Phytochemical-Mediated Biosynthesis of Silver Nanoparticles from Strobilanthes glutinosus: Exploring Biological Applications

The application of green synthesis for silver nanoparticles in nanomedicine has experienced significant growth. Strobilanthes glutinosus, a plant primarily located in the Himalayas, remains largely unexplored. Considering the biomedical value of S. glutinosus, phytochemicals from this plant were used for the biosynthesis of silver nanoparticles. Silver nanoparticles were synthesized from aqueous extract of root and leaves of Strobilanthes glutinosus. The synthesized silver nanoparticles were characterized using UV–Vis spectrophotometry, Fourier-transform infrared spectroscopy, transmission electron microscopy, and X-ray diffraction. Total phenolic and flavonoid contents of plants were determined and compared with nanoparticles. The biomedical efficacy of plant extracts and silver nanoparticles was assessed using antioxidant and antibacterial assays. The UV–Vis spectra of leaf- and root-extract-mediated AgNPs showed characteristic peaks at 428 nm and 429 nm, respectively. TEM images revealed the polycrystalline and spherical shapes of leaf- and root-extract-mediated AgNPs with size ranges of 15–60 nm and 20–52 nm, respectively. FTIR findings shown the involvement of phytochemicals of root and leaf extracts in the reduction of silver ions into silver nanoparticles. The crystalline face-centered cubic structure of nanoparticles is depicted by the XRD spectra of leaf and root AgNPs. The plant has an ample amount of total phenolic content (TPC) and total flavonoid content (TFC), which enhance the scavenging activity of plant samples and their respective AgNPs. Leaf and root AgNPs have also shown good antibacterial activity, which may enhance the medicinal value of AgNPs.


Introduction
Nanotechnology is a rapidly growing field with diverse applications in various disciplines such as health, drug delivery, cosmetics and environment. It also shows great

Optical Observations
When Strobilanthes glutinosus was used to prepare silver nanoparticles at 70 • C, a color change of the reaction mixture from greenish brown to dark brown was observed, which is a visual indication of silver nanoparticle synthesis from the leaves of Strobilanthes glutinosus (as shown in Figure 1). Root Ag NPs were fabricated; with frequent color changes from light yellow to grayish yellow and finally dark brown ( Figure 1). This color change is due to the optical properties of silver nanoparticles. The color changes during the synthesis process are the first indication of the synthesis of nanoparticles, as observed in many previous studies, such as deep yellow to dark brown [18], greenish yellow to brown, greenish to dark brown [19], and light yellow to brown [20].

UV-Vis Analysis
UV-Vis spectroscopy is a technique used to examine the formation of nanomaterials. The peak of silver nanoparticles produced from aqueous leaf extract of Strobilanthes glutinosus (leaf AgNPs) was shown at ~429 nm, while the peak of silver nanoparticles produced from aqueous root extract (root AgNPs) was shown at ~428 nm due to surface plasmon resonance ( Figure 2). In many previous studies, maximum absorbance peaks were recorded at 417 nm, 430 nm, and 428 nm for silver nanoparticles from the plants Ajuga brac-

UV-Vis Analysis
UV-Vis spectroscopy is a technique used to examine the formation of nanomaterials. The peak of silver nanoparticles produced from aqueous leaf extract of Strobilanthes glutinosus (leaf AgNPs) was shown at~429 nm, while the peak of silver nanoparticles produced from aqueous root extract (root AgNPs) was shown at~428 nm due to surface plasmon resonance ( Figure 2). In many previous studies, maximum absorbance peaks were recorded at 417 nm, 430 nm, and 428 nm for silver nanoparticles from the plants Ajuga bracteosa [21], Pedalium murex [22], and Erythrina suberosa [23], respectively. In another study conducted by Salayová and colleagues, silver nanoparticles had absorbance peaks at 421 nm, 422 nm, and 426 nm from the plants Berberis vulgaris, Capsella bursa-pastoris, and Origanum vulgare [24]. Similar results are also reported by Noukelag et al., 2020 [25].

UV-Vis Analysis
UV-Vis spectroscopy is a technique used to examine the formation of nanom The peak of silver nanoparticles produced from aqueous leaf extract of Strobilant nosus (leaf AgNPs) was shown at ~429 nm, while the peak of silver nanoparticles p from aqueous root extract (root AgNPs) was shown at ~428 nm due to surface resonance ( Figure 2). In many previous studies, maximum absorbance peaks w orded at 417 nm, 430 nm, and 428 nm for silver nanoparticles from the plants Aj teosa [21], Pedalium murex [22], and Erythrina suberosa [23], respectively. In anoth conducted by Salayová and colleagues, silver nanoparticles had absorbance pea nm, 422 nm, and 426 nm from the plants Berberis vulgaris, Capsella bursa-pastoris, ganum vulgare [24]. Similar results are also reported by Noukelag et al., 2020 [25]

Transmission Electron Microscopy
Transmission electron microscopy (TEM) depicts the shape, size, crystallinity, and morphology of silver nanoparticles. The TEM images revealed that the silver nanoparticles (both leaf AgNPs and root AgNPs) are polycrystalline and spherical in shape. The sizes of nanoparticles were measured using image J software. The size of leaf AgNPs range from 15-60 nm with average size of 32 ± 12 nm (Figure 3), while root AgNPs range from 20-52 nm with average size of 35 ± 8 nm (Figure 3). In a study conducted by Syed et al. on the synthesis of silver nanoparticles, particles ranging in size from 5 to 50 nm were obtained [26]. In a review article, the sizes of silver nanoparticles produced from various green sources were reported, such as silver nanoparticles produced from coffee, gelatin, glucose, tea, olive extract (1 mL), olive extract (5 mL), Leptadenia reticulate, Elaeagnus latifolia, and Chrysanthemum indicum L., which were 60 nm, 3.68 nm, 5.28 nm, 60 nm, 30 nm, 15 nm, 50-70 nm, 30-50 nm, and 38-72 nm in size, respectively [27]. In another study, 16 nm AgNPs were produced from Ficus benghalensis leaf extract [28]. the synthesis of silver nanoparticles, particles ranging in size from 5 to 50 nm were obtained [26]. In a review article, the sizes of silver nanoparticles produced from various green sources were reported, such as silver nanoparticles produced from coffee, gelatin, glucose, tea, olive extract (1 mL), olive extract (5 mL), Leptadenia reticulate, Elaeagnus latifolia, and Chrysanthemum indicum L., which were 60 nm, 3.68 nm, 5.28 nm, 60 nm, 30 nm, 15 nm, 50-70 nm, 30-50 nm, and 38-72 nm in size, respectively [27]. In another study, 16 nm AgNPs were produced from Ficus benghalensis leaf extract [28].

FTIR Analysis
FTIR is carried out to identify the functional groups of biomolecules that are linked to silver nanoparticles. The peaks of pure leaf extract, pure root extract, root AgNPs, and

X-ray Diffraction Analysis
The XRD analysis of silver nanoparticles synthesized from Strobilanthes glutinosus roots and leaves revealed nine peaks at 27

Antibacterial Activity
The anti-bacterial activity of silver nanoparticles was evaluated using the well-diffusion method on two Gram-positive bacterial strains, Staphylococcus aureus and Bacillus pumilus, and two Gram-negative bacterial strains, Klebsiella pneumoniae and Escherichia coli. Rifampicin was used as a positive control. These extracts did not have any antibacterial activity at all, while leaf AgNPs and root AgNPs showed remarkable results in comparison to positive controls ( Figure 6). The average of three replicates of the diameters of zones of inhibition were measured in millimeters. The aqueous root extract and aqueous leaf extract of Strobilanthes glutinosus had no activity against any of the bacterial species. In a study conducted by Ajaib and colleagues, it was also reported that the plant extracts of Strobilanthes glutinosus have remarkable antibacterial activity in organic solvents like methanol, petroleum ether, and chloroform, but in the case of aqueous extracts, no antibacterial activity was found [17]. In many studies, similar antibacterial activity of silver nanoparticles has been reported against these strains of bacteria. In two different studies conducted by Diana Gabrio and Laura Carson, the silver nanoparticles prepared from Lysiloma acapulcensis exhibited zones of inhibition of 18 ± 13 mm against E. coli and 16 ± 10 mm against S. aureus [49], and the silver nanoparticles prepared from Phyla dulcis exhibited zones of inhibition of 12 mm against both E. coli and S. aureus [50]. Violeta Morales-Lozoya conducted a study to prepare silver nanoparticles from the extracts of different parts of Morinda citrifolia, such as leaf, fruit, and dried seeds, and compared their activities against E. coli and S. aureus. Zones of inhibition were observed at 18.13 mm, 9.81 mm, and 20.45 mm against E. coli and 14.06 mm, 10.63 mm, and 15.10 mm against S. aureus from the silver nanoparticles prepared from the fruit extract, leaf extract, and dried seed extract of Morinda citrifolia, respectively [51]. A study conducted by Masoud Hussein and colleagues reveals that silver nanoparticles synthesized from onion and ginger extracts against K. pneumoniae have zones of inhibition with diameters of 8.33 ± 0.33 mm and 10.33

Antibacterial Activity
The anti-bacterial activity of silver nanoparticles was evaluated using the well-diffusion method on two Gram-positive bacterial strains, Staphylococcus aureus and Bacillus pumilus, and two Gram-negative bacterial strains, Klebsiella pneumoniae and Escherichia coli. Rifampicin was used as a positive control. These extracts did not have any antibacterial activity at all, while leaf AgNPs and root AgNPs showed remarkable results in comparison to positive controls ( Figure 6). The average of three replicates of the diameters of zones of inhibition were measured in millimeters. The aqueous root extract and aqueous leaf extract of Strobilanthes glutinosus had no activity against any of the bacterial species. In a study conducted by Ajaib and colleagues, it was also reported that the plant extracts of Strobilanthes glutinosus have remarkable antibacterial activity in organic solvents like methanol, petroleum ether, and chloroform, but in the case of aqueous extracts, no antibacterial activity was found [17]. In many studies, similar antibacterial activity of silver nanoparticles has been reported against these strains of bacteria. In two different studies conducted by Diana Gabrio and Laura Carson, the silver nanoparticles prepared from Lysiloma acapulcensis exhibited zones of inhibition of 18 ± 13 mm against E. coli and 16 ± 10 mm against S. aureus [49], and the silver nanoparticles prepared from Phyla dulcis exhibited zones of inhibition of 12 mm against both E. coli and S. aureus [50]. Violeta Morales-Lozoya conducted a study to prepare silver nanoparticles from the extracts of different parts of Morinda citrifolia, such as leaf, fruit, and dried seeds, and compared their activities against E. coli and S. aureus. Zones of inhibition were observed at 18.13 mm, 9.81 mm, and 20.45 mm against E. coli and 14.06 mm, 10.63 mm, and 15.10 mm against S. aureus from the silver nanoparticles prepared from the fruit extract, leaf extract, and dried seed extract of Morinda citrifolia, respectively [51]. A study conducted by Masoud Hussein and colleagues reveals that silver nanoparticles synthesized from onion and ginger extracts against K. pneumoniae have zones of inhibition with diameters of 8.33 ± 0.33 mm and 10.33 ± 0.33 mm, respectively [52]. Another study proclaims that silver nanoparticles with a size range of 20 nm to 70 nm have a zone of inhibition of 22 mm diameter against K. pneumoniae [53]. Silver nanoparticles against various strains of K. pneumoniae (MF953599 and 40 MF95353600) have similar results as in the present study with a 500 µg/L concentration and the diameter of the zone of inhibition being 34 ± 1 mm and 37 ± 0.5 mm, respectively [54].

Phytochemical Analysis
The phytochemicals are bioactive nutrient plant materials with antioxidant properties that help prevent many chronic diseases and oxidative damage. Phytochemical molecules include carotenoids, phytosterols, limonoids, polyphenols, glucosinolates, phytoestrogens, terpenoids, fibers, polysaccharides, saponins, etc. A phytochemical analysis (total phenolic content, total flavonoid content) of Strobilanthes glutinosus and its nanoparticles was performed to evaluate the medicinal and nutritional potential of nanoparticles synthesized from Strobilanthes glutinosus extract.

Total Phenolic Content
The total phenolic contents of the leaf and root parts of Strobilanthes glutinosus and their nanoparticles (leaf AgNPs and root AgNPs) were assessed using the calibration curve of gallic acid. The total phenolic contents present in methanolic leaf and root extracts are 8 ± 0.02 mgGAE/g and 1 ± 0.01 mgGAE/g. The methanolic suspensions of leaf AgNPs and root AgNPs have total phenolic contents of 21 ± 0.02 mgGAE/g and 26 ± 0.04 mgGAE/g ( Table 1). The calibration curve for gallic acid was obtained with R2 = 0.9701. A study conducted by Prabha and colleagues reports the amount of total phenolic contents as 1.423 mgGAE/g, which is very close to our results for root extract, which are 1 ± 0.01 mgGAE/g [55]. Geethalakshami reported that the total phenolic content of Sphaeranthus amaranthoides is 2.15 ± 0.26 mg/g dry weight, which is similar to our results for root and leaf extracts [56]. Malik and colleagues described the total phenolic content of Arisaema jacquemontii Blume as 45.17 ± 1.70 mgGAE/g, which is in accordance with our results for root and leaf AgNPs: 26 ± 0.04 mgGAE/g and 21 ± 0.02 mgGAE/g, respectively [57].

Total Flavonoid Content
The calibration curve of rutin was used to evaluate the total flavonoid contents of root and leaf AgNPs. The determination of total flavonoid contents was carried out via the formation of a flavonoid-aluminum complex, where rutin was used as a standard to establish a calibration curve. The total flavonoid content in methanolic leaf and root ex-

Phytochemical Analysis
The phytochemicals are bioactive nutrient plant materials with antioxidant properties that help prevent many chronic diseases and oxidative damage. Phytochemical molecules include carotenoids, phytosterols, limonoids, polyphenols, glucosinolates, phytoestrogens, terpenoids, fibers, polysaccharides, saponins, etc. A phytochemical analysis (total phenolic content, total flavonoid content) of Strobilanthes glutinosus and its nanoparticles was performed to evaluate the medicinal and nutritional potential of nanoparticles synthesized from Strobilanthes glutinosus extract.

Total Phenolic Content
The total phenolic contents of the leaf and root parts of Strobilanthes glutinosus and their nanoparticles (leaf AgNPs and root AgNPs) were assessed using the calibration curve of gallic acid. The total phenolic contents present in methanolic leaf and root extracts are 8 ± 0.02 mgGAE/g and 1 ± 0.01 mgGAE/g. The methanolic suspensions of leaf AgNPs and root AgNPs have total phenolic contents of 21 ± 0.02 mgGAE/g and 26 ± 0.04 mgGAE/g ( Table 1). The calibration curve for gallic acid was obtained with R2 = 0.9701. A study conducted by Prabha and colleagues reports the amount of total phenolic contents as 1.423 mgGAE/g, which is very close to our results for root extract, which are 1 ± 0.01 mgGAE/g [55]. Geethalakshami reported that the total phenolic content of Sphaeranthus amaranthoides is 2.15 ± 0.26 mg/g dry weight, which is similar to our results for root and leaf extracts [56]. Malik and colleagues described the total phenolic content of Arisaema jacquemontii Blume as 45.17 ± 1.70 mgGAE/g, which is in accordance with our results for root and leaf AgNPs: 26 ± 0.04 mgGAE/g and 21 ± 0.02 mgGAE/g, respectively [57]. The calibration curve of rutin was used to evaluate the total flavonoid contents of root and leaf AgNPs. The determination of total flavonoid contents was carried out via the formation of a flavonoid-aluminum complex, where rutin was used as a standard to establish a calibration curve. The total flavonoid content in methanolic leaf and root extracts is 16.9 ± 0.02 mgRE/g and 15.8 ± 0.04 mgRE/g; while the total flavonoid amount that went into the silver nanoparticles is 482.6 ± 0.02 mgRE/g and 504.2 ± 0.04 mgRE/g in leaf AgNPs and root AgNPs ( Table 2). A study by Aryal has reported that Solanum nigrum, and Digera muricata have shown similar amounts of total flavonoid content, i.e., 16.42 ± 0.39 mgQE/g and 18.00 ± 0.68 mgQE/g, respectively [58], which is very close to our results. The results of total flavonoid content and total phenolic contents show that the plant Strobilanthes glutinosus and the nanoparticles synthesized from it have high phenolic and flavonoid contents.

Antioxidant Activity
The antioxidant activity of silver nanoparticles (synthesized from roots and leaves) was compared with plant samples (root and leaf extracts) via DPPH scavenging activity. The IC 50 values have shown good antioxidant potential in plant samples and biofabricated silver nanoparticles. The methanolic extracts of plants (roots and leaves) show good antioxidant potential, while the antioxidant potential of leaf AgNPs and root AgNPs was good but less in comparison to the plant samples. The IC 50 value of ascorbic acid is 20 µg/mL; leaf extract is 23.2 µg/mL, root extract is 24.9 µg/mL; leaf AgNPs are 56.2 µg/mL, and root AgNPs are 60.8 µg/mL. The IC 50 values of samples and scavenging activity are graphically presented in Figures 7 and 8. The antioxidant activity of silver nanoparticles was observed in comparison to plant (root and leaf) extracts by a most commonly used methods, the DPPH scavenging assay. A lower IC 50 value depicts high antioxidant activity; the IC 50 values of ascorbic acid, leaf extract, root extract, leaf AgNPs, and root AgNPs were 20 µg/mL, 41 23 µg/mL, 23.9 µg/mL, 56 µg/mL, and 60.8 µg/mL, respectively, showing that the extracts had better scavenging activity compared to silver nanoparticles. In 2019, a study was conducted by Ahn and colleagues on thirty Chinese plants and silver nanoparticles derived from their extracts to examine their antioxidant, cytotoxic, apoptotic, and wound healing properties. Among those thirty plants, seven (Cratoxylum formosum, Phoebe lanceolata, Scurrula parasitica, Ceratostigma minus, Mucuna birdwoodiana, Myrsine africana, and Lindera strychnifolia) had higher antioxidant properties as compared to their green synthesized silver nanoparticles [59]. Silver nanoparticles of similar antioxidant potential as ours were also reported by Wang (IC 50 = 65 µg/mL) [60] and by Netala (IC 50 = 63.3 µg/mL) [61].

Green Synthesis of AgNPs from Leaf Extract
Firstly, 1 g of dried leaf powder of Strobilanthus glutinous was added to 250 mL of distilled water to prepare leaf extract at 70 °C; it was constantly stirred for 20-25 min and filtered. Then, 750 mL of 1 mM silver nitrate solution were prepared. Next, 245 mL of leaf extract was added to a silver nitrate solution at 70 °C, and an instant color change was observed, indicating the synthesis of silver nanoparticles. The reaction mixture was continuously stirred for 1 h to complete the reaction. dera strychnifolia) had higher antioxidant properties as compared to their green synthesized silver nanoparticles [59]. Silver nanoparticles of similar antioxidant potential as ours were also reported by Wang (IC50 = 65 µg/mL) [60] and by Netala (IC50 = 63.3 µg/mL) [61].

Green Synthesis of AgNPs from Leaf Extract
Firstly, 1 g of dried leaf powder of Strobilanthus glutinous was added to 250 mL of distilled water to prepare leaf extract at 70 °C; it was constantly stirred for 20-25 min and filtered. Then, 750 mL of 1 mM silver nitrate solution were prepared. Next, 245 mL of leaf extract was added to a silver nitrate solution at 70 °C, and an instant color change was observed, indicating the synthesis of silver nanoparticles. The reaction mixture was continuously stirred for 1 h to complete the reaction.

Green Synthesis of AgNPs from Leaf Extract
Firstly, 1 g of dried leaf powder of Strobilanthus glutinous was added to 250 mL of distilled water to prepare leaf extract at 70 • C; it was constantly stirred for 20-25 min and filtered. Then, 750 mL of 1 mM silver nitrate solution were prepared. Next, 245 mL of leaf extract was added to a silver nitrate solution at 70 • C, and an instant color change was observed, indicating the synthesis of silver nanoparticles. The reaction mixture was continuously stirred for 1 h to complete the reaction.

Green Synthesis of AgNPs from Root Extract
For the preparation of AgNPs from root extract, the dried root powder was pre-soaked for 3-7 days. The soaked root powder was boiled for 25-30 min and filtered. The root extract and 1 mM silver nitrate solutions were mixed slowly at 70 • C at 1:3, and the instant color changes depicted the synthesis of nanoparticles, as shown in Figure 1.

Antibacterial Activity of Strobilanthes Glutinous Synthesized AgNPs
The antibacterial study was examined using the well-diffusion method, LB Agar media was poured into the Petri plates, and when the agar media solidified, streaking of the bacterial culture was performed. For digging wells, a well borer was used, and the sample was loaded into the wells. The Petri plates were sealed with parafilm and placed in an incubator at 37 • C for 24 h [61]. The study was conducted against two Gram-positive strains (S. aureus and B. pumilus) and two Gram-negative strains (E. coli and K. pneumoniae).

Antioxidant Activity of Strobilanthes Glutinous Synthesized AgNPs
The antioxidant activity of root AgNPs and leaf AgNPs was evaluated using the DPPH scavenging method. The free-radical scavenging activity of silver nanoparticles and plants (root and leaf) was assessed. The samples of plant, ascorbic acid, and silver nanoparticles with concentrations ranging from 50 to 250 µg/mL were dissolved in methanol. Ascorbic acid was used as a control. Each of these solutions was individually added to 1 mL of 0.2 mM DPPH and incubated in the dark for 30 min at room temperature [21]. Absorbance was recorded at 517 nm, and the scavenging ability of each sample was estimated with the following equation: % Inhibition = Abs. control − Abs. sample/Abs. control × 100 (1)

Determination of Total Phenolic Content
The total phenolic content (TPC) of plant extract (i.e., leaf and root separately) and silver nanoparticles (leaf AgNPs and root AgNPs) was determined using the Folin-Ciocalteu reagent method. The phenolic content was determined by the gallic acid calibration curve with its concentrations of 31.62, 62.5, 125, 250, and 500 µg/mL in methanol. Then, 0.5 mL of sample was added to 2.5 mL of 10% FC reagent and 2.5 mL of 7.5% sodium carbonate. The reaction ice was incubated in the dark at room temperature for 30 min, and three consistent readings were taken. The absorbance was measured at 765 nm. The reaction was performed in triplicate. The TPC was calculated using the following formula: TPC = (XxV)/m; or (2) TPC = Gallic acid concentration mg mL × Extract volume(mL) Weight of tissue extract (g)

Determination of Total Flavonoid Content
The total flavonoid content (TFC) was determined using a spectrophotometric method based on the formation of the flavonoid-AlCl 3 complex. Rutin was used as a standard solution to obtain the calibration curve. The methanolic solution of 0.5 mL of sample was added to 0.5 mL of 10% AlCl 3 solution and 0.75 mL of 5% sodium acetate solution. The reaction mixture was incubated in the dark at room temperature for 2.5 h, and the absorbance was recorded at 440 nm. Three concordance readings were taken.

Conclusions
Herbal medicines are widely accepted in the medical sector due to their various advantages for human health and their low risk of adverse consequences. According to the current outcomes of phytochemical and antioxidant studies of the samples, Strobilanthes glutinosis botanical extracts and metallic nanoparticles may be one of the most therapeutic agents for treating diseases brought on by rising levels of oxidative stress. The rich amounts of phenolics and flavonoids present in both root-based and leaf-based Ag NPs enhanced the antioxidant and antibacterial activities of metallic nanoparticles. The bio fabricated Ag NPs proved to be an effective agent against various types of both Gram-positive and Gram-negative bacterial strains. The green fabrication of Ag NPs may prove a fast, cost-effective, and appropriate alternative to synthetic antibiotics against various multidrugresistant bacteria.