# Exploration of the Antimicrobial and Catalytic Properties of Gold Nanoparticles Greenly Synthesized byCryptolepis buchanani Roem. and Schult Extract

Authors: Kamonpan Wongyai; Phitchayapak Wintachai; Rasimate Maungchang; Parawee Rattanakit
Journal: Journal of Nanomaterials
 (2020)

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## Abstract

A green, simple, and rapid synthesis of gold nanoparticles using plant extract,Cryptolepis buchanani Roem. and Schult, and their applications are first described in this paper. The formation of gold nanoparticles was visually observed by the appearance of a ruby red color, which was further indicated by an absorption peak at 530 nm in UV-Vis spectroscopy. Optimization of reaction parameters for the gold nanoparticles was also investigated. Various analytical techniques were employed as part of the process of characterizing the resulting gold nanoparticles. Fourier transform infrared (FTIR) analysis revealed that the phenol compounds present in the extract were responsible for gold(III) reduction and stabilization of gold nanoparticles. Transmission electron microscopy (TEM) analysis showed that the gold nanoparticles were spherical in shape with an average diameter of 11 nm. Powder X-ray diffraction (XRD) pattern indicated that the green synthesis approach produced highly crystalline, face-centered cubic gold nanoparticles. Energy-dispersive X-ray spectroscopy (EDS) measurements confirmed the presence of elemental gold in the prepared nanoparticles. The negative zeta potential value of gold nanoparticles was found to be -30.28 mV. The green synthesized gold nanoparticles expressed effective antibacterial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii and exhibited an excellent catalytic property in terms of its reduction ability of methylene blue.


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## Body

## 1. Introduction

There has been a gradual emergence of investigation into the potential of metal nanoparticles in a variety of areas; this is due to their unique optical, electromagnetic, and area/volume properties [1, 2]. One of the most applicable metal nanoparticles is gold nanoparticles (AuNPs). Several synthetic pathways have been developed in order to produce metal nanoparticles [3–8]. Of these, conventional physical and chemical methods are commonly used as they provide uniform, stable, and size-controllable metal nanoparticles [9]. Nevertheless, the physical process has a high production cost since it requires high pressure and high energy during the synthesis procedure [10]. Also, toxic and hazardous chemicals are involved in the chemical process, affecting human health and the environment [11]. Therefore, finding an alternative biological synthesis approach is in demand, especially one that is simple, less expensive, and more environmentally friendly [5, 9, 12].Extracts from plants are candidates for preparation of metal nanoparticles as part of the biological method due to their general availability, cost effectiveness, safety, shorter time of synthesis, and the ability to increase production volumes [13, 14]. Moreover, the use of natural resources often provides mild reaction conditions and in situ capping abilities. Active biocompounds found in plant extracts can act as both reducing and stabilizing agents in the preparation of metal nanoparticles. Many research papers have reported on plant-mediated biosynthesis of metal nanoparticles. For AuNP synthesis, various plant extracts such as Olea europaea [15], Ipomoea carnea [16], Salix alba [17], Genipa americana [18], Dalbergia coromandeliana [19], Piper longum [20], Eucommia ulmoides bark [21], Butea monosperma [22], Momordica charatia [23], and Lagerstroemia speciosa [24] have been proposed. Polyphenolic, terpenoids, alkaloids, and sugars have been reported as being very well-known functional groups responsible for metal reducibility and nanoparticle stability [25]. Hence, plants that contain these groups may be utilized as biobased resources.Cryptolepis buchanani Roem. and Schutt; C. buchanani (known as Thao-En-On in Thailand) is an indigenous plant which belongs to the Asclepiadaceae family [26]. It is widely used in folk medicine in Southeast Asia for treating muscle tension and arthritis [27]. The pharmacological properties of C. buchanani include antibacterial, anti-inflammatory, analgesic, chondroprotective, and hepatoprotective effects [28]. Bioactive phenolic compounds like flavonoids, alkaloids, saponins, and tannins are major phytochemicals found in the C. buchanani extract [28–30]. These phenolic groups participate in redox reactions by forming quinones, subsequently releasing electrons which reduce the number of gold ions (Au3+ to Au0) and stabilize AuNPs [22, 31].The aims of this study are to prepare the AuNPs with aqueous extract ofC. buchanani and to assess their performance on the catalytic activity of methylene blue dye and antibacterial activities. To our best knowledge, using C. buchanani extract as a both reducing and stabilizing agents for the AuNP synthesis has not been previously detailed. Thus, this study is the first attempt to investigate the antimicrobial and catalytic properties of gold nanoparticles greenly synthesized by the C. buchanani extract.


## 2. Materials and Methods

### 2.1. Materials

All chemicals used in this study were of analytical reagent grade and deionized water was used throughout the reactions. All glasswares were washed with dilute nitric acid (HNO3) and distilled water, then dried in an oven. A gold standard solution (HAuCl4) of 1000 mg L-1 in 2 mol L-1 hydrochloric acid (HCl) was purchased from BDH (England). Sodium hydroxide (NaOH) was obtained from Loba Chemie Pvt. Ltd. (India). Methylene blue and sodium borohydride (NaBH4) were purchased from Ajax Finechem (Australia).


### 2.2. Green Synthesis of AuNPs

#### 2.2.1. Preparation ofC. buchanani Aqueous Extract

CommercialC. buchanani tea powder was acquired from a local supermarket in Nakhon Si Thammarat Province, Thailand in 2019. Following a typical procedure, about 1 g of the C. buchanani powder was heated in 50 mL deionized water at 60°C for 15 min. After the solution was cooled, it was filtered through Whatman No.1 filter paper. The bright yellow filtrate was then kept in a refrigerator at 4°C. The extract was diluted to a final concentration of 60% (v/v) before usage.


#### 2.2.2. Synthesis of AuNPs

The synthesis of AuNPs was performed using an aqueous solution of HAuCl4 (1 mM) and C. buchanani extract in a 2 : 5 v/v ratio. After the gold solution was slowly added to the extract solution, the pH was adjusted to 7 using 0.1 M NaOH. It was then heated at 80°C for 30 min with continuous stirring. During this step, the emergence of a ruby red color indicated the formation of AuNPs.


### 2.3. Optimization of Reaction Parameters for the Green Synthesis of AuNPs

Experimental conditions used for the synthesis are essential since they affect the size, shape, yield, and agglomeration state of the nanoparticles [32]. Herein, different reaction parameters, including pH, reaction time, reaction temperature, and the concentration of HAuCl4 and the extract were optimized using the univariate method.The pH was studied over the range of 2-14 in order to investigate its effect on the AuNP synthesis. In order to evaluate the effect of temperature on the AuNP formation, the mixture temperature was controlled in a water bath at 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for 30 min. The influence of reaction time was studied by monitoring the absorption spectra of the solution as a function of reaction time from 0 min to 80 min. The effect of HAuCl4 concentration was optimized in the range of 1.25×10−4 to 7.5×10−4 M for biosynthesis reaction. The influence of various C. buchanani aqueous extract concentrations on the production of AuNPs was studied by increasing the concentration from 20 to 100 (% (v/v)). Absorption spectra of the AuNP formation were recorded in the range 450-750 nm with a UV-Vis spectrophotometer.


### 2.4. Characterizations of AuNPs

For UV-Vis spectroscopy, the reaction was monitored by recording the UV-Vis spectrum in wavelengths ranging from 450 to 750 nm by using a Jasco, V-630 spectrophotometer. Measurements of Fourier transform infrared spectroscopy (FTIR) were carried out with KBr disk using a Tensor 27, Bruker spectrometer in the range of 4,000-500 cm-1. The elemental compositions of the synthesized AuNPs were investigated using an EDS (Zeiss Merlin Compact instrument) at an acceleration voltage of 10 kV. Transmission electron microscopy (TEM) images were collected on a JEOL TEM-2010 to analyse the size and morphology of the prepared AuNPs. The surface charge and stability of the AuNPs were measured by a Brookhaven ZetaPlus Zeta potential analyser. Crystalline metallic AuNPs were examined by an X-ray diffractometer (Rigaku, XtaLAB Supernova) operated at a voltage of 40 kV and a current of 30 mA using CuKα radiation. For the preparation of the solid samples, the colloidal AuNPs were centrifuged at 15,000 rpm for 30 min, washed twice with DI water, and dried at 60°C for 4 h in a vacuum oven before they were analysed.


### 2.5. Applications

#### 2.5.1. Catalytic Degradation of Methylene Blue Dye

The catalytic activity of green synthesized AuNPs was demonstrated by degrading methylene blue (MB). Stock solutions of NaBH4 and MB were freshly prepared before usage. MB (1 mM, 200 μL) and NaBH4 (0.1 M, 400 μL) were mixed in a quartz cuvette. Then, 50 μL of AuNPs was added, and the final volume was adjusted to 4 mL with the addition of deionized water. The reaction progress was monitored by UV-visible spectrophotometry in a range of 450 to 750 nm at room temperature. A control set was maintained without adding the AuNPs. The MB degradation percentage was calculated according to equation (1) below, where A0 is the absorbance at t=0and At is the absorbance at timet.
(1)%D=A0−AtA0×100.


#### 2.5.2. MIC and MBC Determination

The antibacterial activity of the AuNPs towards three common bacterial pathogens, namely,Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii was studied. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined using a modified broth microdilution method which was adopted from [33]. Briefly, five colonies were diluted in the Mueller Hinton Broth (MBH) and cultured at 37°C for 7 h. Then, the bacterial suspension was adjusted to a final concentration of around 1×108 CFU/mL. One hundred microliters of bacterial suspension was added to the plate which contained 100 μL of serial twofold dilutions in MHB of AuNPs. The plates were incubated at 37°C for 16 h. Turbidity can be used to observe the growth of bacteria, and the MIC value was set at the lowest concentration that would prevent visible growth. To determine the MBC, 10 μL of the broth with the concentration of test material of MIC was dropped onto the Mueller Hinton Agar (MHA) plates before being incubated at 37°C for 16 h. The concentration that showed no bacteria growth was subsequently used as the MBC value. This experiment was undertaken independently in triplicate.


## 2.1. Materials

All chemicals used in this study were of analytical reagent grade and deionized water was used throughout the reactions. All glasswares were washed with dilute nitric acid (HNO3) and distilled water, then dried in an oven. A gold standard solution (HAuCl4) of 1000 mg L-1 in 2 mol L-1 hydrochloric acid (HCl) was purchased from BDH (England). Sodium hydroxide (NaOH) was obtained from Loba Chemie Pvt. Ltd. (India). Methylene blue and sodium borohydride (NaBH4) were purchased from Ajax Finechem (Australia).


## 2.2. Green Synthesis of AuNPs

### 2.2.1. Preparation ofC. buchanani Aqueous Extract

CommercialC. buchanani tea powder was acquired from a local supermarket in Nakhon Si Thammarat Province, Thailand in 2019. Following a typical procedure, about 1 g of the C. buchanani powder was heated in 50 mL deionized water at 60°C for 15 min. After the solution was cooled, it was filtered through Whatman No.1 filter paper. The bright yellow filtrate was then kept in a refrigerator at 4°C. The extract was diluted to a final concentration of 60% (v/v) before usage.


### 2.2.2. Synthesis of AuNPs

The synthesis of AuNPs was performed using an aqueous solution of HAuCl4 (1 mM) and C. buchanani extract in a 2 : 5 v/v ratio. After the gold solution was slowly added to the extract solution, the pH was adjusted to 7 using 0.1 M NaOH. It was then heated at 80°C for 30 min with continuous stirring. During this step, the emergence of a ruby red color indicated the formation of AuNPs.


## 2.2.1. Preparation ofC. buchanani Aqueous Extract

CommercialC. buchanani tea powder was acquired from a local supermarket in Nakhon Si Thammarat Province, Thailand in 2019. Following a typical procedure, about 1 g of the C. buchanani powder was heated in 50 mL deionized water at 60°C for 15 min. After the solution was cooled, it was filtered through Whatman No.1 filter paper. The bright yellow filtrate was then kept in a refrigerator at 4°C. The extract was diluted to a final concentration of 60% (v/v) before usage.


## 2.2.2. Synthesis of AuNPs

The synthesis of AuNPs was performed using an aqueous solution of HAuCl4 (1 mM) and C. buchanani extract in a 2 : 5 v/v ratio. After the gold solution was slowly added to the extract solution, the pH was adjusted to 7 using 0.1 M NaOH. It was then heated at 80°C for 30 min with continuous stirring. During this step, the emergence of a ruby red color indicated the formation of AuNPs.


## 2.3. Optimization of Reaction Parameters for the Green Synthesis of AuNPs

Experimental conditions used for the synthesis are essential since they affect the size, shape, yield, and agglomeration state of the nanoparticles [32]. Herein, different reaction parameters, including pH, reaction time, reaction temperature, and the concentration of HAuCl4 and the extract were optimized using the univariate method.The pH was studied over the range of 2-14 in order to investigate its effect on the AuNP synthesis. In order to evaluate the effect of temperature on the AuNP formation, the mixture temperature was controlled in a water bath at 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for 30 min. The influence of reaction time was studied by monitoring the absorption spectra of the solution as a function of reaction time from 0 min to 80 min. The effect of HAuCl4 concentration was optimized in the range of 1.25×10−4 to 7.5×10−4 M for biosynthesis reaction. The influence of various C. buchanani aqueous extract concentrations on the production of AuNPs was studied by increasing the concentration from 20 to 100 (% (v/v)). Absorption spectra of the AuNP formation were recorded in the range 450-750 nm with a UV-Vis spectrophotometer.


## 2.4. Characterizations of AuNPs

For UV-Vis spectroscopy, the reaction was monitored by recording the UV-Vis spectrum in wavelengths ranging from 450 to 750 nm by using a Jasco, V-630 spectrophotometer. Measurements of Fourier transform infrared spectroscopy (FTIR) were carried out with KBr disk using a Tensor 27, Bruker spectrometer in the range of 4,000-500 cm-1. The elemental compositions of the synthesized AuNPs were investigated using an EDS (Zeiss Merlin Compact instrument) at an acceleration voltage of 10 kV. Transmission electron microscopy (TEM) images were collected on a JEOL TEM-2010 to analyse the size and morphology of the prepared AuNPs. The surface charge and stability of the AuNPs were measured by a Brookhaven ZetaPlus Zeta potential analyser. Crystalline metallic AuNPs were examined by an X-ray diffractometer (Rigaku, XtaLAB Supernova) operated at a voltage of 40 kV and a current of 30 mA using CuKα radiation. For the preparation of the solid samples, the colloidal AuNPs were centrifuged at 15,000 rpm for 30 min, washed twice with DI water, and dried at 60°C for 4 h in a vacuum oven before they were analysed.


## 2.5. Applications

### 2.5.1. Catalytic Degradation of Methylene Blue Dye

The catalytic activity of green synthesized AuNPs was demonstrated by degrading methylene blue (MB). Stock solutions of NaBH4 and MB were freshly prepared before usage. MB (1 mM, 200 μL) and NaBH4 (0.1 M, 400 μL) were mixed in a quartz cuvette. Then, 50 μL of AuNPs was added, and the final volume was adjusted to 4 mL with the addition of deionized water. The reaction progress was monitored by UV-visible spectrophotometry in a range of 450 to 750 nm at room temperature. A control set was maintained without adding the AuNPs. The MB degradation percentage was calculated according to equation (1) below, where A0 is the absorbance at t=0and At is the absorbance at timet.
(1)%D=A0−AtA0×100.


### 2.5.2. MIC and MBC Determination

The antibacterial activity of the AuNPs towards three common bacterial pathogens, namely,Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii was studied. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined using a modified broth microdilution method which was adopted from [33]. Briefly, five colonies were diluted in the Mueller Hinton Broth (MBH) and cultured at 37°C for 7 h. Then, the bacterial suspension was adjusted to a final concentration of around 1×108 CFU/mL. One hundred microliters of bacterial suspension was added to the plate which contained 100 μL of serial twofold dilutions in MHB of AuNPs. The plates were incubated at 37°C for 16 h. Turbidity can be used to observe the growth of bacteria, and the MIC value was set at the lowest concentration that would prevent visible growth. To determine the MBC, 10 μL of the broth with the concentration of test material of MIC was dropped onto the Mueller Hinton Agar (MHA) plates before being incubated at 37°C for 16 h. The concentration that showed no bacteria growth was subsequently used as the MBC value. This experiment was undertaken independently in triplicate.


## 2.5.1. Catalytic Degradation of Methylene Blue Dye

The catalytic activity of green synthesized AuNPs was demonstrated by degrading methylene blue (MB). Stock solutions of NaBH4 and MB were freshly prepared before usage. MB (1 mM, 200 μL) and NaBH4 (0.1 M, 400 μL) were mixed in a quartz cuvette. Then, 50 μL of AuNPs was added, and the final volume was adjusted to 4 mL with the addition of deionized water. The reaction progress was monitored by UV-visible spectrophotometry in a range of 450 to 750 nm at room temperature. A control set was maintained without adding the AuNPs. The MB degradation percentage was calculated according to equation (1) below, where A0 is the absorbance at t=0and At is the absorbance at timet.
(1)%D=A0−AtA0×100.


## 2.5.2. MIC and MBC Determination

The antibacterial activity of the AuNPs towards three common bacterial pathogens, namely,Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii was studied. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined using a modified broth microdilution method which was adopted from [33]. Briefly, five colonies were diluted in the Mueller Hinton Broth (MBH) and cultured at 37°C for 7 h. Then, the bacterial suspension was adjusted to a final concentration of around 1×108 CFU/mL. One hundred microliters of bacterial suspension was added to the plate which contained 100 μL of serial twofold dilutions in MHB of AuNPs. The plates were incubated at 37°C for 16 h. Turbidity can be used to observe the growth of bacteria, and the MIC value was set at the lowest concentration that would prevent visible growth. To determine the MBC, 10 μL of the broth with the concentration of test material of MIC was dropped onto the Mueller Hinton Agar (MHA) plates before being incubated at 37°C for 16 h. The concentration that showed no bacteria growth was subsequently used as the MBC value. This experiment was undertaken independently in triplicate.


## 3. Results and Discussion

### 3.1. Optimizing Synthesis Parameters of AuNPs Formation

The effect of pH, temperature,C. buchanani, and precursor gold ion concentrations and reaction time were studied to find the optimum experimental conditions for the synthesis of AuNPs using the C. buchanani aqueous extract as reducing and stabilizing agents. In the present study, the optimum conditions were considered with regard to position, symmetry, and the narrowness or broadness of the surface plasmon resonance (SPR) band. The characteristics of the SPR band can indicate the qualities as well as quantities of the nanoparticles, i.e., morphology, size, and agglomeration [34]. Typically, a symmetrical and narrow SPR band indicates the presence of nanoparticles with a narrow range of size and that are uniform in shape. Other dominant factors are shifting or shape changes to the SPR band, increasing or decreasing of absorbance ability and changes to the maximum absorbance wavelength values [35].


#### 3.1.1. Effect of pH

The pH value of the reaction solution has a critical impact on the morphology and size of the synthesized AuNPs. The synthesis of AuNPs in acidic, neutral, and alkaline conditions was carried out. As can be seen in Figure1, the absorbance increases significantly as pH increases from 2 to 7, followed by a dramatic decrease in the pH from 8 to 14. In the acidic to neutral range, λmax was observed at around 530 nm and the highest intensity was obtained at pH 7. In contrast to the alkaline condition, λmax was shifted from 530 to 600 nm. It was observed that the nanoparticles aggregated to larger particles, resulting in a large shift in the SPR band [36]. Moreover, lower intensity and broadening SPR bands occurred when compared to acidic and neutral pH, indicating that the AuNPs formed inefficiently. A colorimetric visualization of the formed AuNPs at different pH levels is presented in Figure 1 (inset). It should be pointed out that different pH levels caused differently colored solutions. The light yellow of the C. buchanani extract solution turned into violet (pH 2 and 4), ruby red (pH 6-8), and black (pH 10-14) after mixing with the HAuCl4. Unfortunately, the AuNPs were precipitated at pH 14. In general, the electric charges of biomolecules present in C. buchanani extract can be changed by altering the pH of the solution. This may affect both the capping and stabilizing ability, and subsequently, the growth of the nanoparticles [37, 38]. This study has revealed that a neutral pH is favorable for the content of the biomolecules in the extract to efficiently reduce and stabilize the AuNPs. Therefore, pH 7 was selected as the optimum pH for AuNP synthesis.Figure 1
UV-Vis spectra of AuNPs at different pH values. The inset shows the photographic image of the colors of the AuNP solutions.It should be noted that some previous studies have reported that a basic pH was favorable for AuNP production using various plant extracts such as olive leaf extract [39], Capsicum annuum var. grossum pulp extract [40], Momordica charantia fruit extract [23], and Lotus Leguminosae [41]. However, in this study, a neutral pH was found to be more favorable for the synthesis of AuNPs. The result is in a good agreement with [36] where AuNPs were synthesized using Dracocephalum kotschyi leaf extract.


#### 3.1.2. Effect of Reaction Temperature and Time

In order to explore the impact of temperature on the AuNP formation, seven different temperatures were used in our experiment. As might be expected, the absorption intensity of AuNPs increased as temperature increased up to around about 80°C, above which it then started to slowly decrease. Thus, 80°C was applied as the optimal heating temperature for the preparation of AuNPs. The reaction times for the synthesis of the AuNPs (from 0 to 80 min) were also evaluated. The maximum absorbance value was saturated at 20 min, indicating the completion of the reduction process. However, the optimum reaction time selected for this study was 30 min. Figure2(a) shows the UV-Vis absorption spectra which highlight the effect of heat treatment on the synthesis of AuNPs. The result demonstrated that the SPR peak of heated AuNPs increased significantly and became narrower; moreover, it appeared at a lower wavelength in comparison to the absorption peak of AuNPs prepared at room temperature. Increasing the reaction temperature from room temperature to 80°C decreased the formation time of the nanoparticles from 2 h to 20 min. Increasing the reaction temperature enhanced the formation rate of nanoparticles, resulting in the decrease of formation time [40] and formation of more small-sized nanoparticles [39].Figure 2
UV-Vis spectrum of green synthesized AuNPs prepared fromC. buchanani aqueous extract (a) with and without heat treatment, (b) HAuCl4 concentration, and (c) C. buchanani concentration.
(a)(b)(c)


#### 3.1.3. Effect of HAuCl4 Concentration

The formation of AuNPs was examined by adding different HAuCl4 concentrations (1.25×10−4 to 7.5×10−4 M) to a constant concentration of the extract solution at 80°C for 30 min. The UV-Vis absorption spectra of the synthesized AuNPs at different HAuCl4 concentrations are presented in Figure 2(b). As the HAuCl4 concentration increased, the shifts of the SPR band from 570 to 530 nm were observed. This indicates that the size of the nanoparticles decreased, resulting in them becoming more spherical in shape, as well as the distribution of the nanoparticles being more homogeneous [42, 43]. Additionally, the peaks at higher concentrations were seen to be more intense. Therefore, a concentration of 5.0×10−4 M HAuCl4 was chosen as the optimum concentration for AuNP formation since it gave the highest absorption intensity.


#### 3.1.4. Effect of Plant Extract Concentration

The effect ofC. buchanani concentration on the synthesis of AuNPs was studied using different extract quantities (varying between 1 and 5 mL). The concentrations of the extract were 20-100% (v/v). Figure 2(c) shows the UV-Vis absorption spectra of greenly synthesized AuNPs using different concentrations of the extract. The absorption intensity gradually increased when the extract concentration increased from 20 to 60% (v/v). Besides, the absorption peak becomes sharper, and a blue shift was observed from 560 nm to 530 nm, suggesting a reduction in the mean diameter of the nanoparticles. However, when the concentration of the extract exceeded 60% (v/v), the absorption band became broader and shifted considerably back towards the red region, appearing at 540 and 550 nm for 80 and 100% (v/v), respectively. This is probably due to the rapid reduction of gold ions by large quantities of biomolecules or reducing agents [44]. Thus, the extract concentration at 60% (v/v) was chosen.


### 3.2. Characterization of Green Synthesized AuNPs

In this study, an aqueous extract ofC. buchanani was utilized as a green reducing agent and stabilizer to synthesize AuNPs. The obtained AuNPs were characterized by employing UV-Vis spectroscopy, TEM, EDS, XRD, FTIR, and zeta potential techniques. The optimum conditions used for preparing the AuNPs were pH 7, 30 min reaction time, 80°C synthesis temperature, 60% (v/v) extract concentration, and 5×10−4 M HAuCl4.


#### 3.2.1. Visualizing the Formation of AuNPs and UV-Vis Spectrum Analysis

The ability of the aqueousC. buchanani extracts to produce AuNPs was firstly monitored by the naked eye and UV-Vis absorption spectroscopy. After adding a gold solution to the C. buchanani extract, the color of the solution changed from light yellow to ruby red while no change in color was observed in the absence of HAuCl4 (Figure 3, inset). The appearance of the characteristic ruby red color indicates the reduction of Au3+ ions to gold nanoparticles. This visual observation could be attributed to the excitation of surface plasmon vibrations. Hence, it was used as a spectroscopic signature to show the formation of AuNPs. In general, the characteristic surface plasmon band from 500 to 550 nm indicates the spherical shape of AuNPs [45].Figure 3
UV-Vis spectra of the green synthesized AuNPs using theC. buchanani extract. Inset: a digital image of the (a) C. buchanani extract solution and (b) AuNPs.To retrieve information on the optical properties of the green synthesized AuNPs, the SPR was monitored by UV-Vis spectroscopy. The UV-Vis spectra of green synthesized AuNPs in which the SPR band was located at 530 nm confirmed the formation of spherical AuNPs in solution, as shown in Figure3. This clearly demonstrates that the C. buchanani extract can perform as an excellent reducing and stabilizing agent for AuNP synthesis. Phytochemicals present in the C. buchanani extract such as flavonoid, alkaloids, saponins, and tannins are believed to play a key role in redox reactions by forming quinones and subsequently releasing electrons which reduce the number of gold ions (Au3+ to Au0) and stabilize the AuNPs [46].


#### 3.2.2. TEM Analysis and Zeta Potential

The morphology of the greenly synthesized AuNPs using theC. buchanani extract was examined using a TEM. Figure 4 shows that the dispersed AuNPs were spherical in shape with an average diameter of around 11.1±1.3 nm. The zeta potential of the AuNPs was found to be -30.28 mV, indicating high stability, good colloidal nature, and high dispersibility due to negative-negative repulsion [47].Figure 4
TEM image of the green synthesized AuNPs.


#### 3.2.3. XRD and EDS Analysis

The crystalline characteristic of the synthesized AuNPs was determined by XRD analysis. Figure5(a) shows the XRD pattern of AuNPs at 2θ ranging between 10° and 80°. Four characteristic diffraction peaks can be seen at two theta values of 38.02°, 44.9°, 65.26°, and 78.3°, which correspond to the (111), (200), (220), and (311) planes of the face-centered cubic crystalline lattice of AuNPs, respectively. These peaks collaborated with the Joint Committee on Powder Diffraction Standard, JCPDS no. 04-0784 [48]. The peak corresponding to the (111) plane is more intense than the other planes, which indicates that (111) is the predominant orientation. The XRD spectrum strongly confirms the high crystalline nature of the prepared AuNPs. The elemental composition of the AuNPs was measured by EDS analysis. The presence of strong signals around 2 keV, as shown in Figure 5(b), confirmed the presence of metallic Au. The presence of C and O is due to the elemental composition of the C. buchanani extract [49].Figure 5
(a) XRD pattern and (b) EDS analysis of the synthesized AuNPs.
(a)(b)


#### 3.2.4. FTIR Analysis

FTIR measurements were carried out to identify the potential functional groups of the biomolecules in theC. buchanani extract, which can be involved in the reduction of Au3+ and capping/stabilization of AuNPs. The major active components present in C. buchanani are known to be tannins, alkaloids, saponins, and flavonoids [28]. Figure 6 shows the FTIR spectra of the extract and the synthesized AuNPs. For the C. buchanani spectrum, the bands observed at 3419 and 2926 cm-1 are attributed to O-H and C-H stretching modes. The peak found at 1625 cm-1 is assigned to C=C and C=O stretching of the phytoconstituents. The strong absorption band at 1032 cm-1 corresponds to the vibration of the -C-O group of constituents. These observations indicate the presence of polyphenol in the extract. For FTIR spectra of the AuNPs, the characteristic absorption bands were found to be quite similar to those of the extract, but the vibration of the O-H group and the C=O group shifted from 3419 cm-1 to 3406 cm-1 and 1625 cm-1 to 1599 cm-1, respectively. The small shifting and decreasing in intensities imply that the biomolecules in the extract, especially polyphenol, may be involved in facilitating the AuNPs, mainly through their oxygen functionalities. The results are in good agreement with those obtained by [50, 51]. All characterization results confirm that the aqueous extract of C. buchanani can be effectively used in the biosynthesis of the AuNPs.Figure 6
FTIR spectra of the synthesized AuNPs.


#### 3.2.5. Comparison of the Properties of AuNPs Prepared withC. buchanani Extract with Select Previous Literature

The uses of various plant extracts for the green synthesis of AuNPs have been previously investigated in published studies, and the properties of the obtained AuNPs are listed in Table1. As can be seen, different sizes and shapes of the prepared AuNPs are reported. The features of the produced AuNPs are dependent on the plant extracts and the reaction conditions. According to a careful examination of the synthesis conditions in this work, well-dispersed, small-sized AuNPs that had a uniform spherical shape were produced.Table 1
A partial list of the properties of AuNPs synthesized by plant extracts reported in the select literature.
PlantMeasurement techniqueSize (nm)ShapeZP (mV)RefAbutilon indicumTEM1-20Sph-25[52]Aegle marmelosTEM18Sph—[53]Eugenia jambolana28Soursop16Bacopa monnieriTEM3-45Sph—[54]Capsicum annuum var. grossumTEM6-37Sph, Tri, Hex—[40]Corchorus olitoriusTEM27-35Sph—[43]Cornus masTEM19Sph-33[47]Couroupita guianensis Aubl.TEM25±6Sph, Tri, Hex—[55]C. buchananiTEM11Sph-30.3This workCroton caudatus geiselTEM20-50Sph—[56]Dalbergia coromandelianaHRTEM10.5Sph—[19]Dracocephalum kotschyiTEM8-23Sph, Tri, Pen, Hex-29.3[36]Elaeis guineensisTEM35-75Sph, Tri, Pen-17[57]Eucommia ulmoidesTEM16.4Sph-21.3[21]Genipa americanaTEM15-40Sph—[18]Hibiscus sabdariffaTEM10-60Sph—[58]Ipomoea carneaTEM3-100Sph, Tri, Pen, Hex, R—[16]Nepenthes khasianaSEM50-80Tri, Sph—[59]Nerium oleanderHRTEM2-10Sph—[60]Olea europaeaTEM50-100Tri, hex, Sph—[39]Justicia adhatodaSEM13-57Sph—[61]Sansevieria roxburghianaTEM5-31Sph, Tri, Hex, Dec, R—[62]Note: Dec: decahedral; Hex: hexagonal; Pen: pentagonal; R: rod; Sph: spherical; Tri: triangular; ZP: zeta potential.


### 3.3. Applications

#### 3.3.1. Catalytic Degradation of Methylene Blue

To evaluate the catalytic activity of the AuNPs synthesized by theC. buchanani extract, the reduction reaction of methylene blue (MB) dye by NaBH4 was carried out as a model system. The experiment was conducted at room temperature, and the reactions were monitored by UV-visible spectroscopy. Typically, an aqueous solution of MB imparting a blue color shows two characteristic absorption peaks at 664 nm with a shoulder at 612 nm [63]. The prominent band at 664 nm is assigned to the n→π∗ transitions and a shoulder band at 612 nm is attributed to the dimer (MB)2 due to its dimerization in aqueous medium [64]. When the prepared AuNPs were added to the reaction mixture containing MB dye and NaBH4, the color of the dye gradually altered over time from deep blue to light blue before finally disappearing. The lack of color in the MB solution was due to the reduction of the MB to leucomethylene blue [65]. In the absence of catalyst AuNPs, no degradation of MB could be seen, although after 90 min of reaction between MB with a strong reducing agent, NaBH4, it could be suggested that MB had not reduced effectively by NaBH4 and reaction rate was very slow. When compared with the solution in the presence of the catalyst, the complete degradation (dye degradation at 95%) of MB was accomplished within 4 min (Figures 7(a) and 7(b)). The result proves that the C. buchanani extract-mediated AuNPs possess a significant catalytic activity.Figure 7
UV-visible spectra of reduction of MB by sodium borohydride in (a) the presence of AuNPs as the catalyst, (b) only NaBH4, and (c) linear plot of MB over the time.
(a)(b)(c)A report on the catalytic mechanism of the degradation reaction of MB by AuNPs in the NaBH4 system has been previously published [66]. Borohydride ions (BH4−) and the MB dye serve their roles as donors and an acceptor, respectively. The large redox potential difference between them kinetically hinders the reduction of dyes [67]. The AuNPs act as an electron relay and initiate shifting of the electrons from the BH4− ions (donor B2H4/BH4−) to the acceptor (acceptor LMB/MB), thus causing a reduction of the dye. BH4− ions are simultaneously adsorbed on the surface of the NPs; thus, electron transfer occurs from the BH4− ions to the dye through AuNPs [68].According to the presence of excess reducing agent, the pseudofirst-order kinetic was applied herein. The slope of the linear regression plot oflnA0/At versus reaction time, t, is the rate of degradation, which was found to be 0.3628 min-1 (Figure 7(c)). The effect of the catalyst AuNP quantity on the degradation rate was also studied. The rate constant obtained from varying the volumes of the AuNPs is given in Table 2. The result shows that the rate constant increases with an increasing amount of AuNPs, suggesting that the reduction of MB occurs on the surface of the catalyst. A comparison of the catalytic activity of the AuNPs toward the degradation of MB is presented in Table 3. Additionally, the amounts of substrates (MB), reductants (NaBH4), and catalysts (AuNPs) in each reduction of MB were carefully examined. The results obtained in our study are similar or superior to the reference presented in the last column of Table 3. The excellent catalytic efficiency of MB could be due to the small size of the synthesized AuNPs which plays an essential role in catalytic reduction of dye. Therefore, the greenly synthesized AuNPs using C. buchanani could act as a promising material for the degradation of toxic MB dye and could be useful for developing organic dye-containing wastewater treatment.Table 2
The rate constant of different amount of AuNPs.
Volume of AuNP solution (μL)Rate constant (min-1)200.1634300.2555400.3398500.3628Table 3
Comparison of the catalytic activity of green synthesized AuNPs towards the degradation of MB.
(NaBH4) (M)(MB) (mM)AuNPs (μL)AuNPs size (nm)Degradation time (min)Degradation (%)Rate constant (min-1)R2Ref.0.151100176049.620.01180.9852[62]0.0311000≤4014>900.19800.9600[24]0.050.110011895.620.07200.9786[69]0.115011494.810.36280.9953This work


#### 3.3.2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

Generally, MIC and MBC values provide quantitative data on the antimicrobial efficacy of the AuNPs. The lowest AuNP concentration that inhibited visible growth of bacterial strains was defined as “MIC,” whereas the lowest concentration of the AuNP that inhibited growth of ≥99.99% of bacterial strains was defined as “MBC.. The MIC and MBC values ofC. buchanani extract mediating AuNPs were evaluated using a microbroth dilution method against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii. The results are shown in Table 4. The MIC values for all tested strains were equal to 0.209 μg/mL. The MBC values for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus were both 0.418 μg/mL while a value greater than 0.835 μg/mL was observed for Acinetobacter baumannii (2-fold higher). According to the MBC values, Gram-positive bacteria exhibited higher sensitivity to that of Gram-negative bacteria. This might be due to the presence of capsules on the cell walls of bacteria, which prevent the attachment of the AuNPs [70]. Therefore, the biosynthesized AuNPs show significant potential in terms of their antimicrobial application.Table 4
MIC and MBC values of the AuNPs against the three pathogenic strains.
Test organismsGram classMIC (μg/mL)MBC (μg/mL)Staphylococcus aureusGram positive0.2090.418Methicillin-resistantStaphylococcus aureusGram positive0.2090.418Acinetobacter baumanniiGram negative0.209>0.835


## 3.1. Optimizing Synthesis Parameters of AuNPs Formation

The effect of pH, temperature,C. buchanani, and precursor gold ion concentrations and reaction time were studied to find the optimum experimental conditions for the synthesis of AuNPs using the C. buchanani aqueous extract as reducing and stabilizing agents. In the present study, the optimum conditions were considered with regard to position, symmetry, and the narrowness or broadness of the surface plasmon resonance (SPR) band. The characteristics of the SPR band can indicate the qualities as well as quantities of the nanoparticles, i.e., morphology, size, and agglomeration [34]. Typically, a symmetrical and narrow SPR band indicates the presence of nanoparticles with a narrow range of size and that are uniform in shape. Other dominant factors are shifting or shape changes to the SPR band, increasing or decreasing of absorbance ability and changes to the maximum absorbance wavelength values [35].


### 3.1.1. Effect of pH

The pH value of the reaction solution has a critical impact on the morphology and size of the synthesized AuNPs. The synthesis of AuNPs in acidic, neutral, and alkaline conditions was carried out. As can be seen in Figure1, the absorbance increases significantly as pH increases from 2 to 7, followed by a dramatic decrease in the pH from 8 to 14. In the acidic to neutral range, λmax was observed at around 530 nm and the highest intensity was obtained at pH 7. In contrast to the alkaline condition, λmax was shifted from 530 to 600 nm. It was observed that the nanoparticles aggregated to larger particles, resulting in a large shift in the SPR band [36]. Moreover, lower intensity and broadening SPR bands occurred when compared to acidic and neutral pH, indicating that the AuNPs formed inefficiently. A colorimetric visualization of the formed AuNPs at different pH levels is presented in Figure 1 (inset). It should be pointed out that different pH levels caused differently colored solutions. The light yellow of the C. buchanani extract solution turned into violet (pH 2 and 4), ruby red (pH 6-8), and black (pH 10-14) after mixing with the HAuCl4. Unfortunately, the AuNPs were precipitated at pH 14. In general, the electric charges of biomolecules present in C. buchanani extract can be changed by altering the pH of the solution. This may affect both the capping and stabilizing ability, and subsequently, the growth of the nanoparticles [37, 38]. This study has revealed that a neutral pH is favorable for the content of the biomolecules in the extract to efficiently reduce and stabilize the AuNPs. Therefore, pH 7 was selected as the optimum pH for AuNP synthesis.Figure 1
UV-Vis spectra of AuNPs at different pH values. The inset shows the photographic image of the colors of the AuNP solutions.It should be noted that some previous studies have reported that a basic pH was favorable for AuNP production using various plant extracts such as olive leaf extract [39], Capsicum annuum var. grossum pulp extract [40], Momordica charantia fruit extract [23], and Lotus Leguminosae [41]. However, in this study, a neutral pH was found to be more favorable for the synthesis of AuNPs. The result is in a good agreement with [36] where AuNPs were synthesized using Dracocephalum kotschyi leaf extract.


### 3.1.2. Effect of Reaction Temperature and Time

In order to explore the impact of temperature on the AuNP formation, seven different temperatures were used in our experiment. As might be expected, the absorption intensity of AuNPs increased as temperature increased up to around about 80°C, above which it then started to slowly decrease. Thus, 80°C was applied as the optimal heating temperature for the preparation of AuNPs. The reaction times for the synthesis of the AuNPs (from 0 to 80 min) were also evaluated. The maximum absorbance value was saturated at 20 min, indicating the completion of the reduction process. However, the optimum reaction time selected for this study was 30 min. Figure2(a) shows the UV-Vis absorption spectra which highlight the effect of heat treatment on the synthesis of AuNPs. The result demonstrated that the SPR peak of heated AuNPs increased significantly and became narrower; moreover, it appeared at a lower wavelength in comparison to the absorption peak of AuNPs prepared at room temperature. Increasing the reaction temperature from room temperature to 80°C decreased the formation time of the nanoparticles from 2 h to 20 min. Increasing the reaction temperature enhanced the formation rate of nanoparticles, resulting in the decrease of formation time [40] and formation of more small-sized nanoparticles [39].Figure 2
UV-Vis spectrum of green synthesized AuNPs prepared fromC. buchanani aqueous extract (a) with and without heat treatment, (b) HAuCl4 concentration, and (c) C. buchanani concentration.
(a)(b)(c)


### 3.1.3. Effect of HAuCl4 Concentration

The formation of AuNPs was examined by adding different HAuCl4 concentrations (1.25×10−4 to 7.5×10−4 M) to a constant concentration of the extract solution at 80°C for 30 min. The UV-Vis absorption spectra of the synthesized AuNPs at different HAuCl4 concentrations are presented in Figure 2(b). As the HAuCl4 concentration increased, the shifts of the SPR band from 570 to 530 nm were observed. This indicates that the size of the nanoparticles decreased, resulting in them becoming more spherical in shape, as well as the distribution of the nanoparticles being more homogeneous [42, 43]. Additionally, the peaks at higher concentrations were seen to be more intense. Therefore, a concentration of 5.0×10−4 M HAuCl4 was chosen as the optimum concentration for AuNP formation since it gave the highest absorption intensity.


### 3.1.4. Effect of Plant Extract Concentration

The effect ofC. buchanani concentration on the synthesis of AuNPs was studied using different extract quantities (varying between 1 and 5 mL). The concentrations of the extract were 20-100% (v/v). Figure 2(c) shows the UV-Vis absorption spectra of greenly synthesized AuNPs using different concentrations of the extract. The absorption intensity gradually increased when the extract concentration increased from 20 to 60% (v/v). Besides, the absorption peak becomes sharper, and a blue shift was observed from 560 nm to 530 nm, suggesting a reduction in the mean diameter of the nanoparticles. However, when the concentration of the extract exceeded 60% (v/v), the absorption band became broader and shifted considerably back towards the red region, appearing at 540 and 550 nm for 80 and 100% (v/v), respectively. This is probably due to the rapid reduction of gold ions by large quantities of biomolecules or reducing agents [44]. Thus, the extract concentration at 60% (v/v) was chosen.


## 3.1.1. Effect of pH

The pH value of the reaction solution has a critical impact on the morphology and size of the synthesized AuNPs. The synthesis of AuNPs in acidic, neutral, and alkaline conditions was carried out. As can be seen in Figure1, the absorbance increases significantly as pH increases from 2 to 7, followed by a dramatic decrease in the pH from 8 to 14. In the acidic to neutral range, λmax was observed at around 530 nm and the highest intensity was obtained at pH 7. In contrast to the alkaline condition, λmax was shifted from 530 to 600 nm. It was observed that the nanoparticles aggregated to larger particles, resulting in a large shift in the SPR band [36]. Moreover, lower intensity and broadening SPR bands occurred when compared to acidic and neutral pH, indicating that the AuNPs formed inefficiently. A colorimetric visualization of the formed AuNPs at different pH levels is presented in Figure 1 (inset). It should be pointed out that different pH levels caused differently colored solutions. The light yellow of the C. buchanani extract solution turned into violet (pH 2 and 4), ruby red (pH 6-8), and black (pH 10-14) after mixing with the HAuCl4. Unfortunately, the AuNPs were precipitated at pH 14. In general, the electric charges of biomolecules present in C. buchanani extract can be changed by altering the pH of the solution. This may affect both the capping and stabilizing ability, and subsequently, the growth of the nanoparticles [37, 38]. This study has revealed that a neutral pH is favorable for the content of the biomolecules in the extract to efficiently reduce and stabilize the AuNPs. Therefore, pH 7 was selected as the optimum pH for AuNP synthesis.Figure 1
UV-Vis spectra of AuNPs at different pH values. The inset shows the photographic image of the colors of the AuNP solutions.It should be noted that some previous studies have reported that a basic pH was favorable for AuNP production using various plant extracts such as olive leaf extract [39], Capsicum annuum var. grossum pulp extract [40], Momordica charantia fruit extract [23], and Lotus Leguminosae [41]. However, in this study, a neutral pH was found to be more favorable for the synthesis of AuNPs. The result is in a good agreement with [36] where AuNPs were synthesized using Dracocephalum kotschyi leaf extract.


## 3.1.2. Effect of Reaction Temperature and Time

In order to explore the impact of temperature on the AuNP formation, seven different temperatures were used in our experiment. As might be expected, the absorption intensity of AuNPs increased as temperature increased up to around about 80°C, above which it then started to slowly decrease. Thus, 80°C was applied as the optimal heating temperature for the preparation of AuNPs. The reaction times for the synthesis of the AuNPs (from 0 to 80 min) were also evaluated. The maximum absorbance value was saturated at 20 min, indicating the completion of the reduction process. However, the optimum reaction time selected for this study was 30 min. Figure2(a) shows the UV-Vis absorption spectra which highlight the effect of heat treatment on the synthesis of AuNPs. The result demonstrated that the SPR peak of heated AuNPs increased significantly and became narrower; moreover, it appeared at a lower wavelength in comparison to the absorption peak of AuNPs prepared at room temperature. Increasing the reaction temperature from room temperature to 80°C decreased the formation time of the nanoparticles from 2 h to 20 min. Increasing the reaction temperature enhanced the formation rate of nanoparticles, resulting in the decrease of formation time [40] and formation of more small-sized nanoparticles [39].Figure 2
UV-Vis spectrum of green synthesized AuNPs prepared fromC. buchanani aqueous extract (a) with and without heat treatment, (b) HAuCl4 concentration, and (c) C. buchanani concentration.
(a)(b)(c)


## 3.1.3. Effect of HAuCl4 Concentration

The formation of AuNPs was examined by adding different HAuCl4 concentrations (1.25×10−4 to 7.5×10−4 M) to a constant concentration of the extract solution at 80°C for 30 min. The UV-Vis absorption spectra of the synthesized AuNPs at different HAuCl4 concentrations are presented in Figure 2(b). As the HAuCl4 concentration increased, the shifts of the SPR band from 570 to 530 nm were observed. This indicates that the size of the nanoparticles decreased, resulting in them becoming more spherical in shape, as well as the distribution of the nanoparticles being more homogeneous [42, 43]. Additionally, the peaks at higher concentrations were seen to be more intense. Therefore, a concentration of 5.0×10−4 M HAuCl4 was chosen as the optimum concentration for AuNP formation since it gave the highest absorption intensity.


## 3.1.4. Effect of Plant Extract Concentration

The effect ofC. buchanani concentration on the synthesis of AuNPs was studied using different extract quantities (varying between 1 and 5 mL). The concentrations of the extract were 20-100% (v/v). Figure 2(c) shows the UV-Vis absorption spectra of greenly synthesized AuNPs using different concentrations of the extract. The absorption intensity gradually increased when the extract concentration increased from 20 to 60% (v/v). Besides, the absorption peak becomes sharper, and a blue shift was observed from 560 nm to 530 nm, suggesting a reduction in the mean diameter of the nanoparticles. However, when the concentration of the extract exceeded 60% (v/v), the absorption band became broader and shifted considerably back towards the red region, appearing at 540 and 550 nm for 80 and 100% (v/v), respectively. This is probably due to the rapid reduction of gold ions by large quantities of biomolecules or reducing agents [44]. Thus, the extract concentration at 60% (v/v) was chosen.


## 3.2. Characterization of Green Synthesized AuNPs

In this study, an aqueous extract ofC. buchanani was utilized as a green reducing agent and stabilizer to synthesize AuNPs. The obtained AuNPs were characterized by employing UV-Vis spectroscopy, TEM, EDS, XRD, FTIR, and zeta potential techniques. The optimum conditions used for preparing the AuNPs were pH 7, 30 min reaction time, 80°C synthesis temperature, 60% (v/v) extract concentration, and 5×10−4 M HAuCl4.


### 3.2.1. Visualizing the Formation of AuNPs and UV-Vis Spectrum Analysis

The ability of the aqueousC. buchanani extracts to produce AuNPs was firstly monitored by the naked eye and UV-Vis absorption spectroscopy. After adding a gold solution to the C. buchanani extract, the color of the solution changed from light yellow to ruby red while no change in color was observed in the absence of HAuCl4 (Figure 3, inset). The appearance of the characteristic ruby red color indicates the reduction of Au3+ ions to gold nanoparticles. This visual observation could be attributed to the excitation of surface plasmon vibrations. Hence, it was used as a spectroscopic signature to show the formation of AuNPs. In general, the characteristic surface plasmon band from 500 to 550 nm indicates the spherical shape of AuNPs [45].Figure 3
UV-Vis spectra of the green synthesized AuNPs using theC. buchanani extract. Inset: a digital image of the (a) C. buchanani extract solution and (b) AuNPs.To retrieve information on the optical properties of the green synthesized AuNPs, the SPR was monitored by UV-Vis spectroscopy. The UV-Vis spectra of green synthesized AuNPs in which the SPR band was located at 530 nm confirmed the formation of spherical AuNPs in solution, as shown in Figure3. This clearly demonstrates that the C. buchanani extract can perform as an excellent reducing and stabilizing agent for AuNP synthesis. Phytochemicals present in the C. buchanani extract such as flavonoid, alkaloids, saponins, and tannins are believed to play a key role in redox reactions by forming quinones and subsequently releasing electrons which reduce the number of gold ions (Au3+ to Au0) and stabilize the AuNPs [46].


### 3.2.2. TEM Analysis and Zeta Potential

The morphology of the greenly synthesized AuNPs using theC. buchanani extract was examined using a TEM. Figure 4 shows that the dispersed AuNPs were spherical in shape with an average diameter of around 11.1±1.3 nm. The zeta potential of the AuNPs was found to be -30.28 mV, indicating high stability, good colloidal nature, and high dispersibility due to negative-negative repulsion [47].Figure 4
TEM image of the green synthesized AuNPs.


### 3.2.3. XRD and EDS Analysis

The crystalline characteristic of the synthesized AuNPs was determined by XRD analysis. Figure5(a) shows the XRD pattern of AuNPs at 2θ ranging between 10° and 80°. Four characteristic diffraction peaks can be seen at two theta values of 38.02°, 44.9°, 65.26°, and 78.3°, which correspond to the (111), (200), (220), and (311) planes of the face-centered cubic crystalline lattice of AuNPs, respectively. These peaks collaborated with the Joint Committee on Powder Diffraction Standard, JCPDS no. 04-0784 [48]. The peak corresponding to the (111) plane is more intense than the other planes, which indicates that (111) is the predominant orientation. The XRD spectrum strongly confirms the high crystalline nature of the prepared AuNPs. The elemental composition of the AuNPs was measured by EDS analysis. The presence of strong signals around 2 keV, as shown in Figure 5(b), confirmed the presence of metallic Au. The presence of C and O is due to the elemental composition of the C. buchanani extract [49].Figure 5
(a) XRD pattern and (b) EDS analysis of the synthesized AuNPs.
(a)(b)


### 3.2.4. FTIR Analysis

FTIR measurements were carried out to identify the potential functional groups of the biomolecules in theC. buchanani extract, which can be involved in the reduction of Au3+ and capping/stabilization of AuNPs. The major active components present in C. buchanani are known to be tannins, alkaloids, saponins, and flavonoids [28]. Figure 6 shows the FTIR spectra of the extract and the synthesized AuNPs. For the C. buchanani spectrum, the bands observed at 3419 and 2926 cm-1 are attributed to O-H and C-H stretching modes. The peak found at 1625 cm-1 is assigned to C=C and C=O stretching of the phytoconstituents. The strong absorption band at 1032 cm-1 corresponds to the vibration of the -C-O group of constituents. These observations indicate the presence of polyphenol in the extract. For FTIR spectra of the AuNPs, the characteristic absorption bands were found to be quite similar to those of the extract, but the vibration of the O-H group and the C=O group shifted from 3419 cm-1 to 3406 cm-1 and 1625 cm-1 to 1599 cm-1, respectively. The small shifting and decreasing in intensities imply that the biomolecules in the extract, especially polyphenol, may be involved in facilitating the AuNPs, mainly through their oxygen functionalities. The results are in good agreement with those obtained by [50, 51]. All characterization results confirm that the aqueous extract of C. buchanani can be effectively used in the biosynthesis of the AuNPs.Figure 6
FTIR spectra of the synthesized AuNPs.


### 3.2.5. Comparison of the Properties of AuNPs Prepared withC. buchanani Extract with Select Previous Literature

The uses of various plant extracts for the green synthesis of AuNPs have been previously investigated in published studies, and the properties of the obtained AuNPs are listed in Table1. As can be seen, different sizes and shapes of the prepared AuNPs are reported. The features of the produced AuNPs are dependent on the plant extracts and the reaction conditions. According to a careful examination of the synthesis conditions in this work, well-dispersed, small-sized AuNPs that had a uniform spherical shape were produced.Table 1
A partial list of the properties of AuNPs synthesized by plant extracts reported in the select literature.
PlantMeasurement techniqueSize (nm)ShapeZP (mV)RefAbutilon indicumTEM1-20Sph-25[52]Aegle marmelosTEM18Sph—[53]Eugenia jambolana28Soursop16Bacopa monnieriTEM3-45Sph—[54]Capsicum annuum var. grossumTEM6-37Sph, Tri, Hex—[40]Corchorus olitoriusTEM27-35Sph—[43]Cornus masTEM19Sph-33[47]Couroupita guianensis Aubl.TEM25±6Sph, Tri, Hex—[55]C. buchananiTEM11Sph-30.3This workCroton caudatus geiselTEM20-50Sph—[56]Dalbergia coromandelianaHRTEM10.5Sph—[19]Dracocephalum kotschyiTEM8-23Sph, Tri, Pen, Hex-29.3[36]Elaeis guineensisTEM35-75Sph, Tri, Pen-17[57]Eucommia ulmoidesTEM16.4Sph-21.3[21]Genipa americanaTEM15-40Sph—[18]Hibiscus sabdariffaTEM10-60Sph—[58]Ipomoea carneaTEM3-100Sph, Tri, Pen, Hex, R—[16]Nepenthes khasianaSEM50-80Tri, Sph—[59]Nerium oleanderHRTEM2-10Sph—[60]Olea europaeaTEM50-100Tri, hex, Sph—[39]Justicia adhatodaSEM13-57Sph—[61]Sansevieria roxburghianaTEM5-31Sph, Tri, Hex, Dec, R—[62]Note: Dec: decahedral; Hex: hexagonal; Pen: pentagonal; R: rod; Sph: spherical; Tri: triangular; ZP: zeta potential.


## 3.2.1. Visualizing the Formation of AuNPs and UV-Vis Spectrum Analysis

The ability of the aqueousC. buchanani extracts to produce AuNPs was firstly monitored by the naked eye and UV-Vis absorption spectroscopy. After adding a gold solution to the C. buchanani extract, the color of the solution changed from light yellow to ruby red while no change in color was observed in the absence of HAuCl4 (Figure 3, inset). The appearance of the characteristic ruby red color indicates the reduction of Au3+ ions to gold nanoparticles. This visual observation could be attributed to the excitation of surface plasmon vibrations. Hence, it was used as a spectroscopic signature to show the formation of AuNPs. In general, the characteristic surface plasmon band from 500 to 550 nm indicates the spherical shape of AuNPs [45].Figure 3
UV-Vis spectra of the green synthesized AuNPs using theC. buchanani extract. Inset: a digital image of the (a) C. buchanani extract solution and (b) AuNPs.To retrieve information on the optical properties of the green synthesized AuNPs, the SPR was monitored by UV-Vis spectroscopy. The UV-Vis spectra of green synthesized AuNPs in which the SPR band was located at 530 nm confirmed the formation of spherical AuNPs in solution, as shown in Figure3. This clearly demonstrates that the C. buchanani extract can perform as an excellent reducing and stabilizing agent for AuNP synthesis. Phytochemicals present in the C. buchanani extract such as flavonoid, alkaloids, saponins, and tannins are believed to play a key role in redox reactions by forming quinones and subsequently releasing electrons which reduce the number of gold ions (Au3+ to Au0) and stabilize the AuNPs [46].


## 3.2.2. TEM Analysis and Zeta Potential

The morphology of the greenly synthesized AuNPs using theC. buchanani extract was examined using a TEM. Figure 4 shows that the dispersed AuNPs were spherical in shape with an average diameter of around 11.1±1.3 nm. The zeta potential of the AuNPs was found to be -30.28 mV, indicating high stability, good colloidal nature, and high dispersibility due to negative-negative repulsion [47].Figure 4
TEM image of the green synthesized AuNPs.


## 3.2.3. XRD and EDS Analysis

The crystalline characteristic of the synthesized AuNPs was determined by XRD analysis. Figure5(a) shows the XRD pattern of AuNPs at 2θ ranging between 10° and 80°. Four characteristic diffraction peaks can be seen at two theta values of 38.02°, 44.9°, 65.26°, and 78.3°, which correspond to the (111), (200), (220), and (311) planes of the face-centered cubic crystalline lattice of AuNPs, respectively. These peaks collaborated with the Joint Committee on Powder Diffraction Standard, JCPDS no. 04-0784 [48]. The peak corresponding to the (111) plane is more intense than the other planes, which indicates that (111) is the predominant orientation. The XRD spectrum strongly confirms the high crystalline nature of the prepared AuNPs. The elemental composition of the AuNPs was measured by EDS analysis. The presence of strong signals around 2 keV, as shown in Figure 5(b), confirmed the presence of metallic Au. The presence of C and O is due to the elemental composition of the C. buchanani extract [49].Figure 5
(a) XRD pattern and (b) EDS analysis of the synthesized AuNPs.
(a)(b)


## 3.2.4. FTIR Analysis

FTIR measurements were carried out to identify the potential functional groups of the biomolecules in theC. buchanani extract, which can be involved in the reduction of Au3+ and capping/stabilization of AuNPs. The major active components present in C. buchanani are known to be tannins, alkaloids, saponins, and flavonoids [28]. Figure 6 shows the FTIR spectra of the extract and the synthesized AuNPs. For the C. buchanani spectrum, the bands observed at 3419 and 2926 cm-1 are attributed to O-H and C-H stretching modes. The peak found at 1625 cm-1 is assigned to C=C and C=O stretching of the phytoconstituents. The strong absorption band at 1032 cm-1 corresponds to the vibration of the -C-O group of constituents. These observations indicate the presence of polyphenol in the extract. For FTIR spectra of the AuNPs, the characteristic absorption bands were found to be quite similar to those of the extract, but the vibration of the O-H group and the C=O group shifted from 3419 cm-1 to 3406 cm-1 and 1625 cm-1 to 1599 cm-1, respectively. The small shifting and decreasing in intensities imply that the biomolecules in the extract, especially polyphenol, may be involved in facilitating the AuNPs, mainly through their oxygen functionalities. The results are in good agreement with those obtained by [50, 51]. All characterization results confirm that the aqueous extract of C. buchanani can be effectively used in the biosynthesis of the AuNPs.Figure 6
FTIR spectra of the synthesized AuNPs.


## 3.2.5. Comparison of the Properties of AuNPs Prepared withC. buchanani Extract with Select Previous Literature

The uses of various plant extracts for the green synthesis of AuNPs have been previously investigated in published studies, and the properties of the obtained AuNPs are listed in Table1. As can be seen, different sizes and shapes of the prepared AuNPs are reported. The features of the produced AuNPs are dependent on the plant extracts and the reaction conditions. According to a careful examination of the synthesis conditions in this work, well-dispersed, small-sized AuNPs that had a uniform spherical shape were produced.Table 1
A partial list of the properties of AuNPs synthesized by plant extracts reported in the select literature.
PlantMeasurement techniqueSize (nm)ShapeZP (mV)RefAbutilon indicumTEM1-20Sph-25[52]Aegle marmelosTEM18Sph—[53]Eugenia jambolana28Soursop16Bacopa monnieriTEM3-45Sph—[54]Capsicum annuum var. grossumTEM6-37Sph, Tri, Hex—[40]Corchorus olitoriusTEM27-35Sph—[43]Cornus masTEM19Sph-33[47]Couroupita guianensis Aubl.TEM25±6Sph, Tri, Hex—[55]C. buchananiTEM11Sph-30.3This workCroton caudatus geiselTEM20-50Sph—[56]Dalbergia coromandelianaHRTEM10.5Sph—[19]Dracocephalum kotschyiTEM8-23Sph, Tri, Pen, Hex-29.3[36]Elaeis guineensisTEM35-75Sph, Tri, Pen-17[57]Eucommia ulmoidesTEM16.4Sph-21.3[21]Genipa americanaTEM15-40Sph—[18]Hibiscus sabdariffaTEM10-60Sph—[58]Ipomoea carneaTEM3-100Sph, Tri, Pen, Hex, R—[16]Nepenthes khasianaSEM50-80Tri, Sph—[59]Nerium oleanderHRTEM2-10Sph—[60]Olea europaeaTEM50-100Tri, hex, Sph—[39]Justicia adhatodaSEM13-57Sph—[61]Sansevieria roxburghianaTEM5-31Sph, Tri, Hex, Dec, R—[62]Note: Dec: decahedral; Hex: hexagonal; Pen: pentagonal; R: rod; Sph: spherical; Tri: triangular; ZP: zeta potential.


## 3.3. Applications

### 3.3.1. Catalytic Degradation of Methylene Blue

To evaluate the catalytic activity of the AuNPs synthesized by theC. buchanani extract, the reduction reaction of methylene blue (MB) dye by NaBH4 was carried out as a model system. The experiment was conducted at room temperature, and the reactions were monitored by UV-visible spectroscopy. Typically, an aqueous solution of MB imparting a blue color shows two characteristic absorption peaks at 664 nm with a shoulder at 612 nm [63]. The prominent band at 664 nm is assigned to the n→π∗ transitions and a shoulder band at 612 nm is attributed to the dimer (MB)2 due to its dimerization in aqueous medium [64]. When the prepared AuNPs were added to the reaction mixture containing MB dye and NaBH4, the color of the dye gradually altered over time from deep blue to light blue before finally disappearing. The lack of color in the MB solution was due to the reduction of the MB to leucomethylene blue [65]. In the absence of catalyst AuNPs, no degradation of MB could be seen, although after 90 min of reaction between MB with a strong reducing agent, NaBH4, it could be suggested that MB had not reduced effectively by NaBH4 and reaction rate was very slow. When compared with the solution in the presence of the catalyst, the complete degradation (dye degradation at 95%) of MB was accomplished within 4 min (Figures 7(a) and 7(b)). The result proves that the C. buchanani extract-mediated AuNPs possess a significant catalytic activity.Figure 7
UV-visible spectra of reduction of MB by sodium borohydride in (a) the presence of AuNPs as the catalyst, (b) only NaBH4, and (c) linear plot of MB over the time.
(a)(b)(c)A report on the catalytic mechanism of the degradation reaction of MB by AuNPs in the NaBH4 system has been previously published [66]. Borohydride ions (BH4−) and the MB dye serve their roles as donors and an acceptor, respectively. The large redox potential difference between them kinetically hinders the reduction of dyes [67]. The AuNPs act as an electron relay and initiate shifting of the electrons from the BH4− ions (donor B2H4/BH4−) to the acceptor (acceptor LMB/MB), thus causing a reduction of the dye. BH4− ions are simultaneously adsorbed on the surface of the NPs; thus, electron transfer occurs from the BH4− ions to the dye through AuNPs [68].According to the presence of excess reducing agent, the pseudofirst-order kinetic was applied herein. The slope of the linear regression plot oflnA0/At versus reaction time, t, is the rate of degradation, which was found to be 0.3628 min-1 (Figure 7(c)). The effect of the catalyst AuNP quantity on the degradation rate was also studied. The rate constant obtained from varying the volumes of the AuNPs is given in Table 2. The result shows that the rate constant increases with an increasing amount of AuNPs, suggesting that the reduction of MB occurs on the surface of the catalyst. A comparison of the catalytic activity of the AuNPs toward the degradation of MB is presented in Table 3. Additionally, the amounts of substrates (MB), reductants (NaBH4), and catalysts (AuNPs) in each reduction of MB were carefully examined. The results obtained in our study are similar or superior to the reference presented in the last column of Table 3. The excellent catalytic efficiency of MB could be due to the small size of the synthesized AuNPs which plays an essential role in catalytic reduction of dye. Therefore, the greenly synthesized AuNPs using C. buchanani could act as a promising material for the degradation of toxic MB dye and could be useful for developing organic dye-containing wastewater treatment.Table 2
The rate constant of different amount of AuNPs.
Volume of AuNP solution (μL)Rate constant (min-1)200.1634300.2555400.3398500.3628Table 3
Comparison of the catalytic activity of green synthesized AuNPs towards the degradation of MB.
(NaBH4) (M)(MB) (mM)AuNPs (μL)AuNPs size (nm)Degradation time (min)Degradation (%)Rate constant (min-1)R2Ref.0.151100176049.620.01180.9852[62]0.0311000≤4014>900.19800.9600[24]0.050.110011895.620.07200.9786[69]0.115011494.810.36280.9953This work


### 3.3.2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

Generally, MIC and MBC values provide quantitative data on the antimicrobial efficacy of the AuNPs. The lowest AuNP concentration that inhibited visible growth of bacterial strains was defined as “MIC,” whereas the lowest concentration of the AuNP that inhibited growth of ≥99.99% of bacterial strains was defined as “MBC.. The MIC and MBC values ofC. buchanani extract mediating AuNPs were evaluated using a microbroth dilution method against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii. The results are shown in Table 4. The MIC values for all tested strains were equal to 0.209 μg/mL. The MBC values for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus were both 0.418 μg/mL while a value greater than 0.835 μg/mL was observed for Acinetobacter baumannii (2-fold higher). According to the MBC values, Gram-positive bacteria exhibited higher sensitivity to that of Gram-negative bacteria. This might be due to the presence of capsules on the cell walls of bacteria, which prevent the attachment of the AuNPs [70]. Therefore, the biosynthesized AuNPs show significant potential in terms of their antimicrobial application.Table 4
MIC and MBC values of the AuNPs against the three pathogenic strains.
Test organismsGram classMIC (μg/mL)MBC (μg/mL)Staphylococcus aureusGram positive0.2090.418Methicillin-resistantStaphylococcus aureusGram positive0.2090.418Acinetobacter baumanniiGram negative0.209>0.835


## 3.3.1. Catalytic Degradation of Methylene Blue

To evaluate the catalytic activity of the AuNPs synthesized by theC. buchanani extract, the reduction reaction of methylene blue (MB) dye by NaBH4 was carried out as a model system. The experiment was conducted at room temperature, and the reactions were monitored by UV-visible spectroscopy. Typically, an aqueous solution of MB imparting a blue color shows two characteristic absorption peaks at 664 nm with a shoulder at 612 nm [63]. The prominent band at 664 nm is assigned to the n→π∗ transitions and a shoulder band at 612 nm is attributed to the dimer (MB)2 due to its dimerization in aqueous medium [64]. When the prepared AuNPs were added to the reaction mixture containing MB dye and NaBH4, the color of the dye gradually altered over time from deep blue to light blue before finally disappearing. The lack of color in the MB solution was due to the reduction of the MB to leucomethylene blue [65]. In the absence of catalyst AuNPs, no degradation of MB could be seen, although after 90 min of reaction between MB with a strong reducing agent, NaBH4, it could be suggested that MB had not reduced effectively by NaBH4 and reaction rate was very slow. When compared with the solution in the presence of the catalyst, the complete degradation (dye degradation at 95%) of MB was accomplished within 4 min (Figures 7(a) and 7(b)). The result proves that the C. buchanani extract-mediated AuNPs possess a significant catalytic activity.Figure 7
UV-visible spectra of reduction of MB by sodium borohydride in (a) the presence of AuNPs as the catalyst, (b) only NaBH4, and (c) linear plot of MB over the time.
(a)(b)(c)A report on the catalytic mechanism of the degradation reaction of MB by AuNPs in the NaBH4 system has been previously published [66]. Borohydride ions (BH4−) and the MB dye serve their roles as donors and an acceptor, respectively. The large redox potential difference between them kinetically hinders the reduction of dyes [67]. The AuNPs act as an electron relay and initiate shifting of the electrons from the BH4− ions (donor B2H4/BH4−) to the acceptor (acceptor LMB/MB), thus causing a reduction of the dye. BH4− ions are simultaneously adsorbed on the surface of the NPs; thus, electron transfer occurs from the BH4− ions to the dye through AuNPs [68].According to the presence of excess reducing agent, the pseudofirst-order kinetic was applied herein. The slope of the linear regression plot oflnA0/At versus reaction time, t, is the rate of degradation, which was found to be 0.3628 min-1 (Figure 7(c)). The effect of the catalyst AuNP quantity on the degradation rate was also studied. The rate constant obtained from varying the volumes of the AuNPs is given in Table 2. The result shows that the rate constant increases with an increasing amount of AuNPs, suggesting that the reduction of MB occurs on the surface of the catalyst. A comparison of the catalytic activity of the AuNPs toward the degradation of MB is presented in Table 3. Additionally, the amounts of substrates (MB), reductants (NaBH4), and catalysts (AuNPs) in each reduction of MB were carefully examined. The results obtained in our study are similar or superior to the reference presented in the last column of Table 3. The excellent catalytic efficiency of MB could be due to the small size of the synthesized AuNPs which plays an essential role in catalytic reduction of dye. Therefore, the greenly synthesized AuNPs using C. buchanani could act as a promising material for the degradation of toxic MB dye and could be useful for developing organic dye-containing wastewater treatment.Table 2
The rate constant of different amount of AuNPs.
Volume of AuNP solution (μL)Rate constant (min-1)200.1634300.2555400.3398500.3628Table 3
Comparison of the catalytic activity of green synthesized AuNPs towards the degradation of MB.
(NaBH4) (M)(MB) (mM)AuNPs (μL)AuNPs size (nm)Degradation time (min)Degradation (%)Rate constant (min-1)R2Ref.0.151100176049.620.01180.9852[62]0.0311000≤4014>900.19800.9600[24]0.050.110011895.620.07200.9786[69]0.115011494.810.36280.9953This work


## 3.3.2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

Generally, MIC and MBC values provide quantitative data on the antimicrobial efficacy of the AuNPs. The lowest AuNP concentration that inhibited visible growth of bacterial strains was defined as “MIC,” whereas the lowest concentration of the AuNP that inhibited growth of ≥99.99% of bacterial strains was defined as “MBC.. The MIC and MBC values ofC. buchanani extract mediating AuNPs were evaluated using a microbroth dilution method against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii. The results are shown in Table 4. The MIC values for all tested strains were equal to 0.209 μg/mL. The MBC values for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus were both 0.418 μg/mL while a value greater than 0.835 μg/mL was observed for Acinetobacter baumannii (2-fold higher). According to the MBC values, Gram-positive bacteria exhibited higher sensitivity to that of Gram-negative bacteria. This might be due to the presence of capsules on the cell walls of bacteria, which prevent the attachment of the AuNPs [70]. Therefore, the biosynthesized AuNPs show significant potential in terms of their antimicrobial application.Table 4
MIC and MBC values of the AuNPs against the three pathogenic strains.
Test organismsGram classMIC (μg/mL)MBC (μg/mL)Staphylococcus aureusGram positive0.2090.418Methicillin-resistantStaphylococcus aureusGram positive0.2090.418Acinetobacter baumanniiGram negative0.209>0.835


## 4. Conclusions

Highly crystalline and monodispersed spherical AuNPs were successfully produced by using a rapid and straightforward green approach utilizing theC. buchanani aqueous extract. Using plant extract as reducing and stabilizing agents for synthesis offers advantages, such as the fact that plant extract is readily available, sustainable, ecofriendly, cost effective, and nonhazardous. The prepared AuNPs exhibited efficient antimicrobial activities against both Gram-positive and Gram-negative bacteria. Moreover, they were also proven as efficient catalysts with enhanced rates of reduction of MB dye. Therefore, the green synthesized AuNPs are potentially useful for environmental remediation applications.



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*Source: 1320274-2020-07-20.xml*