PHYTOSYNTHESIS AND CHARACTERIZATION OF SILVER NANOPARTICLES FROM RUELLIA TUBEROSA (L.): EFFECT OF PHYSICOCHEMICAL PARAMETERS

Objective: The current study focused on synthesizing silver nanoparticles (AgNPs) using tuberosa aqueous tuber extract and silver nitrate (AgNO 3 ) solution. Methods: AgNPs were synthesized using an aqueous tuber extract of the medicinal herb R. tuberosa (L.). The existence of significant phytoconstituents involved in synthesizing the AgNPs was determined using the gas chromatography–mass spectrometry (GC–MS) study. We evaluated the physical and chemical parameters such as the effect of time, temperature, metal ion concentration, crude aqueous tuber extract concentration, and pH in the synthesis of nanoparticles. The AgNPs were characterized using ultraviolet (UV)–Vis spectroscopy, field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDX), high-resolution transmission electron microscope (HRTEM), selected area electron diffraction (SAED), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) techniques. Results: R. tuberosa tuber extract was rich in various phytochemical constituents which were identified by GC–MS. For biosynthesis, the optimal values were 1 mM AgNO 3 concentration, 0.1 mL of aqueous tuber extract, and a 40 min incubation temperature of 70°C. The existence of a characteristic surface plasmon resonance (SPR) peak at 421 nm indicated the biosynthesis of AgNPs using UV–Vis spectroscopy. At higher temperatures and alkaline pH, the development of AgNPs increased overtime and remained stable up to 4 weeks. FESEM, EDX, HRTEM, SAED, and XRD analysis revealed that most AgNPs were spherical, with an average size distribution of 34.9 nm and a crystalline phase, face-centered cubic lattice. Infrared (FTIR) spectroscopic analysis revealed that hydroxyl and amino functional groups were involved in the biosynthesis and stabilization of AgNPs. Conclusion: The synthesis of AgNPs from R. tuberosa aqueous tuber extract was a cost-effective process and environmental friendly.


INTRODUCTION
In the field of nanobiotechnology, there had been significant advancements in recent years. Materials classified as nanoscaled structures usually had one dimension less than 100 nm. Relative surface area and size were the two main factors that influenced the chemical reactivity, electrical, optical, and thermal properties of nanoparticles [1]. Metal nanoparticles such as silver, gold, palladium, zinc, copper, and iron oxide were synthesized using a variety of physical, chemical, and biological methods [2][3][4]. A variety of metal nanoparticles were synthesized using biological agents such as plants and their pure compounds, algae, bacteria, fungi, and yeast. In the case of microorganisms, however, the intricate process of maintaining cell culture was complicated [5,6].
Ruellia tuberosa L. was a low-growing perennial herb with tuberous roots in the Acanthaceae family. R. tuberosa, a native of America, had a common name, Minnie root, popping pod. Tuber powder was given with milk to treat abdominal pain after delivery [15]. This plant's roots had been used to reduce toxicity and promote healing in cases of urinary tract inflammation [16]. The reported biological activities of the plant's tubers were antioxidant, antispermatogenic, anti-inflammatory, and antimicrobial [17][18][19][20]. Previously, there was a report on synthesis of silver and iron oxide nanoparticles from leaf extracts of R. tuberosa [20,21]. In this present study, R. tuberosa aqueous tuber extract was used to synthesize AgNPs and to study their antiproliferative activity for the 1 st time.
approximately 70 g of dry weight, was filled in the thimble and extracted successively with ethanol using a Soxhlet extractor. A rotary flash evaporator was used to concentrate the extracts, which were then stored at 4°C.
Phytochemical evaluation of both aqueous tuber extract and ethanolic extracts was done to determine the active phytoconstituents such as alkaloids, phenolics, flavonoids, steroids, terpenoids, saponins, coumarins, carbohydrates, cardiac glycosides, amino acids, and protein compounds using standard methods [22].

GC-MS analysis of R. tuberosa
A GC-MS study was used to investigate the phytochemical constituents of an ethanolic extract of R. tuberosa using using GC-MS system model (Thermo GC-TRACE ultraversion 2.2, Thermo TSQ QUANTUM XLS (Thermo Scientific Co.). The following were the GC-MS system's experimental conditions: Dimensions: 30 Mts, ID: 0.25 mm, film thickness: 0.25 μm, DB 5-MS capillary standard non-polar column. The mobile phase flow rate was set to 1.0 mL/min (carrier gas: He). In the gas chromatography section, the oven temperature was 40°C, which was gradually increased to 290°C at a 5°C/min rate, and the injection volume was 1.0 L. A scan interval of 0.5 s was used to maintain a range of 40-600 m/z. The GC took 35 min to complete. The compounds were identified by comparing their mass spectra to those of the WILEY and NIST libraries.

Synthesis of AgNPs
AgNO 3 in Milli-Q water was used as a source of silver. All the experiments were done in duplicates. Synthesis of AgNPs was carried out at 70°C in a boiling water bath by taking a standard reaction mixture contains 1 mL of R. tuberosa aqueous tuber extract in 8 mL of 1mM AgNO 3 . At room temperature, various optimization studies were carried out to determine the optimal reaction ratio (1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, and 1:14) for AgNP formation and to study their stability [23]. The golden yellow color in the reaction mixtures (1:10, 1:12, and 1:14) was taken as the primary indicator of AgNP synthesis. Even after 20 h, the ratios 1:1, 1:2, 1:4, 1:6, and 1:8 did not achieve the same level of color transition. This significant time lag showed that AgNPs need additional energy for bioreduction.

Study of the effect of physicochemical parameters on AgNPs synthesis
The extract concentration was 0.1 mL, and the AgNO 3 concentration was 0.8 mL to form reaction mixture (1:8). The effect of temperature on nanoparticle synthesis was investigated by incubating the reaction mixture at various temperatures (40°, 50°, 60°, 70°, 80°, 90°, and 100°C). The effect of metal ions was also monitored by adjusting the AgNO 3 concentration (1, 2, 3, 4, and 5 mM) with a constant volume of aqueous tuber extract. By keeping 1 mM AgNO 3 at a constant range, the effect of crude aqueous tuber extract concentration (0.1, 0.2, 0.3, 0.4, and 0.5 mL) was analyzed. The effect of pH (4-12) on nanoparticle synthesis investigated using different buffers. In addition, stability studies were carried out in the presence or absence of a stabilizing agent. Following the study, the optimum concentration was chosen for bulk nano colloid preparation. Centrifugation at 10,000 rpm for 15 min was done to extract AgNPs. The pellet was redispersed 2-3 times in Milli-Q water. Further, the characterizations were carried out on the dried nanoparticles.

Characterization of AgNPs
The biosynthesis of AgNPs was first confirmed using ultraviolet (UV)-Vis spectroscopy (SHIMADZU, UV-1800) in the range of 300-700 nm. UV-Vis spectroscopy was used to measure the characteristic optical properties of AgNPs in the colloidal reaction mixture. Field emission scanning electron microscopy (FESEM) (JOEL, JSM-7100F) was used to determine the particle morphology at a 6 kV accelerating voltage. Carbon tape was coated with AgNPs, which were then marked. Energy-dispersive X-ray spectroscopy (EDX), which was connected with FESEM, was used to determine the purity of the AgNPs. The morphology of the synthesized AgNPs was further investigated using a high-resolution transmission electron microscope (HRTEM) on a Tecnai G2, F30 HRTEM 300 kV. For HRTEM analysis, a drop coating of ultrasonically scattered AgNPs was applied to the transmission electron microscope (TEM) grid and dried under UV light. HRTEM images were analyzed with the "image J" tool to determine the average particle size distribution. An X-ray diffractometer analysis was also used to conduct X-ray diffraction (XRD) (Panalytical, X-Pert pro, and Netherland) analysis. The functional groups involved in AgNP biosynthesis and stability were screened using Fourier transform infrared (FTIR) spectroscopy in the range of 4000-400 cm -1 using Bruker (ALPHA-T) IR by KBr pelleting.

Antiproliferative activity using sulforhodamine B assay
For the present study, five human cancer cell lines (A549, HeLa, MDA-MB 231, PANC-1, and SK-OV-3) were selected. The National Centre for Cell Sciences in Pune, India, provided the cell lines. The cells were maintained in Dulbecco's modified eagle medium, minimal essential medium, and Roswell Park Memorial Institute Medium with glutamine, and were passaged using conventional trypsinization. In a 96-well plate, cells were plated at a density of about 10,000 cells per well. Cells were treated with different concentrations of RTAgNPs (10, 20, 40, 60, 80, and 100 μg/ml) in triplicates for potent compounds and incubated for 48 h. After the incubation time, cell monolayers were fixed in a 10% trichloroacetic acid solution at 4°C for 1 h, after which the cells were washed with Milli-Q water to extract the trichloroacetic acid and held for drying. Dried cell plates were dyed for 30 min, after which the excess dye was washed away with 1% acetic acid. In a 10 mM tris base solution, the protein bound dye was dissolved. In a Varioskan flash Multimode reader, the OD was measured at 510 nm (Thermo Scientific) [24].

Phytochemical analysis
The phytochemical evaluation of aqueous and ethanolic tuber extracts showed the presence of phenols, flavonoids, terpenoids, saponins, carbohydrates, and amino acids. Table 1 shows the results of GC-MS analysis of ethanolic tuber extract, which revealed a total 16 compounds. These compounds were identified using the NIST library and information from the literature. Some of the compounds had simple ring structures similar to those seen in the literature, but they varied in that they had different functional groups. The compounds were classified using retention time (RT), molecular structure, and peak area percentage. Fig. 1 depicts the GC-MS spectrum. Flavone, E, E, Z-1, 3, 12-nonadecatriene 5,14-diol, Phytol, and Methyl-6-octadecenoate were the major compounds in ethanolic tuber extract. The retention times (RT) with relative peak areas of the compounds were 17 (50%), 19.52 (41.8%), 18.68 (41.4%), and 18.9 min (40.2%), respectively [25][26][27][28].

Synthesis of AgNPs
The formation of AgNPs was primarily indicated by a transition in the color of the solution from light yellow to golden yellow. The presence of color in the reaction mixture was caused by mutual oscillations of electron-free conduction induced by an interacting electromagnetic field [29]. Noble metals were found to have unique optical properties due to the surface plasmon resonance (SPR), which indicates the synthesis of AgNPs by reducing silver metal ions Ag + into AgNPs Ag° by the active phytoconstituents present in the tuberosa aqueous tuber extract [30]. UV-Vis spectroscopy was used to screen the reduction process (Fig. 2a).

Effect of reaction time
The reaction time (Fig. 2b) was optimized by maintaining the reaction mixtures at room temperature and continuously observed. The samples were taken at regular intervals and analyzed using UV-Vis spectroscopy. Variations in reduction potentials were more likely to be responsible for the slower reduction rate of silver ions at room temperature. The redox potential for Ag + to Ag° was significantly smaller. After 24 h, AgNPs formed with less color intensity and fewer nanoparticles. Basing on the redox potential of plants, the time required for the completion of the reaction ranged from minutes to hours [23].

Effect of temperature
The temperature had an effect on the reduction process as well. The optimized reaction mixtures containing test tubes were kept in a water bath with an interval of 10°C rise in temperature at 40°, 50°, 60°, 70°, 80°, 90°, and 100°C. At 40-60°C, no noticeable rise in size or number of AgNPs was detected. At higher incubation temperatures (70-100°C),

Harika and Radhika
an increase in color intensity and more intense SPR peaks were revealed. The maximum SPR peak was detected at 421 nm at 70°C for 40 min [31]. Narrow peaks developed the smaller sized nanoparticles in the lower wavelength area (397 nm at 100°C) as the temperature was increased (Fig. 2c and d). When the temperature was raised, the reactants were quickly absorbed, resulting in the production of smaller nanoparticles [32,33].

Effect of metal salt concentration
At 0.1 mL of R. tuberosa aqueous tuber extract and 0.8 mL of AgNO 3 , at 70 °C for 40 min, the effect of AgNO 3 concentration on AgNPs biosynthesis was monitored in the range of 1-5 mM. The SPR peaks for AgNPs at various metal ion concentrations are shown in Fig. 2e. For the specified reaction time, the color of the reaction mixture darkened as the concentration of AgNO 3 in the solution increased. According to the previous research studies, the size of nanoparticles was closely related to the color of the reaction mixture [33,34]. The number of synthesized AgNPs per unit volume of the reaction mixture increased with the band intensity increased. As a result, 1 mM AgNO 3 was chosen as the optimum concentration for achieving smaller particle sizes and stable growth [35].

Effect of crude aqueous tuber extract concentration
The aqueous tuber extract concentration was optimized by varying the R. tuberosa aqueous tuber extract volume from 0.1 mL to 0.5 mL, while the other parameters were kept constant (temperature and concentration of AgNO 3 ). The number of nanoparticles was found to be greater in 0.2 mL of R. tuberosa aqueous tuber extract volume than in 0.1 mL. This increased nucleation at a higher reductant volume would result in a more significant number of nanoparticles and a more intense SPR band. When R. tuberosa aqueous tuber extract volumewas increased from 0.3 to 0.5 mL (Fig. 2f), the SPR band becomes more prominent and less concentrated with a blue shift in wavelength. Since there was more reductant available than free Ag + ions for the growth of stable nuclei, the reaction rate increased, resulting in a more significant number of smallsized nanoparticles [33,36].
Effect of pH pH influenced the shape, size, and stability of the AgNPs. These three different buffer solutions were used to carry out the reactions, ranging the pH from 4 to 12 were Tris HCl (7,8,9), phosphate buffer (4, 5, 6),

Harika and Radhika
and glycine-NaOH (10,11,12) [37]. There was no color transition or characteristic SPR peak in acidic conditions. When the solution becomes more alkaline, the color intensity increased from dark yellow to reddishorange. The development of AgNPs was instantaneous at alkaline pH, and an extreme SPR peak was observed at pH11 (Fig. 2g). At higher pH, alkalinity promoted AgNPs reduction and capping at various facets and the eventual accumulation of silver atoms on these facets [38]. However, because of the high concentration of hydrogen ions, the functional groups responsible for reducing the precursor to nanoparticles had positive charges at acidic pH, lowering their reducing potential.

Stability studies
Silver nanoparticles synthesized from the optimum reaction mixture (1mL of R. tuberosa aqueous tuber extract in 8 mL of 1mM AgNO 3 ) were studied for stability at 70°C for 40 min, as shown in Fig. 2h. The visual precipitation and the shift in SPR peak intensity were all taken into consideration when examining the stability of AgNPs. Even 4 weeks after their synthesis, the AgNPs were shown to be stable in solution [23]. The particles remained stable for 95 days at 4°C. Following that, AgNPs precipitation was found at the tube's bottom, attributed to AgNP overgrowth and agglomeration overtime.
To compare the stability of biologically synthesized nanoparticles, a stabilizing agent like polyvinylpyrrolidone (PVP) (0.2% and 0.5%) aqueous solution was used at pH 5. PVP concentration significantly influenced the size distribution as well as the dispersion of the resulting nanoparticles. Particles were stable for up to 45 days at room temperature (Fig. 2i), AgNPs were stable for more than 6 months at 4°C. PVP was considered an excellent dispersant as it possess favorable protected properties. At low PVP concentration, some agglomerated particles were observed. However, when the PVP concentration was increased (0.5%), well isolated with controlled size and monodispersed nanoparticles were formed. Therefore, PVP regulated bond formation between individual AgNPs and protected the particles from agglomeration by forming a layer of the stabilizing agent on the surface of nanoparticles [39].

Characterization of AgNPs FESEM analysis
FESEM was used to analyze the shape of the synthesized AgNPs. A powdered AgNPs sample was used for the analysis. An agglomeration of nanoparticles was visible in the FESEM image ( Fig. 3a and b). The nanoparticles were formed spherical and polydispersed. The purity and elemental composition of AgNPs were determined using an EDX detector connected with a FESEM. According to the energy-dispersive spectrum  Harika and Radhika many nearly spherical AgNPs ranging in size from 11.6 nm to 69.7 nm. The onset of Fig. 4c showed the selected area electron diffraction (SAED) sequence of AgNPs. The bright circular rings indicated the crystalline nature of synthesized AgNPs in the SAED pattern. A size distribution histogram defined the average size distribution of nanoparticle (34.9 nm) (Fig. 4d), which was derived from many micrographs of AgNPs corresponding to HRTEM images. Similar findings were observed in the synthesis of AgNPs mediated through mangosteen (Garcinia mangostana) leaf extract with an average size of 35 nm [36].

Fig. 5: X-ray diffractogram of Ruellia tuberosa tuber mediated silver nanoparticles
image, the nanoparticle was mainly composed of silver (Fig. 3b). In EDX spectra, a silver peak recorded at 3 keV. Apart from silver signal, spectral signals for oxygen and carbon were also observed [40]. Carbon spectra had the most prominent peak due to the carbon sheet on which the sample smear was made [41]. Gardea-Torresdey et al. used alfalfa (M. sativa) plant to form individual spherical-shaped AgNPs in the range of 2.5-4 keV [7].

HRTEM analysis
HRTEM data were used to investigate the detailed surface morphology of biosynthesized AgNPs. The HRTEM image ( Fig. 4a and b) showed  . By comparing the JCPDS values, the typical pattern of green synthesized AgNPs was found to possess a face-centered cubic (FCC) structure [9][10][11]. Additional unassigned peaks were found in addition to the Bragg peaks representative of FCC silver nanocrystals, implied that bio-organic phase crystallization occurred on the surface of the AgNPs. The average crystallite size of the synthesized AgNP was determined using the Debye Scherrer equation.
Where, D is the average particle size, k is the shape factor (constant 0.9), λ is the X-ray wavelength (1.5406A°), β is the full width at half maximum of the peak, and is the diffraction angle. The average crystallite size of the AgNP synthesized by aqueous tuber extract of R. tuberosa was arrived at 39.8 nm, which was nearer to TEM size.

FTIR analysis
The possible biomolecules in R. tuberosa aqueous tuber extract responsible for the synthesis and stabilization of AgNPs were investigated using FTIR analysis of both  (Fig. 6a). The band at 3281 cm −1 represents -OH groups of tannins, flavonoids (phenolic compounds), glucose, and -NH stretching of proteins [10,42].  (Fig. 6b). According to these results, the hydroxyl, carboxyl, amino, and amide groups of biomolecules present in R. tuberosa aqueous tuber extract served as reducing and capping agents for the biosynthesis of AgNPs. Flavonoids, phenolic compounds, tannins, carbohydrates, and amino acids found in R. tuberosa aqueous tuber extract may contribute to these groups [44].

Possible mechanism of AgNPs formation
The exact process by which plants produce nanoparticles was unknown. Phenolics, flavonoids, terpenoids, sugars, and amino acids were among the phytochemical constituents contained in the aqueous tuber extract [44]. These compounds could help to reduce metal salt solutions (Ag + to Ag°) and act as capping agents, resulted in stable AgNPs. The formation and size of nanoparticles were effected by several optimization parameters (Fig. 7).

Antiproliferative activity
No significant results were observed after performing the in vitro antiproliferative activity on A549, HeLa, MDA-MB 231, PANC-1, and SK-OV-3 cell lines.

CONCLUSION
In this study, AgNPs were synthesized using a crude aqueous tuber extract of R. tuberosa. GC-MS data revealed that the tuber of R. tuberosa was rich in various phytochemical constituents. These phytochemicals could aid in nanoparticle synthesis by acting as reducing and capping agents. After studying the effect of various physicochemical parameters, the optimized conditions were found to be 0.1 mL aqueous tuber extract, 1 mM AgNO 3 at 70°C, at pH 11. These particles were stable for up to 4 weeks. AgNPs synthesized by R. tuberosa were spherical in shape and crystalline in nature, with an average particle size of 34.9 nm.