Biosynthesis of Silver and Gold Nanoparticles Using Aqueous Extract of Codonopsis pilosula Roots for Antibacterial and Catalytic Applications

Faculty of Chemical Engineering, Industrial University of Ho Chi Minh City, Ho Chi Minh City, Vietnam Institute of Chemical Technology, Vietnam Academy of Science and Technology, Ho Chi Minh City, Vietnam Institute of Biotechnology and Food Technology, Industrial university of Ho Chi Minh City, Ho Chi Minh City, Vietnam Faculty of Electrical Engineering Technology, Industrial University of Ho Chi Minh City, Ho Chi Minh City, Vietnam Center for Advanced Chemistry, Institute of Research and Development, Duy Tan University, Da Nang City 550000, Vietnam The Faculty of Environmental and Chemical Engineering, Duy Tan University, Da Nang City 550000, Vietnam


Introduction
In recent years, there has been a growing interest in the synthesis of metal nanoparticles (MNPs) such as silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) due to their useful properties for applications in different areas of medicine, biology, catalysis, and antibacterial [1][2][3][4]. Along with the rapid development of nanotechnology, several promising approaches were utilized to synthesize AgNPs and AuNPs [5][6][7]. Owing to the simplicity and rapid operation, chemical reduction methods using commercial chemicals such as hydrazine [8], sodium borohydride [9], ascorbic acid [10], and ethylene glycol [11] are widely applied to convert gold and silver ions into metals at nanoscale. However, these methods have a major drawback related to the toxicity originated from the excess amount of the used chemicals that can affect the quality of nanoproducts. Recently, green approaches have been preferred to use. Among them, the way of using extracts of plants as reducing agents is quite popular, especially in developing countries, with several advantages such as high efficiency and potential for practical applications [12][13][14]. As reported in literatures [15][16][17][18], different parts of plants such as leaves, stems, roots, tubers, and flowers that contain a high amount of bioactive molecules with water-soluble polyol components responsible in reduction and stabilization of biogenic AgNPs and AuNPs were deployed already for the synthesis.
Codonopsis pilosula (Franch.) Nannf. (CP) belonging to Campanulaceae family is a perennial herb grows naturally in mountains of Vietnam, China, and India with bellflower and wire stem. Commonly, the extracts of CP roots were used as a traditional medicine for health promotion, prevention, and treatment of many diseases [25]. The main chemical constituents of CP roots include phytosteroids, sesquiterpenes, triterpenes, alkaloids, alkyl alcohol glycosides, phenylpropanoid glycosides, polyacetylene glycosides, and polysaccharides that could be an excellent source for synthesizing AgNPs and AuNPs [26,27].
In this study, the aqueous extract of CP roots was used as a reducing and stabilizing agent simultaneously for the biosynthesis of AgNPs and AuNPs. The biosynthesized MNPs were studied for antimicrobial activity toward four bacterial strains including two Gram-positive bacteria (B. subtilis and S. aureus) and two Gram-negative bacteria (Salmonella Enteritidis (S. Enteritidis) and E. Coli), and for catalytic degradation of 1,4-DNB, 2-NP, 3-NP, and 4NP in aqueous medium.

Preparation of CP Aqueous
Extract. The dried CP roots were finely ground up powder using an electronic blender. The CP root powder (5 g) was boiled in distilled water (300 mL) with reflux for 1 h. The obtained mixture was filtered with Whatman filter paper No.1 to remove the solid, and the extract was stored in a refrigerator at 4-10°C for further experiments.
2.3. Synthesis of Biogenic AuNPs and AgNPs. Synthesis of biogenic AuNPs and AgNPs was performed with HAuCl 4 , AgNO 3 solutions, and aqueous CP extract. Briefly, 10 mL of CP extract was mixed with 10 mL of metallic ion solutions under vigorous stirring in the dark. The change in the solution color after reactions complete acts as a visual sign for the success of the synthesis process. Factors that affect the synthesis process, such as concentration (0.5-2 mmol/L), temperature (60-100°C), and time (15-180 min) were also investigated to determine the optimal conditions using UV-Vis measurements on an Evolution 300 UV-Vis spectrophotometer with characterized maximum absorption peaks of AgNPs and AuNPs at around 420 and 540 nm, respectively. The obtained MNPs under optimal conditions were centrifuged, dried, and then used to study their physico-chemical characteristics and antimicrobial and catalytic activities. The procedure for the biosynthesis of CP-AgNPs and CP-AuNPs with their applications can be illustrated in Figure 1.

Characterization of Biogenic AgNPs and AuNPs
Nanoparticles. Fourier transform infrared spectroscopy (FTIR) in the range of 4000-500 cm -1 on a Bruker Tensor 27 (Germany) was applied to detect covalent bonds of possible functional groups presented in the dried CP extract and powdered MNPs. Powder X-ray diffraction (XRD) analysis on a Shimadzu 6100 X-ray diffractometer (Japan) operating at the voltage of 40 kV, the current of 30 mA with CuKα radiation at the wavelength of 1.5406 nm, scanning speed of 0.05°/s and step size of 0.02 o in the range 2θ from 10°to 80°w as used to determine the crystalline structure and composition of MNPs. The morphology of the MNPs in colloidal solution was determined by transmission electron microscope (TEM) and high-resolution transmission electron microscope (HR-TEM) on a JEOL JEM-2100 (Japan) at an accelerated voltage of 120 and 200 kV, respectively. The selected area electron diffraction pattern (SAED) of the nanoparticles was also recorded. Morphology of biosynthesized MNPs in the aggregation form after centrifugation was also examined by field-emission scanning electron microscopy (FE-SEM) on a Hitachi S-4800 HI-9057-0006 (Japan) at an accelerating voltage of 10 kV. A Horiba EMAX Energy EX-400 analyzer (Japan) was used to perform energy dispersive X-ray spectroscopy (EDX) for the determination of the chemical elemental composition of the powder nanoparticles. Finally, the dynamic diameter of MNPs in colloidal solution was examined by a Horiba SZ-100 (Japan) using dynamic light scattering (DLS) technology. 2 Journal of Nanomaterials 2.5. Antibacterial Activity. Antibacterial performance of the CP-AgNPs and CP-AuNPs in the form of optimized stable colloidal solution was studied by the agar disk diffusion method. The standard antibiotic ampicillin (0.1 μg/mL) was used as a positive control, while the aqueous CP-extract was used as a negative control. The used concentrations of MNPs were of 80, 40, 20, 10, 5 ppm for CP-AgNPs, and 120, 60, 30, 15, 7.5 ppm for CP-AuNPs. In the study, four bacterial strains including two Gram-positive bacteria (B. subtilis and S. aureus) and two Gram-negative bacteria (S. Enteritidis and E. Coli) were applied to evaluate the antibacterial activity of the biosynthesized CP-AgNPs and CP-AuNPs. Briefly, aliquots (50 μL) of CP-AgNPs and CP-AuNPs suspension were put into 6 mm-diameter paper disks on Petri plates with the bacterial culture of brainheart infusion (100 μL, 10 6 CFU/mL) by Mueller Hinton agar. The plates were kept at 37°C for 24 h, and the antibacterial activity was determined via the inhibition zone diameter of tested bacteria.
2.6. Catalytic Activity of CP-AgNPs and CP-AuNPs. It has been reported that AgNPs and AuNPs exhibit strong catalytic activity for the complete hydrogenation of toxic nitrophenolic compounds to unharmful substances of respective aminophenols [28,29]. The catalytic performance of CP-AgNPs and CP-AuNPs was investigated via reduction reaction of contaminated organic substances (1,4-DNB, 2-NP, 3-NP, and 4-NP) using NaBH 4 solution as reducing agent in a cuvette at room temperature. The pollutants (2.5 mL of 0.1 mmol/L) was mixed with the excess amount of NaBH 4 (0.5 mL of 0.1 mol/L) and CP-AgNPs and CP-AuNPs (3 mg) added then. After the reaction completed, MNPs were recovered by centrifugation, washed thoroughly with ethanol for reuse. The catalytic performance and kinetics were evaluated using UV-Vis spectroscopy at the wavelength of 380, 410, 390, and 400 nm for 2-NP, 3-NP, 4-NP, and 1,4-DNB, respectively. In the context, the amount of NaBH 4 was used far beyond the concentration of pollutants, so the concentra-tion of NaBH 4 could be considered as a constant during the reaction. In this regard, the reaction could be pseudo-firstorder one which the kinetics described by the linear equation ln ðAtÞ = −kt + ln ðAoÞ [30], where k is the rate constant, t is the reaction time, At and Ao are the concentrations of pollutants at the time t and initial concentration, respectively. The rate constant k is determined from the slope of the straight line using linear regression of ln ðAtÞ over the reaction time t. All catalytic and antibacterial experiments were performed in triplicate to confirm the reproducibility of the results and data are presented as mean and standard deviation.

Optimization of CP-AgNPs and CP-AuNPs Synthesis.
The optimization procedure is extremely necessary to ensure the stability of any preparation process in general, as well as the quality of MNPs obtained under optimum conditions using aqueous extract of plants as reducing and capping agents in particular [31]. In this study, the significant synthesis conditions including the concentration of metal ions, reaction temperature, and reaction time were optimized through UV-Vis measurements based on the surface plasmon resonance phenomenon in MNPs [32]. The optimum concentrations of metal ions were investigated by adjusting the respective concentrations of the HAuCl 4 and AgNO 3 solutions in the range of 0.5-1.5 mmol/L for Au 3+ and 0.5-2 mmol/L for Ag + , while the reaction temperature and reaction time were kept constant at 80°C and 60 min, respectively (Figures 2(a) and 2(a')). The results show that the concentration of metal ions strongly affects the formation of MNPs, avoiding the use of the excess amount of expensive precious metals. In fact, in small concentration range, the increase in metal ion concentration led to the higher UV-Vis absorbance. In the high concentration range, MNPs have been partially coagulated, resulting in a decrease in its UV-Vis absorbance. It has been found that the    Journal of Nanomaterials appropriate concentrations of HAuCl 4 and AgNO 3 necessary to form stable CP-AuNPs and CP-AgNPs with maximum yield are 1.25 mmol/L and 1.5 mmol/L, respectively. To find the suitable time for the formation of MNPs, the synthesis mixture (2.5 mL) was taken out to perform UV-Vis measurement for every 15 min toward CP-AuNPs and every 20 min toward CP-AgNPs while fixing the two other parameters (80°C, 1.25 mmol/L of Au 3+ , and 1.5 mmol/L of Ag + ). As seen in Figures 2(b) and 2(b'), the reaction time plays an important role in the formation of CP-AgNPs but has less influence on the CP-AuNPs during the survey period due to the superiority in reducing the ability of Au 3+ (E o Au 3+ /Au = +1:5 V) as compared to Ag + ions (E o Ag + /Ag = +0:799 V) [33]. For CP-AuNPs, in the first stage (0-45 min), the longer the reaction time, the higher the maximum UV-Vis absorption was observed. In the case of reaction time longer than 45 min, the decrease in UV-Vis absorbance and the slight shift of maximum wavelength toward larger values indicated the formation of larger size CP-AuNPs. Therefore, 45 min was chosen as the optimum reaction time for the synthesis of CP-AuNPs. For CP-AgNPs, with the reaction time of 40 to 120 min, the maximum UV-Vis absorbance slightly increased. However, it reached stable values after 120 min. Therefore, it can be concluded that the reaction time of 120 min is the best choice to perform the synthesis of CP-AgNPs.
Finally, the optimum reaction temperature was inves-  At higher temperatures, the ions could move faster, and the number of effective collisions might increase rapidly, resulting in partial coagulation of newly formed nanoparticles with the larger size, causing a decrease in optical density. Therefore, the optimal temperatures of 90°C and 80°C were chosen for CP-AuNPs CP-AgNPs, respectively.   Journal of Nanomaterials 3.2. Characterizations of CP-AgNPs and CP-AuNPs. The features of crystalline structures of CP-AgNPs and CP-AuNPs were determined by XRD patterns as presented in Figure 3(a  1540 nm) is the wavelength of used CuKα X-ray radiation and "θ" (degree) is the Bragg diffraction angle. Accordingly, the average crystal size of CP-AgNPs and CP-AuNPs is 16 ± 0:8 nm and 19:8 ± 1:2 nm, respectively. The FTIR spectra of CP-AgNPs, CP-AuNPs, and dried CP extract as presented in Figure 3   Journal of Nanomaterials [37,38]. Therein, the water-soluble polyol compounds are mainly responsible for the reduction and stabilization of biosynthesized CP-AgNPs and CP-AuNPs [16] [17]. In addition, the absorption bands at 2928, 1396, and 1026 cm -1 characterized for -CH, -NH, and -CN groups, respectively, were also observed in the FTIR spectra of all samples [39].
The sharp peaks at 1596 and 1701 cm -1 are assigned to C=C and C=O groups in aromatic compounds [23]. Consequently, the FTIR spectra indicate that the organic constituents of the CP extract acted as an effective reducing agent and stabilizer for CP-AgNPs and CP-AuNPs nanoparticles. The surface morphology and element composition of biosynthesized MNPs after undergoing coagulation were expressed by SEM images and EDX analysis, respectively ( Figure 4). In this work, SEM microscopy was applied for dried solid CP-AgNPs and CP-AuNPs samples. For this purpose, colloidal stable CP-AgNPs and CP-AuNPs solutions were centrifuged to separate the solids. As presented in SEM images of CP-AuNPs (Figure 4(a)) and CP-AgNPs (Figure 4(b)), both obtained MNPs are spherical in shape and fairly uniform in size. In powdered form, that may be a part of nanoparticles was agglomerated because after the centrifugation at high speed, the organic layer acting as a capping agent could be separated from the metal cores, leading to partial agglomeration. In the EDX spectrum for CP-AuNPs (Figure 4(c)), the strong peaks appeared at 1.65, 2.2, (c)  [40]. The high content of silver element was certified by characteristic signals at 2.65, 2.85, and 2.95 keV (Figure 4(d)). It should be noted that the average content of gold (29.78 w%) is much lower than that of the silver element (65.57 w%); meanwhile, the average total content of carbon and oxygen in CP-AuNPs (66 w%) is superior to that of CP-AgNPs (21.01 w%). This can be understood because CP-AgNPs contained more capping agents of organic molecules in CP-extract than CP-AuNPs.
The particle size, shape, and distribution of MNPs were evaluated by TEM, SAED, and DLS measurements, and the obtained results are presented in Figure 5. It can be seen from Figures 5(a) and 5(a'), the CP-AgNPs and CP-AuNPs crystals were mostly formed in uniform spheres dispersed well in colloidal solution with the respective average sizes of about 10 ± 2:5 nm and 20 ± 3:2 nm determined by Debye-Scherrer equation. The crystalline nature of CP-AuNPs and CP-AgNPs can be visually observed by HR-TEM and SEAD images (Figures 5(b) and 5(b')). The lattice fringe of CP-AuNPs and CP-AgNPs corresponding to the (111) plane had d-spacing of 0.24 and 0.22 nm, respectively. The bright circular rings in SAED images related to (111), (200), (220), and (311) lattice planes also indicated the existence of MNPs in cubic crystal form. Furthermore, the particle size distribution diagrams of CP-AuNPs and CP-AgNPs (Figures 5(c) and 5(c')) indicated dynamic diameters of 89 ± 4:5 nm and 112 ± 5:8 nm, respectively. The difference in particle size between TEM and DLS measurements confirmed that CP-AuNPs and CP-AgNPs in colloidal solutions were covered with a thick layer of organic molecules acted as a stabilizing agent. In fact, both CP-AgNPs and CP-AuNPs samples in colloidal form were stable for more than 3 weeks under normal room conditions at 25°C. The thickness of this organic matter layer covering CP-AgNPs sample is larger than that of CP-AuNPs sample. This is confirmed by the difference in EDX results of total content of carbon and oxygen presented in the two MNPs.  Figures 6 and 7, respectively. It has been found that CP-AuNPs did not exhibit bioactivity against any bacterial strain even at the highest concentration (120 ppm), while the colloidal sample of CP-AgNPs possessed a good antibacterial activity against three tested bacterial strains. The observed nonantibacterial activity of CP-AuNPs might be due to its minimum concentration required for antibacterial performance is still higher than the optimum. In addition, the surface area, size, and shape of MNPs can also affect bacterial inhibition [22,41,42]. For CP-AgNPs, a high antibacterial activity against three bacteria strains including B. subtilis, S. aureus, and E. coli was observed; however, it did not inhibit S. enteritidis at tested  Journal of Nanomaterials  concentrations. Moreover, the antibacterial activity increased with an increase in the concentration of the biosynthesized CP-AgNPs. The comparison of antibacterial activity for the CP-AgNPs with those reported in previous works are listed in Table 1. It is evident that the antibacterial activity of CP-AgNPs depends not only on AgNPs size but also strongly on the stabilizing agents.

Catalytic Performance for Reduction of Nitrophenols.
Resistant substituted phenols, especially nitrophenols, are widely used in chemical industries for many applications. However, they can cause a serious threat to the aquatic animals even at low concentrations due to high toxicity and difficulty in degradation [46]. In this work, the catalytic performance of CP-AgNPs and CP-AuNPs was evaluated by degradation of 2-NP, 3-NP, 4-NP, and 1,4-DNB using NaBH 4 as reductant. It is well known that the reduction of those organic substances by NaBH 4 without a catalyst is a thermodynamically favorable reaction but kinetically unfavorable due to the kinetic barrier between the BH 4 and nitrophenolate ions [14]. This barrier can be quickly overcome by using AgNPs and AuNPs via an electron transfer mechanism [28]. The results for the reduction of 1,4-DNB, 2-NP, 3-NP, and 4-NP by NaBH 4 in the presence of the biosynthesized CP-AgNPs and CP-AuNPs are shown in Figures 8-11, respectively.

Catalytic Activity of CP-AgNPs and CP-AuNPs for
Reduction of 1,4-DNB. After adding NaBH 4 to the yellowish 1,4-DNB solution, the color of the solution turns into dark red, and the maximum absorbance peak at 380 nm appeared regardless of that the decomposition reaction has not started 12 Journal of Nanomaterials yet. As soon as CP-AgNPs and CP-AuNPs were added, the color of the solution gradually disappears. Figure 8 shows the UV-Vis spectra and first-order kinetics for the degradation of 1,4-DNB by NaBH 4 in the presence of CP-AuNPs and CP-AgNPs. A gradual decrease in UV-Vis maximum absorbance at the wavelength of 380 nm and a simultaneous increase in peak at 300 nm demonstrated the occurrence of 1,4-DNB decomposition process with the formation of 1,4-diaminobenzene. The results indicated that the 1,4-diaminobenzene decomposition in the presence of CP-AgNPs and CP-AuNPs was completed within 15 minutes as evidenced by near-zero absorbance at 380 nm (Figures 8(a) and 8(b)). The linear relationship can be observed from the equation ln ðAtÞ over the reaction time (Figures 8(b) and 8(d)), wherein the first-order reaction rate constants k were determined as ð2:58 ± 0:15Þ × 10 − 3 sec − 1 for CP-AuNPs and ð2:34 ± 0:18Þ × 10 − 3 sec − 1 for CP-AgNPs.

Catalytic Activity of CP-AgNPs and CP-AuNPs for
Reduction of 2-NP. Quite similarly as above, the color of 2-NP solution was changed from light to dark yellow after the addition of NaBH 4 due to the formation of 2nitrophenolate ions in a slightly alkaline environment without reaction. UV-Vis spectroscopy (Figures 9(a) and 9(b)) shows a gradual decrease in absorbance at 410 nm and a simultaneous increase in a new peak at 290 nm, suggesting that 2-NP was reduced to 2-aminophenol. The reduction reaction was also completed within 15 min with the reaction rate constants k = ð2:54 ± 0:13Þ × 10 − 3 sec − 1 for

Catalytic Activity of CP-AgNPs and CP-AuNPs for
Reduction of 3-NP. For 3-NP, the color of the solution turned from pale yellow to yellow by the addition of NaBH 4 and simultaneously disappeared in the presence of CP-AgNPs and CP-AuNPs after 13 min. UV-Vis spectroscopy shows changes in UV-Vis maximum absorbance during the reaction (Figures 10(a) and 10(c)), wherein a gradual decrease in absorbance at 390 nm and a simultaneous increase in a new peak at 300 nm suggests that the decomposition of 3-NP occurred to form 3-aminophenol with the first-order reaction rate constants k = ð1:71 ± 0:075Þ × 10 − 3 sec − 1 for CP-AuNPs and k = ð4:09 ± 0:16Þ × 10 − 3 sec − 1 for CP-AgNPs (Figures 10(b) and 10(d)).

Catalytic Activity of CP-AgNPs and CP-AuNPs for
Reduction of 4-NP. For the reduction of 4-NP by NaBH 4 using MNPs as a catalyst, UV-Vis spectroscopy showed a decrease in absorbance at 400 nm characterized for the dark yellow 4-nitrophenolate solution and the simultaneous increase in the peak at 300 nm related to transparent 4aminophenol (Figures 11(a) and 11(b)). It has been found that the 4-NP reduction in the presence CP-AuNPs and CP-AgNPs completed within 14 minutes with reaction rate constant values of ð3:84 ± 0:32Þ × 10 − 3 sec − 1 and ð2:88 ± 0:34Þ × 10 − 3 sec − 1, respectively (Figures 11(c) and 11(d)). Thus, the catalytic ability of CP-AuNPs showed the best performance for the reduction of 4-NP, almost double that of 3-NP. Meanwhile, CP-AgNPs exhibited the best catalytic activity with 3-NP by 3.35 times greater than that of 2-NP. The CP-AuNPs revealed better catalytic performance than 14 Journal of Nanomaterials CP-AgNPs for all nitrophenols, while the reduction time was only slightly different. The CP-AuNPs and CP-AgNPs biosynthesized by the extract of CP also demonstrated a good catalyst performance in comparison with those of AuNPs and AgNPs prepared by the extract from different plants ( Table 2).
3.4.5. The Reusability of MNPs. The reusability of catalysts based on MNPs is especially necessary for practical applications by means of confirming their stability for long-term use and reducing the cost. In this work, the recyclable performance was tested for four reaction recycles toward the reduction of 4-NP as representative for nitrophenols. After the initial use, the MNPs were recovered by centrifugation, washed carefully with ethanol, and applied for reuse according to the mentioned above procedure. In the first reuse for both CP-AuNPs and CP-AgNPs, the UV-Vis absorbance peak at 400 nm corresponded to yellow 4-NP disappeared in about 20 minutes and a peak at 300 nm increased over time, indicating the formation of colorless 4-aminophenol. For the subsequent reuses, the same results were also observed. However, the reduction of 4-NP lasted longer, about 30 and 35 min, respectively. The recycling process confirmed a good recyclability of biosynthesized MNPs in the reduction of 4-NP after 4 successive recycles with the yield greater than 96% for CP-AuNPs and 95% for CP-AuNPs ( Figure 12).

Conclusions
In this study, the aqueous extract from Codonopsis pilosula roots was successfully used as both reducing and stabilizing agents for the synthesis of AgNPs and AuNPs. The obtained biosynthesized MNPs were formed in a spherical 15 Journal of Nanomaterials   Journal of Nanomaterials shape with an average size of about 10-20 nm. The colloidal CP-AgNPs exhibited selective, strong antibacterial activity against three bacterial strains including two Gram-positive B. subtilis and S. aureus, and one Gramnegative E. coli, but there was no bacterial activity detected toward Gram-negative S. enteritidis at all tested concentrations. The biogenic MNPs also possessed a high catalytic activity in degradation of 1,4-DNB, 2-NP, 3-NP, and 4-NP. Therefore, the novel CP-AgNPs and CP-AuNPs synthesized by the aqueous extract of Codonopsis pilosula roots can be considered as perspective nanomaterials for large-scale biological and catalytic applications.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no conflicts of interest.