Green Synthesis of Gold and Silver Nanoparticles Using Opuntia dillenii Aqueous Extracts: Characterization and Their Antimicrobial Assessment

Department of Chemistry, University of Swabi, Anbar, Swabi, KPK, Pakistan Department of Medical Laboratory Technology, College of Applied Medical Sciences, Taibah University, P.O. Box 344, Al-Medinah Al-Monawara 41411, Saudi Arabia Department of Rasa Shastra and Bhaishajya Kalpana, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005 Uttar Pradesh, India Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University, Buraydah, Saudi Arabia Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah, Saudi Arabia Department of Pharmacy, Faculty of Allied Health Sciences, Daffodil International University, Dhaka 1207, Bangladesh


Introduction
The past decades have witnessed rising importance of nanotechnology in medicine and healthcare [1][2][3]. Keeping this in view, the green synthesis methods which are having abilities of producing the environment friendly nanoparticles NPs are being adapted by the various field experts [4]. Therefore, the importance is being given to such technologies which are not only environment friendly but also have the wide range of the nutrients. The NPs are crystallographic with high surface area and they are very small sized structures having high reactivity [5]. So, the use of green technology is now becoming more popular than ever before.
The crops at subtropical areas are difficult to grow because of the lack of water and salinity in soil. The natural beauty of the subtropical areas may be enhanced by using the species of the Opuntia which may be grown in the subtropical areas. Opuntia is a genus which is medically important as well and it is required to be explored in term of the medical characteristics. Opuntia specie was first grown in America as there are many subtropical areas in America where this Opuntia specie may be easily grown. It is mostly found in dry and scarce water condition as it is a xerophytic plant. Specific species including O. dillenii belongs to the cactus family and is commonly called as prickly pear plant, with 1500 known species across the globe [6]. Hence, the scholars emphasized on cultivation of the O. dillenii in the tropical and subtropical areas for the purpose of animal feeding and medical implications [7]. In the zones of South Africa, Egypt, and South America, its fruit is also used for feeding the animals [8]. Similar types of the researches related to the O. dillenii proved that it may be used in the production of the nectar, sweeteners, jellies, jams and beverages of various types [9,10]. O. dillenii is also grown in the sandy areas of Egypt as it has high drought resistance quality is widely used source of animal food and also act as air resistance during the storm [11]. O. dillenii has been used as a folk medicine to treat health disorders in several countries. This botanical is also used to prepare several cosmetic products like creams, lotions, and shampoos. In food industry it is being used to prepare as wines, jams [12]. Researchers have proved that many phytochemicals are present in different parts of cactus plant. O. dillenii have the potential against diverse environmental conditions [13]. The alcoholic extract of O. dillenii has antibacterial, anti-inflammatory, hypoglycemic activities. It has been used as anti-diabetic agent for the treatment of diabetes due to its hypoglycemic property [14]. The scientists are researching on the phytochemical as well as other pharmacological actions of this botanical in order to explore the use of this plant for prevention, management and treatment of many dreadful diseases [8]. Different plants were utilized for the synthesis of NPs including Aloe vera [15], Cicer arietinum [16], Cymbopogon citratus [3], and Argemone mexicana [17].
Plant crude extracts and their phytoconstituents with proven biocidal properties in therapeutic medicine are extremely important. In more recent years, various surveys have been conducted in various countries to demonstrate this efficacy. The secondary metabolites produced by the plants are thought to be responsible for the plants' antibacterial properties. As a result, these plants are commonly employed for therapeutic purposes. The antimicrobial ability of plant bioactive substances has been proven through phytochemical screening that vascular plants could be a source of unique antimicrobial properties [18]. Phytochemicals are preferable to manufactured biomolecules because they have no or low toxicity in humans [19]. This feature makes them ideal candidates for drug manufacture and development [20]. The discovery of novel antifungal medicines relies heavily on phytochemicals without endangering human health [21]. The present study is aimed at developing gold (Au) and silver (AgNPs) from the aqueous extract of O. dillenii and at exploring their antimicrobial potential. The powder was soaked in organic solvent methanol for a week and then filtered. The filtrate was concentrated in rotatory evaporator. The crude methanolic extract was suspended in water and successively partitioned with n-hexane and chloroform, ethyl acetate and methanol.

Extract Preparation.
The fine powder (1 kg) of the plant material was divided into three parts 550 gm, 350 gm, and 100 gm. About 550 gm of plant material were dipped in methanol, 350 gm in distilled water and 100 gm in n-hexane. The plant material was kept for one week in order to obtain crude extract. The obtain extract will be concentrated in rotatory evaporator to obtain crude n-hexane, methanolic and aqueous extract. Three different fractions were prepared in three different solvents to check the presence of most of the active ingredients, the active ingredients involved in capping and biological activities. As in n-hexane only nonpolar ingredients were determined and they are not involved in the capping during nanoparticle synthesis. The methanolic fraction will be applied next time for the synthesis of Ag and AuNPs, and biological activities. The plant material was stored in refrigerator for further NP synthesis.
2.4. Synthesis of NPs. 1 mM solution of silver salt (AgNO 3 ) and gold salt [HAuCl 4 ] were prepared for the synthesis of Ag and AuNPs by using plant crude extract at different ratio 1 : 1, 1 : 2, 1 : 3, 1 : 4, and 1 : 5. Then, the solution was placed on stirrer with constant stirring at room temperature for 5 hours. After that, characterization of NPs was carried out.

Pharmacological
Activities. The crude extract and synthesized NPs were evaluated for antibacterial and antifungal activities.
2.6. Chemical Used. The analytical grade chemicals were used in the synthesis of Au/AgNPs. AgNO 3 was purchased from Sigma-Aldrich, and HAuCl 4 , methanol, and deionized water were purchased from Merck.

Biosynthesis of AgNPs
Using the Aqueous Extract of O. dillenii. 100 ml of the already prepared extract was taken. 1 mM solution of AgNO 3 was prepared using distilled water. Different fractions of the preparations were prepared using Ag and extract solution in different ratios such as 1 : 1, 2 : 1, Initially the NPs were characterized by UV-visible spectrophotometer in the wavelength ranging from 200 to 800 nm. The spectroscopic analysis for both silver and gold NPs were carried out by using freshly prepared fractions at 37-38°C and by using optical path 1 cm length of quartz cuvettes using spectrometer (300 Plus Optima Japan). The AgNPs solution gave an absorption maximum at 420-450 nm while that of AuNPs was 520-530 nm.
2.9.2. Scanning Electron Microscopy (SEM). The size and morphological surface of NPs were determined using JEM 2100, Jeol CRL Scanning Electron Microscope (University of Peshawar, Pakistan). The size and shape of the AgNPs and AuNPs were determined using SEM images. By using an electron microscope, a layer of AuNPs thin sediment was placed under vacuum pressure of 5-8 Torr.

Fourier Transform Infrared (FTIR) Spectroscopy
Analysis. The functional group involved in the formation of Au and AgNPs was evaluated using Shimadzu FTIR -8400-S (AWKUM) Fourier transform spectrometer. The samples were prepared using the powdered sample. The powdered samples were placed in NaCl cells and were placed in pellet cells of KBr. The bands detected on the computer showed the results. The range of 4000-400 cm -1 was used.

Energy-Dispersive X-Ray (EDX) Spectroscopy Analysis.
Au and AgNPs geometry and morphology were also determined. Using a Bruker X-flash in energy dispersive X-ray spectroscopy the colloids of Au and AgNPs were prepared determined. For EDX and imaging 15 keV energy of the electron beam was maintained.
2.9.5. Antibacterial Activity. The MIC and MBC which are commonly known as minimum inhibitory concentration and Minimum bactericidal concentration, respectively, of AgNPs and AuNPs were also determined in the microbial activity.
The bacteria S. aureus and E. coli were used in the microbial assay. Micro dilutions of the NPs were prepared. The bacterial culture was prepared using nutrient agar and incubated for 24 hours at a temperature of 37°C. NPs dilutions were also employed at a specific area to evaluate the MIC of NPs.
The NPs containing Petri dishes were also incubated at the temperature of 37°C and for 24 hours. MBC or minimum bactericidal concentration evaluation of AuNPs and AgNPs was also determined. The lowest dose or dilutions of Au/AgNPs were used to test the area of inhibition. The process was repeated twice to get accurate results.
2.10. Antifungal Activity. The micro dilution plate assay method was used to determine fungicidal activity. The fungus species of Candida albicans was used in the assay. 20 mM buffer solution of sodium phosphate was mixed with 20 μl of both Au and AgNPs dilution 5, 2.5, 1.25 mg/ml in water. The sample was incubated at a temperature of 37°C for 2 hours.
The NP visibility loss was calculated using the following formula: 1 − colony-forming unit in the presence of NPs CFUs with no particles × 100: 2.11. Stability of NPs. The stability of Au and AgNPs were checked against varying pH, different concentration of NaCl, same concentration of different salt and heat effect. After each treatment UV-visible spectra were recorded.

Kinetic Study of Synthesis of NPs.
For the timedependent synthesis of Au and AgNPs, samples were drawn from reaction mixture at regular interval of time and UVvisible spectra were recorded.
2.13. Phytochemical Analysis. The qualitative screening for the assessment of phytochemical components like flavonoids, terpenoids, alkaloids, carbohydrate and steroids, in the methanol, n-hexane, and distilled water extracts of the plant was carried out.

Results and Discussion
3.1. Phytochemical Screening. The qualitative screening for the assessment of phytochemical components like flavonoids, polyphenols, terpenoids, alkaloids, carbohydrates, and steroids in the methanol in each extract was performed by using the following reagents ( Table 1). The results of qualitative screening for the assessment of bioactive secondary metabolites such as flavonoids, polyphenols, terpenoids, alkaloids, carbohydrates, and steroids in the methanol, n-hexane, and distilled water extracts of the title plant are given in Table 2. The methanol plant extracts of O. dillenii showed presence of steroids, coumarins, 3 Journal of Nanomaterials betacyanin, terpenoids, tannins, flavonoids while the n-hexane plant extract confirms the presence of saponins, terpenoids, flavonoids, and coumarin and the distilled water plant extracts of O. dillenii revealed saponins, terpenoids, flavonoids presence and the absence of carbohydrate, glycosides, anthraquinones, anthocyanin, emodins, and phlobatannins.

Characterization of Au and AgNPs
3.2.1. UV-Visible Spectroscopy. UV-visible spectroscopy is the technique which is considered as the colorimetry extension, it works on the principle of the absorption of light from the test sample, by using various components that are similar to the colorimeter but spectroscopy has the advantage of improved accuracy in a wide range of wavelengths between 190 and 700 nm [22]. This wavelength range was used because the absorption by the Au and Ag is usually observed in this range. In this work, we used a 300 Plus Optima Japan Spectrophotometer, with quartz cells and 1 ml deionized water as a blank. The formation of the NPs was confirmed by the formation of color visually. Scanning the absorption using wavelength ranges of 200 and 800 nm was used to confirm the formation of the Au and AgNPs. Spectra show the band wavelength of the Au and AgNPs obtained using the plant extract. The difference in the bands and the wavelengths indicate the different sizes and the shapes of the Au and AgNPs obtained ( Figure 1).
The prominent UV-visible spectrum was observed at the wavelength of 543 nm and 445 nm which confirmed the synthesis of Ag and AuNPs, respectively ( Figure 1). The UVvisible absorption spectrum of the aqueous extract O. dillenii has not shown any significant bands. But after the extract of O. dillenii with the silver nitrate colorless and chloroauric acid yellow colored solution the color of the solutions were changed to characteristic ruby-red color and reddishbrown showing the convenient excitation due to the surface Plasmon resonance phenomenon, that indicates the silver and gold NPs formation [23]. This research also observed the synthesis of Au and AgNPs within 15 to 20 minutes in the case of silver and 10 to15 minutes in the case of the synthesis of gold NPs, respectively.

Scanning Electron Microscopy (SEM)
. The analysis of the size and shape of the Au and AgNPs was determined using SEM. For this purpose, SEM images and photographs of SEM using JEM 2100, Jeol CRL, Scanning Electron Microscope. By using an electron microscope, a layer of Au and AgNPs thin sediment was placed under vacuum pressure of 5-8 Torr.
The results of the SEM analysis revealed the shape of the NPs were roughly spherical-shaped NPs and in some areas stacked together. The characterization results of the Au and AgNPs by SEM analysis also confirm the method developed was suitable and effective to obtain the silver and AuNPs of different sizes and in diverse shapes (Figure 2). The size distribution of the NPs ranges from 45 nm to 77 nm approximately and was uniformly distributed.   Absorbance (a.u) S1: 2G S1: 1G S1: 3G S1: 4G S1: 5G S1: 6G S1: 7G S1: 8G 5 Journal of Nanomaterials act as the capping or reducing agents of the Au and AgNPs. Correspondingly the results of these studies were observed compared to the study in which a similar phenomenon was observed to evaluate the biological molecules acting as the capping and reduction agents for Au and AgNPs [24].

Energy-Dispersive X-Ray (EDX) Spectroscopy Analysis.
Au and AgNPs geometry and morphology were also determined. Using a Bruker X-flash in energy dispersive X-ray spectroscopy the colloids of Au and AgNPs were prepared. For EDX and imaging 15 keV energy of the electron beam was maintained. The obtained spectra are given in Figure 4.
The synthesized Au and AgNPs were further characterized qualitatively as well as quantitatively using EDX analysis, which revealed the highest signal proportion of Au and Ag in the solutions as shown in Figure 4.
The results of the EDX analysis confirmed the major metal in the precipitate were Au and Ag, respectively. The results obtained were consistent with the outcome of the EDX analysis. The identification of Cl in the spectrum was due to the ions of salt in the sample. The energy bands for a strong signal of Au were in the range of 2 to 2.5 keV, 9.5 to 10 keV, and 10 to 11 keV and for Ag, it was observed in the range of 2.9 to 3.8 keV which were similar to the observations and evaluations of the study of Au and AgNPs [25,26].
The EDX analysis of these NPs also shows the presence of other elements in the precipitate mainly carbon, aluminum, sulfur. Oxygen was also identified in the spectrum of the precipitate, which is assumed to be associated with the metals as the oxides or hydroxide of metals. For the presence of grid composition, Si showed the band. Minor carbon bands were also observed that may be due to the biomolecules which were bound to the NPs surface.
The bands of C and O along with the bands of metal signals suggested the Au and AgNPs may be capped due to the presence of phytochemicals of the plant extract by the atoms of an oxygen atom or may be related due to the existence of consequent oxides of metals which were identified in sample precipitate [25].
3.3. Antibacterial Activity. The MIC and MBC which are commonly known as minimum inhibitory concentration and Minimum bactericidal concentration, respectively, of Au and AgNPs were also determined in the microbial assay using S. aureus, E. coli, P. aeruginosa, S. typhi, and B. subtilis. These results are obtained by comparing with standard.
The antibacterial activity of the extract of O. dillenii was in range of 11 to 14 mm against selected bacterial strains S. typhi, B. subtilis, S. aureus, P. aeruginosa, and E. coli while for AgNPs, the zone of inhibition was 11 to 16 mm and for AuNPs, it was 11 to 17 mm. The Au and AgNPs exhibited good antibacterial property against various strains of bacteria as shown in Table 3.
3.4. Antifungal Activity. The microdilution plate assay method was used to determine fungicidal activity. The fungus species of C. albicans, A. niger, and P. notatum were used in the assay. In this assay of spot plating, the antifungal activity of the O. dillenii extract, and its synthesized Au       9 Journal of Nanomaterials and AgNPs was tested in a growing medium. This assay ascertained whether the division and the growth are prerequisites for Au and AgNP antifungal activity. The results of the antifungal activity are given in Table 4.
These results of the antifungal assay confirmed that extract of O. dillenii contain only mild antifungal activity while the Au and AgNPs possess some antifungal activity. The antifungal activity exhibited by extract of O. dillenii against different fungal strains ranged between 11 and 13 mm. The antifungal activity 13 mm was found against P. notatum, followed by C:albicans ð12 mmÞ > A:niger ð11 mmÞ. The antifungal activity of AuNPs of O. dillenii against different fungal strains ranged between 19 and 22 mm. The antifungal activity 22 mm was noted against C. albicans followed by P:notatum ð21 mmÞ > A:niger ð19 mmÞ. Likewise, the antifungal activity of AgNPs of O. dillenii against different fungal strains ranged between 21 and 27 mm. The antifungal activity 27 mm was found against P. notatum followed by C:albicans ð24 mmÞ > A:niger ð21 mmÞ as shown in Table 4. of NaCl in specific volume of distilled water. 0.5 ml of all the prepared molar solution was added in 2 ml of Au and AgNP solution and was shaken well, and the effect of different NaCl concentrations on the NPs was evaluated using a UV-visible spectrophotometer and spectrum was drawn based on the absorbance values on different wavelengths ranging from 200 to 800 nm as shown in Figure 5.

Stability
The Au and AgNP solution contains Au and Ag atoms, respectively, along with negatively charged chloride ions which allow the Au and Ag atoms to be dispersed in the solution. The NPs then starts forming agglomerate and change the solution color because the NPs absorb wavelength e.g., green, orange, red, and yellow excluding shorter wavelengths of purple and blue. Then, the color of the colloids changes to bluish by absorbing shorter wavelengths: green and blue and green instead of red and orange and red. By increasing the concentration of NaCl, the solution became colorless because of the no suspended particles in the solution they started forming precipitates and aggregates at the bottom therefore no absorbance of light occurred by the solution.

Journal of Nanomaterials
The salts used in the evaluation of the effects on Au and AgNPs were NiCl 2 , CuCl 2 , HgCl 2 , CaCl 2 , ZnCl 2 , and PbCl 2 . The difference in the particle sizes as a function of the ionic strength is shown in Figure 6. As revealed from the spectrum steep gradient it is manifested that the AuNPs are highly susceptible to high salts strength as compared to the AgNPs. As the size of NPs increase it lead to formation of the aggregates in the media. The steepness in the spec-trum shows the particle aggregation caused by ionic strength. The research by NPs evaluated and compared the effects of different electrolytes and concluded that salts with the divalent cations have a great influence on NPs aggregation in comparison to the salts with monovalent cations. The chloride ions also enhanced the NPs aggregation [27]. Likewise, Badawy also analyzed AgNPs and the consequence of different monovalent and divalent cations [28]. The Au      (Figure 7). After specific intervals of time revealed that the production and the uniformity of the NPs increase with the increases in time, the slope bands in the spectrum showed the maximum production of the NPs in the solution.  13 Journal of Nanomaterials shown in Figure 8 and was confirmed by analysis by the UV-visible spectra at three different temperatures 40°C, 60°C, and 80°C.
It can be observed clearly from the spectra of UV-visible spectrophotometer that synthesis of Ag and AuNPs increases with temperature but after certain temperature, it became destabilized which caused the NPs to clump together and agglomerated. It also caused the broadening of peak which indicate the big NPs that settled down. in the formation of Au and AgNPs with an increase in the temperature [30].
3.5.5. Effects of pH on Au and AgNPs. The effect on the Au and AgNPs was also studied due to the change in the pH was studied in different conditions including the pH ranges from 1 to 14. The pH was adjusted by dropwise addition of HCl and NaOH. Figure 9 shows the visual analysis of pH on NPs, and the effect of changes in pH on UV-Vis spectra of Au and AgNPs synthesized is shown in Figure 9.
The effect of pH on stability of NPs can be observed by change in the color of NPs solution [31]. At low pH ranges small the broadening of the bands was formed which indicates the formation of large-sized NPs. In the extract mediated synthesis of NPs alkaline pH shows the narrowing of the band at 400 nm with maximum sharp band production. The formation of the sharp band indicates the formation of the spherical shape of NPs [32]. Several studies reported that pH plays a vital role in the determination of the size and shape control synthesis process of NPs. This research indicates that alkaline pH 6-7 is more suitable for the synthesis of Ag and 8-9 for AuNPs. It was reported that AuNPs at pH 10 showed maximum stability using the extract of M. charantia [33].

Conclusions
The Au and AgNPs were characterized using UV, FTIR, SEM, and EDX spectra and showed the rapid biosynthesis of NPs using O. dillenii. An increase in the awareness of utilizing green chemistry and adopting the green route for the production of metallic nanoparticles leads to the development of efficient and ecofriendly techniques. The advantages 15 Journal of Nanomaterials of the synthesis of Au and AgNPs by the use of plant extracts are being most economical, cost-effective, and energyefficient and provide an efficient application towards the communities, protecting environment and health leading towards the lessening in the production of hazardous wastes and development of safe products. Green-synthesized Au and AgNPs have various significant nanotechnology aspects with matchless applications.

Data Availability
The data produced in this finding has been included in the main text of this paper.