nanoparticles. To achieve the maximum stability of silver nanoparticles formation at optimized conditions, maximum absorbance of synthesized AgNPs was measured at different periods of storage time (up to two months), as shown in Figure-6. Obviously, there is a slight change in the peak at 422 nm even after two months of storage, indicating the high stability of biosynthesized silver nanoparticles.
The optimized conditions played a major role in the stability and aggregation of the nanoparticles. For better understanding of the mechanism of the reaction, various concentrations of the flowers extract and the substrate were prepared. Many studies indicated that the biomolecules present in the plant extract play an important role in reducing silver ions to the nanosize. It is a chemically complicated phenomenon involving an array of biomolecules, such as enzymes/proteins, flavonoids, phenols, vitamins, organic acids (such as citrates), amino acids and polysaccharides. The flowers of L. Rosa were reported to consist of flavonoids, glycosides, and tannins. Quercetin, a well-known flavonol that is found in L. Rosa flowers extract, has strong antiviral, antibacterial, anti-inflammatory and anti-carcinogenic effects. Quercetin, isolated alone, was used to synthesize nanoparticles [23]. Hence, we assume that quercetin may be reliable for silver nitrate reduction into silver nanoparticles. However, bio component products or reduced cofactors in plant extracts may also play a crucial role in the reduction of respective salts to nanoparticles.

Characterization of the synthesized silver nanoparticles
The size, shape and distribution of the green-synthesized silver nanoparticles were characterized by TEM in an average size of 7.5 -50 nm (Figure-7). The FT-IR analysis was utilized to identify the different functional groups present in L. Rosa flowers extract, which can be responsible for the reduction of AgNO 3 to silver nanoparticles and play roles as efficient capping and stabilization agents. Figure-8 shows a typical FT-IRspectrum for L. Rosa flowers extract along with a comparison with Figure-9 which shows AgNPs synthesized using this extract. Comparison of these two spectra indicated that the peak appears at 1514 cm -1 , which corresponds to alkene groups that where shifted to 2945 cm -1 . A peak corresponding to the azo group at (1461-1625) cm -1 was shifted to (3255-3301) cm 1 which corresponds to the formation of the amino group with increased intensity, indicating the binding of silver ion with the hydroxyl, azo, and amide groups of the flowers extract.  Atomic force microscope was used to present the surface morphology and to determine topography of the prepared NPs. AFM gives a three dimensional image of the surface of a nanoparticle. The particle diameter was calculated in nanoscale size, and the results demonstrated a diameter range of 20-50 nm. Figure-10 shows the three-dimensional image of synthesized AgNPs using L. rosa flowers extract.  Science, 2021, Vol. 62, No. 8, pp: 2486-2500 2495 SEM images were used to show the size, shape and distribution of the synthesized silver nanoparticles. As shown in Figure-11, the particles are spherical with an average size between 10.5 to 74 nm.

Antibacterial effects of synthesized silver nanoparticles
The antimicrobial activity of the synthesized silver nanoparticles against gram negative E. coli and gram positive Staphyloccus aureus bacteria were revealed and the zone of inhibition was measured, as shown in Figure-13 and Table-1.
The results indicated that different concentrations of the synthesized silver nanoparticles showed effective antibacterial activities, both in gram negative and gram positive bacteria. Many studies confirmed that effect of silver nanoparticles on bacteria by destructing the membrane integrity [24,25]. These studies indicated that silver nanoparticles may interact with phosphorus and sulphurcontaining compounds, which may cause destruction to the DNA and proteins and, eventually, cell death. Based on the outcomes of this study, it is clear that silver nanoparticles may have significant applications in the agricultural sector and also could be sufficiently used as an effective agent against human pathogens [26]. AgNPs can easily penetrate bacteria through sulfate groups of the membrane protein resulting in the destruction of the structural composition of the bacteria. Also, AgNPs can pass into the cytoplasmic fluid where enzymatic proteins damaging could be occurred [27].  Table-2 for both myocardial infarction patient and control groups. It is obvious that AgNPs have inhibition effects on CK activity. The inhibition percentage values of CK were (84%) and (74.7%) for the patient and control groups, respectively, as shown in Table-2. This decrease in CK activity might be due to the interaction between AgNPs and the thiol group of the amino acids (e.g. cysteine )found in the enzyme structure [28].

Kinetic and thermodynamic studies
The analysis of creatine kinase binding data was performed using Linweaver-Burk equation. The activity of the enzyme was reduced in the presence of AgNPs. AgNPs bind equally well to the enzyme whether or not it has already bound to the substrate. V max, Km and Keq values were calculated using Linweaver-Burk equation (1): ΔH values were unaffected by temperature, as they were constant over the range of temperatures used in this study. They were also positive for all studied groups, as shown in Table-4, indicating exothermic binding reaction between the enzyme and its substrate via the formation of sets of noncovalent interactions that yielded stabilization to the formed structure of enzyme-substrate complex. The association of CK from all studied groups to its substrate seems to proceed spontaneously in view of ΔG° values shown in Table-4. Values of this reaction depend on the molecular pathways of the mechanism of transformation [28]. The results also reveal the positive values of ΔS°, which indicates that the change from the order to the disorder state may be the driving force behind the reaction, which is observed to be unfavored by enthalpy but entropically favored. The differences seen in the value of ΔS° among the studied groups are a good indication of variation in their conformational stability, molecular flexibility, complexity and structure rigidity [29,30].

Conclusions
In the present study, AgNPs were synthesized with greater stability and good yield, using simple, cheap and environmentally green method. This single step procedure is more suitable for large scale production, as it is rapid and eliminates the elaborate processes employed in the other bio-based protocols (e.g. by using fungi and bacteria). Interestingly , silver nanoparticles revealed good antibacterial activities at relatively low concentration. Based on the present findings, it is concluded that silver nanoparticles could be used as anti-bacterial agents in controlling different pathogens. However, it is necessary to conduct further studies to understand the exact mechanisms by which silver nanoparticles enter into the cell wall of bacterial. Silver nanoparticles showed a decrease in creatine kinase activity. The kinetic study showed that the inhibition type was noncompetitive, whereas the thermodynamic study revealed exothermic enthalpy, spontaneous Gipps free energy, and change from order to disorder of entropy for all study groups.