Evidence on temperature and concentration of reducing agents to control the nanoparticles growth and their microbial inhibitory efficacy

The present study deals with the nanoparticles synthesis from Impatiens balsamina L. plant flower extract. The concentration of reducing agent (flower extract) and different temperature is involved in the reaction. Sixteen different silver nanoparticles were synthesized with using different ratios of the reaction mixture and different temperature. The different silver nanoparticles are different color based on the temperature and concentration of reaction mixture. The synthesized silver nanoparticles are characterized by UV/vis Spectrophotometer. The microorganisms Bacillus subtilis are highly inhibited by (90:10/60 °C; 99:1/70 °C; 92:2/70 °C; and 90:10/70 °C) silver nanoparticles. Pseudomonas aeruginosa are highly inhibited by 98:2/50 °C and 90:10/70 °C based silver nanoparticles. The bacterial species Staphylococcus aureus are highly inhibited by 98:2/50 °C and 98:2/70 °C. The E. coli was inhibited by 99:1/70 °C; 98:2/70 °C and 90:10/70 °C based silver nanoparticles. The temperature and concentration of reducing agents can play a significant role in controlling the growth of nanoparticles. This study is one of the evidence on temperature and concentration of reducing agents to control the nanoparticles growth and their microbial inhibitory efficacy.


Introduction
Silver nanoparticles are very small particles of silver that have a size on the nanometer scale, typically ranging from 1 to 100 nanometers in diameter (Khan et al 2019). These particles have unique physical and chemical properties that differ from those of bulk silver, and they have attracted significant interest due to their potential use in a variety of applications, including as antimicrobial agents (Mariselvam et al 2014, Mariselvam et al 2021, catalysts (Mariselvam et al 2016, Mariselvam et al 2019, and in electronic and optical devices (Lee andJun 2019, Majeed et al 2022).
One of the main properties of silver nanoparticles that have attracted attention is their antimicrobial activity (Franci et al 2015). Silver ions and silver nanoparticles have been shown to have strong antimicrobial effects against a wide range of microorganisms, including bacteria, viruses, and fungi (Dakal et al 2016). As a result, silver nanoparticles have been incorporated into a variety of products, such as textiles, plastics, and coatings, to help control the growth of microbes and reduce the risk of infection (Zhang et al 2016). Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
However, the use of silver nanoparticles also raises concerns about their potential environmental and health impacts. There is evidence that silver nanoparticles can be toxic to aquatic organisms and plants, and that they can accumulate in the environment and potentially enter the food chain (Yan and Chen 2019, Ferdous and Nemmar 2020). There is also ongoing debate about the potential health effects of silver nanoparticles on humans, including the potential for skin irritation and the potential for long-term exposure to these particles to have negative health effects (Ferdous and Nemmar 2020). As a result, there is ongoing research into the potential risks and benefits of using silver nanoparticles in different applications, and efforts are being made to identify safer and more sustainable alternatives (Burdușel et al 2018).
Green synthesis refers to the production of nanoparticles using eco-friendly (Mariselvam et al 2014) and sustainable methods that minimize the use of hazardous chemicals and minimize waste. There are several approaches that have been developed for the green synthesis of silver nanoparticles, including the use of plant extracts, microorganisms, and renewable resources as reducing agents and stabilizers (Singh et al 2018).
One example of a green synthesis method for silver nanoparticles involves the use of plant extracts as reducing agents. Variety of plant extracts used to reduced the silver ions to silver nanoparticles (Sandip Kumar et al 2021) through eco friendly approach, some plant examples as following Mentha piperita (Abobakr et al 2022), Salvadora persica (Arshad et al 2021), Mangifera indica (Fuad et al 2019) etc. In this approach, plant extracts containing compounds such as tannins, flavonoids, and phenols are mixed with a solution of silver ions (Melkamu and Bitew 2021). The plant extract acts as a reducing agent, reducing the silver ions to silver metal and forming silver nanoparticles (Rodríguez-León et al 2013). This process can be facilitated by the presence of certain enzymes in the plant extract, such as peroxidases and polyphenol oxidases. Another example of a green synthesis method for silver nanoparticles involves the use of microorganisms, such as bacteria and fungi, as reducing agents. In this approach, the microorganisms are cultured in a medium containing silver ions, and they are able to reduce the silver ions to silver metal and form silver nanoparticles (Iravani et al 2014). This process can be facilitated by the presence of certain enzymes in the microorganisms, such as cytochrome c and nitrogenases. Overall, the green synthesis of silver nanoparticles has the potential to provide a more sustainable and eco-friendly approach to the production of these particles, while also minimizing the potential environmental and health risks associated with the use of hazardous chemicals (Niknejad et al 2015).
The size and shape of silver nanoparticles can be controlled by a variety of factors, including the temperature and concentration of the reducing agents used to synthesize the nanoparticles (Iravani et al 2014). Temperature is an important factor that can affect the rate of reduction and the size of the resulting silver nanoparticles (Hernández-Pinero et al 2016). In general, higher temperatures can increase the rate of reduction, leading to the formation of smaller nanoparticles. On the other hand, lower temperatures can slow down the reduction process, leading to the formation of larger nanoparticles. Therefore, carefully controlling the temperature during the synthesis process can be used to control the size of the resulting silver nanoparticles (Thanh et al 2014).
The concentration of the reducing agent can also affect the size and shape of silver nanoparticles. Higher concentrations of the reducing agent can lead to faster reduction and the formation of smaller nanoparticles, while lower concentrations can result in slower reduction and the formation of larger nanoparticles. Additionally, the specific type of reducing agent used can also influence the size and shape of the resulting nanoparticles, as different reducing agents can have different reactivity and reducing power (Rodríguez-León et al 2013).
Overall, the size and shape of silver nanoparticles can be controlled by carefully manipulating the temperature and concentration of the reducing agents used during the synthesis process. This can be important for optimizing the properties of the nanoparticles for different applications (Iravani et al 2014).
The present study deals with the preparation of silver nanoparticles through green synthesis method with the help of Impatiens balsamina L. flower extract as a reducer.

Materials and methods
2.1. Plant collection Impatiens balsamina L. is a species of flowering plant in the family Balsaminaceae, commonly known as balsam, garden balsam, spotted snapweed, or soldier's woundwort. It is native to tropical regions of Asia and Africa, and is widely cultivated as an ornamental plant in gardens and parks. The plant is known for its attractive, showy flowers, which come in a variety of colors including pink, red, white, and purple. The Impatiens balsamina L plant flowers were collected from Kadayam region at the range of Latitude: 8.82453571479. Longitude: 77.3738524858, Tenkasi district, Tamil Nadu, India during the period of December 2022.

Extraction
The fresh flowers were washed with DD H2O and remove the water content with the help of hot air oven inoculation at 37°C for 1 h. 10 g of flowers were grained with the help of mortar and pestle with added 90 ml of DD H2O. Finally the flower extract were filtered with the help of Whatman No 1 filter paper. The filter was stored in 4°C for further studies.

Preliminary phytochemical study
A preliminary phytochemical study is a type of analysis that is performed to identify the presence or absence of certain chemical compounds in a plant or plant extract. Impatiens balsamina L. flower extract analyzed the following preliminary phytochemical(s) like Carbohydrate, Reducing sugar, Terpenoid, Protein, Xanthoprotein, Phenol, Flavonoid, Saponins, and Aromatic acid with help of standard protocols.

Synthesis of silver nanoparticles
Add the different concentration of plant extracts to the silver nitrate solution. The final volume is 100 ml. The ratio of silver nitrate and plant flower extract as follows 99:1, 98:2, 95:5 and 99:10. The reaction mixture is mixed properly through heating magnetic stirrer. Different temperature (40°C, 50°C, 60°C and 70°C) Totally 16 experiments were carried out. (The ratio of reaction mixture 99:1 in under 40°C, 50°C, 60°C and 70°C and other ratios are carried out the mentioned different temperatures.

Color observation and UV/vis spectral characterization
Silver nanoparticles can exhibit a range of colors, including red, orange, yellow, green, blue, and purple, depending on their size, shape, and surface chemistry. When silver nanoparticles are synthesized, their size, shape, and surface chemistry can be controlled by changing the synthesis conditions. UV/vis spectral characterization of silver nanoparticles is a common method for studying their size, shape, and surface chemistry. The absorption spectrum of silver nanoparticles exhibits a Surface Plasmon Resonance (SPR) peak in the visible region of the electromagnetic spectrum. The position and shape of the SPR peak is sensitive to the size, shape, and surface chemistry of the silver nanoparticles. By measuring the absorption spectrum of silver nanoparticles, it is possible to determine their size, shape, and surface chemistry. The synthesized nanoparticles were characterized using ultraviolet (UV)/visible spectroscopy from 200 to 1000 nM.

Assessment of antibacterial activity
The antibacterial activity of synthesized Ag-NPs was tested against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa using the agar well diffusion method (Mariselvam et al 2014). Wells (8 mm in size) were made in agar plates containing bacterial inoculums. The prepared different silver NPs (25, 50, 75 and 100 μl) were added to the wells of the culture plates. Following incubation for 24 h at 37°C, the plates were observed. The zone of inhibition was measured using a Hi Media measuring scale and expressed in millimeters.

Results and discussion
Impatiens balsamina L, also known as balsam or touch-me-not, is a flowering plant that is native to tropical regions of Asia. The collected flowers of Impatiens balsamina are pink. The extract of Impatiens balsamina flowers is likely to be a pale yellow. A preliminary phytochemical study is a type of analysis that is performed to identify the presence or absence of certain chemical compounds in a plant or plant extract. These chemical compounds, known as phytochemical(s), can include a wide range of substances such as alkaloids, flavonoids, tannins, and saponins, among others. The purpose of a preliminary phytochemical study is to provide a general overview of the chemical makeup of a plant or plant extract, which can be useful for various applications, such as the identification of potential medicinal properties or the development of natural products. To perform a preliminary phytochemical study, the flowers of Impatiens balsamina contains flavonoids, terpenes, alkaloids, tannins and Saponins (table 1). These compounds may have various biological activities, such as antioxidant, anti-inflammatory, or antiviral effects.
Silver nanoparticles can exhibit a range of colors, including red, orange, yellow, green, blue, and purple, depending on their size, shape, and surface chemistry. When silver nanoparticles are synthesized, their size, shape, and surface chemistry can be controlled by changing the synthesis conditions. For example, using a seedmediated growth method, it is possible to synthesize silver nanoparticles with a narrow size distribution and a well-defined shape.
In reaction temperature at 40°C, different ratio of reaction mixtures based silver nanoparticles in represented in figure 1(a). The color of the 99:1 ratio based silver nanoparticles is dark brown. The 98:2 ratio based silver nanoparticles are brown color. The 95:5 ratio based silver nanoparticles are golden yellow color. The 90:10 ratio based silver nanoparticles are red color.
In reaction temperature at 50°C, different ratio of reaction mixtures based silver nanoparticles in represented in figure 1(b). The color of the 99:1, 95:5 and 90:10 ratio based silver nanoparticles is dark brown. The 98:2 ratio based silver nanoparticles are pale yellow color.
In reaction temperature at 60°C, different ratio of reaction mixtures based silver nanoparticles in represented in figure 1(c). The color of the 99:1, 99:2 ratio based silver nanoparticles is dark brown. The 95:5 ratio based silver nanoparticles are brown color. The 90:10 ratio based silver nanoparticles are reddish brown color.
In reaction temperature at 70°C, different ratio of reaction mixtures based silver nanoparticles in represented in figure 1(a). The color of the 99:1 ratio based silver nanoparticles is dark brown. The 98:2 ratio based silver nanoparticles are reddish brown color. The 95:5 ratio based silver nanoparticles are pale yellow color. The 90:10 ratio based silver nanoparticles are golden yellow color.
The comparison of silver nanoparticles synthesized using different temperature in same ratio of the reaction mixture represented in figure 2. The figure 2(a) shows 99:1 ratio of the reaction mixtures in 40°C, 50°C, 60°C  Aromatic acid − and 70°C. In 40°C and 60°C, the colors of the silver nanoparticles are dark brown. Brown color nanoparticles are synthesized at reaction temperature is 50°C and 70°C. The color of silver nanoparticles is brown at reaction temperature is 40°C in the reaction mixture ratio 98:2. The pale yellow colored nanoparticles were obtained in the reaction temperature is 50°C. The reddish brown colored nanoparticles were obtained in the reaction temperature is 60°C. The color of silver nanoparticles is red at reaction temperature is 70°C ( figure 2(b)).
The color of silver nanoparticles is pale yellow at reaction temperature is 40°C in the reaction mixture ratio 95:5. The brown colored nanoparticles were obtained in the reaction temperature is 50°C. The reddish brown colored nanoparticles were obtained in the reaction temperature is 60°C. The color of silver nanoparticles is golden yellow at reaction temperature is 70°C ( figure 2(c)).
The obtained silver nanoparticles is golden yellow at reaction temperature is 40°C and 70°C in the reaction mixture ratio 90:10. The dark brown colored nanoparticles were obtained in the reaction temperature is 50°C. The reddish brown colored nanoparticles were obtained in the reaction temperature is 60°C ( figure 2(d)).
The sixteen different silver nanoparticles were analyzed their spectral characterization using UV/vis spectrophotometer. In reaction temperature at 40°C, different ratio of reaction mixtures based silver nanoparticles spectral characterization in represented in figure 3(a). The maximum absorbance observed in the 99:1 ratio based silver nanoparticles compare than the other ratios based silver nanoparticles synthesis. The maximum absorbance present in 90:10 ratio based silver nanoparticles represented in the figure 3(b) at 50°C temperature in the reaction. In reaction temperature at 60°C, different ratio of reaction mixtures based silver nanoparticles spectral characterization in represented in figure 3(c). The maximum absorbance observed in the 90:10 ratio based silver nanoparticles compare than the other ratios based silver nanoparticles synthesis. The 90:10 ratio based silver nanoparticles are high absorption observed in UV spectral characterization represented in the figures 3(d) (b) at 70°C temperature in the reaction.
The figure 4 despites the comparison of silver nanoparticles synthesized using different temperature in same ratio of the reaction mixture represented in figure 4. The figure 4(a) shows 99:1 ratio of the reaction mixtures in 40°C, 50°C, 60°C and 70°C. The absorption spectra higher in of the silver nanoparticles synthesized from the reaction mixture ratio 99: 1 at the temperature of 40°C. In the reaction mixture ratio 98: 2 based silver nanoparticles higher absorption peaks present in the reaction temperature 70°C ( figure 4(b)). The absorption spectra higher in of the silver nanoparticles synthesized from the reaction mixture ratio 95: 5 at the temperature of 60°C ( figure 4(c)). In the reaction mixture ratio 90: 10 based silver nanoparticles higher absorption peaks present in the reaction temperature 60°C ( figure 4(d)).
In 90:10 ratio of reaction mixture at 60°C reaction temperature based silver nanoparticles having good inhibitory property against Bacillus subtilis (19 mM). In 98:2 ratio of reaction mixture at 50°C based silver nanoparticles having good inhibitory property against Staphylococcus aureus (11 mM) and Pseudomonas aeruginosa (20 mM). In reaction temperature 70°C, the silver nanoparticles prepared in the ratios of 99:1, 98:2 and 90:10 having good inhibitory activity against test all organisms (figure 6). Especially 99:1 ratio based silver nanoparticles good inhibitory activity against Bacillus subtilis (28 mM) and E. coli (11 mM). The 98:2 reaction

Conclusion
The present study concluded nanoparticles growth based on temperature and concentration of reducing agents. To controlling the temperature and concentration of reducing agents, as well as the rate of reduction, can be effective ways to control the growth of nanoparticles. The different temperature and reaction mixture to produced different colors of silver nanoparticles, absorption of silver nanoparticles and properties of silver nanoparticles also varied it was confirmed by antimicrobial assay and UV/vis Spectrophotometer. This study is one of the evidence on temperature and concentration of reducing agents to control the nanoparticles growth and their microbial inhibitory efficacy.

Acknowledgments
This project was supported by Researchers Supporting Project number (RSP2023R230) King Saud University, Riyadh, Saudi Arabia.

Data availability statement
All data that support the findings of this study are included within the article.

Declarations
Ethics Approval and Consent to Participate Not applicable.