Chemicals and Materials
Silver nitrate (AgNO3) and 2,2-diphenyl − 1-picryhydrazyl (DPPH) reagents were procured from the Sisco Research Laboratory (SRL) in Mumbai, India. Other chemicals, including dinitro salicylic acid, ascorbic acid, gallic acid, MEM media, cell culture tested PBS, trypsin EDTA solution, and sodium bicarbonate, were imported from HiMedia, India. Enzyme α-amylase and standard acarbose were purchased from Sigma, USA. The human renal adenocarcinoma (ACHN) cell line was brought from the National Centre for Cell Science (NCCS), Pune, India. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] dye was purchased from Bio Basic Canada Inc., Canada. Analytical grade solvents were used for the total procedure.
Preparation of the A. racemosus Willd aerial extract
Fresh aerial parts of A. racemosus Willd were collected from the Medicinal Plant Garden, University of North Bengal, Darjeeling, India. Further, collected aerial parts were washed, dried under the shed, and ground into powder using a mechanical grinder. The powder was extracted with deionized water. With the help of Whatman No.1 filter paper (Whatman, Fisher Scientific, Pittsburgh, PA, USA), the plant extract was filtered and dried to get the amorphous powdered crude extract.
Green synthesis of A. racemosus-mediated AgNPs
Silver nanoparticles were synthesized from the aerial parts of A. racemosus Willd. This synthesis followed a previously established method with slight modifications [19]. A. racemosus Willd aerial part powder (100 gm) was mixed with the required deionized water. Subsequently, 10 mL of the extract solution was dissolved in 90 ml of 20 mM AgNO3 solution in a 250 mL conical flask. The mixture was continuously stirred on a hot plate magnetic stirrer at 60 ± 5 0C under dark conditions, maintaining a stirring speed of 400 rpm. The formation of A. racemosus extract mediated-AgNPs (AgNPs-AR) was noticed visually by the shift in color from yellow to brown, which was further confirmed by the UV-vis spectrophotometer (Fig. 1A.-1C). The mixture was centrifuged at 16000 rpm for 15 minutes after spectrometric confirmation, yielding a dark brown precipitate. The pellet was washed thrice with deionized water precipitate before being dried using a freeze-dryer. The dried AgNPs-AR powder was used for further characterization and biological evaluation.
Characterization of AgNPs-AR
A UV-vis spectrophotometer (UV-1780, Shimadzu, Double Beam Spectrophotometer, Japan) was used at 200–800 nm with a resolution of 1 nm to measure the reduction of silver ions in AgNPs-AR [15]. FTIR characterization confirmed functional biomolecule participation in reducing silver ions to AgNPs-AR using a compact FTIR spectrometer (Bruker, Alpha II spectrometer, USA) with a region of 400 to 4000 cm− 1 [15]. XRD (Rigaku, Japan, SmartLab 9kW) technique was used to evaluate the crystalline pattern of AgNPs-AR by using Scherer’s equation [D = kλ/βcosθ, β = the width of the observed diffraction line at its half maximum intensity, k = tends to the shape factor (0.9), and λ = the wavelength] where the crystallite diameter of AgNPs-AR operated with Cu-kα radiation (1.5406 A˚). International Centre for Diffraction Data’s Powder Diffraction database was used to compare the pattern of AgNPs-AR stoichiometrically [27]. A DLS particle size analyzer (Litesizer 500, Anton Paar GmbH, Austria) determined AgNPs-AR’s particle size distribution and charge in a colloidal solution. The scattering intensity of AgNPs-AR was measured using Anton Paar’s Omega Cuvettes to determine zeta potential. SEM (JEOL, Tokyo, Japan) and high-resolution TEM (TEM 2100 PLUS, JEOL, Tokyo, Japan) were used to evaluate the size and shape of the AgNPs-AR. EDX was used to determine the elemental composition of AgNPs-AR.
Antioxidant activity of AgNPs-AR
Free radical scavenging activity (DPPH)
For the determination of antioxidant activity, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity is one of the most widely used methods [34]. Briefly, 2 ml of methanolic solution (100–800 µg/mL) was added to 1 mL of 0.3 mM DPPH solution. The mixture was incubated for 30 min in a dark place, followed by measurement of the absorbance at 517 nm against a blank solution. The percentage (%) of DPPH scavenging activity was calculated using the following equation: percentage (%) of DPPH scavenging activity = \(\:\left[\right(\text{S}0\:-\:\text{S}1)/\text{S}0]\:\times\:\:100\). where, S0 = absorbance of control; S1 = absorbance of sample. The IC50 value, or 50% inhibitory concentration, confirmed the antioxidant activity.
Ferric-reducing power assay
The reducing effect of AgNPs-AR was examined using the previously established method. [34]. Ascorbic acid was used as a standard for this method. Sodium phosphate buffer, pH 6.6 (2.5 mL), and 1% potassium ferricyanide (2.5 ml) were added with different dilutions (100–600 µg/mL) of AgNPs-AR solution. The above mixture was incubated in a water bath for 20–30 min by maintaining the temperature at 50 0C, then adding 10% trichloroacetic acid (2.5 mL) and centrifuging it at 1000 g for 10 min. 2.5 mL of distilled water was added to the upper layer of the mixture along with 1% ferric chloride (0.5 mL) and vortex. The final solution’s absorbance was measured at 700 nm, where the increasing absorbance of the reaction mixture suggested a greater capacity for reduction.
α-Amylase Inhibition Assay
To evaluate the anti-diabetic activity of AgNPs-AR, the α-amylase inhibition assay method was performed with slight modification [8]. After mixing 500 µL of the extract solution (pH 6.9) with 13 U/mL of the α-amylase solution followed by incubation for 20 minutes at 37 0C. The mixture was then mixed with 500 µL of 1% starch solution and incubated for 20 minutes at 37 0C. Subsequently, 1 mL of 3,5-dinitrosalicylic acid (DNSA) was added to stop the process. Using a UV-VIS spectrophotometer, the optical density of final solutions was measured at 500 nm. Acarbose was used as a positive control for this assay. The experiment was done in triplicate to ensure the reproducible data. The following formula was used to calculate extract inhibitory activity % inhibitory activity = \(\:\left[\right(\text{C}0\:-\:\text{S}1)/\text{C}0]\:\times\:\:100\), where C0 = absorbance of the control (100% enzyme activity) and S1 = absorbance of the sample. The inhibitory activity was expressed as the half maximal inhibitory concentration (IC50), which measures the effectiveness of a compound in inhibiting biological or biochemical function.
In vitro cell viability assay [MTT assay]
Human renal adenocarcinoma (ACHN) cells were developed in 100 mm petri dishes in a humified incubator with 5% CO2 at 37 0C. The MEM (Minimum Essential Medium Eagle) medium was supplemented with 10% FCS (Foetal Calf Serum), 100 units/mL Penicillin, 100 µg/mL Streptomycin, and 2.2 g/L Sodium bicarbonate. Following trypsinization, a 96-well microtiter plate was filled with roughly 100 µL of medium-containing cells and then planted at a density of 5×103 cells per well. The plate was then incubated for 24 hours at 37 0C with 5% CO2. The next day, AgNPs-AR was added in triplicate at various doses (50, 100, 150, 200, and 250 µg/mL), and it was incubated for another 24 hours in the same environment. Following a 24-hour incubation period, the media was disposed of, and 10 µL of newly made MTT dye (Stock solution, 5 mg/mL) was added to each well. The wells were then incubated for three more hours at 37 0C in a 5% CO2 incubator. Following three hours, each well received 50 µL of isopropanol to dissolve the purple formazan crystals, and the wells were then gently agitated for five minutes. At 620 nm, the Spectro Star Nano Spectrophotometer recorded an absorption. The formula used to compute the percentage of Cytotoxicity was \(\:\frac{B-T}{B}*100\), where T represents the mean OD of cells treated with AgNPs-AR and B represents the mean OD of blank cells [36].
Antimicrobial assay of AgNPs-AR
An agar well diffusion assay was conducted to ascertain the antimicrobial activity of the AgNPs-AR. [33]. The test plates were seeded with 106 freshly grown target cells of E. coli K12 and S. aureus MTCC 96 over nutrient broth soft agar (0.8% agar) over a base layer of nutrient agar (1.5% agar). Using a sterile cork-borer, 8.0 mm diameter holes were punched into each assay plate. 100 µL of the test sample was added to each well. The plates were pre-incubated for three hours at 4 0C to facilitate sample diffusion. They were then incubated for twenty-four hours at 37 0C. By examining the zone of inhibition, the samples' antibacterial activity was ascertained [40]. The minimum inhibitory concentration (MICs) and minimum bactericidal concentration (MBC) of AgNPs-AR were evaluated by the previously published work [40].
Statistical analysis
The information was presented as the average ± standard deviation (SD) across three replicates. Utilizing a one-way ANOVA and a Tukey multiple range test, Graph Pad Instat carried out the statistical study.
Result
Synthesis of A. racemosus-mediated AgNPs
In this characterization study, the A. racemosus Willd aerial part incorporated AgNO3 solution, and the A. racemosus aerial part extract was the main component of green synthesis. The formation of AgNPs of silver nitrate containing A. racemosus Willd aerial part extract was confirmed by visualizing the color change from yellow to dark brown (Fig. 1A-C).
Characterization of AgNPs-AR
The synthesized AgNPs-AR displayed a UV-visible spectra peak at 426nm (λmax) (Fig. 1D) due to the excitation of free electrons.
The FTIR spectrum of the aqueous extract from the aerial part of A. racemosus Willd showed the presence of biomolecules, which are responsible for reduction as well as give stability to AgNPs-AR by acting as a capping agent (Fig. 1E). The intense FTIR vibration peaks of AgNPs-AR recorded at 3455 cm− 1, 2918 cm− 1, 2356 cm− 1, 1628 cm− 1, 1597 cm− 1, 1385 cm− 1, 1346 cm− 1, 1125 cm− 1, 1064 cm− 1, 827 cm− 1, 796 cm− 1, and 613 cm− 1 where the peaks of A. racemosus Willd extracts peaks were observed at 3414 cm− 1, 2961 cm− 1, 2926 cm− 1, 2850 cm− 1, 2383 cm− 1, 1739 cm− 1, 1628 cm− 1, 1595 cm− 1, 1591 cm− 1, 1391 cm− 1, 1259 cm− 1, 1087 cm− 1, 839 cm− 1, 788 cm− 1, and 580 cm− 1 (Fig. 1D). The peaks at 3455 cm− 1 and 3414 cm− 1 of AgNPs-AR and extract were the strong absorbance bands, which represents the presence of free OH phenols and alcohol compounds.
An XRD analysis of AgNPs-AR was represented in Fig. 2A. A range of 100-900 was used to analyze AgNPs-AR diffraction peaks. The analysis showed five diffraction peaks of AgNPs-AR at 2θ = 38.900, 45.800, 64.070, 77.980, and 86.060, which confirmed the existence of Miller indices (111), (200), (220), (311), and (222) as well as the crystallinity of AgNPs-AR (Fig. 2A). The thickness and average size distribution of AgNPs-AR were assessed via dynamic light scattering (DLS) of biosynthesized AgNPs-AR, revealing a particle size of 202.5 nm (Fig. 2B) and a polydispersity index (PDI) of 28.8% (Table 1). The Zeta potential of formulated AgNPs-AR was recorded as -24 mV (Table 1), suggesting more AgNPs-AR stability.
Table 1
Details of surface zeta potential values and particle size distribution of AgNPs-AR.
Conductivity (mS/cm) | Zeta potential (mV) | Zeta deviation (mV) | z-Average Size (nm) | Polydispersity index (PDI %) |
0.028 | -24 | 1.9 | 202.5 | 28.8 |
The SEM image in Fig. 3A illustrates that AgNPs-AR are uniformly distributed, minute, and spherical when aggregated. AgNPs-AR that have not been cryoprotected against freezing may experience stress from ice crystal formation, which could lead to aggregation and particle size variation [15]. The elemental composition and purity of the metal nanoparticles were evaluated using the EDX spectrum. The high-intensity peak of silver was confirmed by the EDX profile (Fig. 4B). Additionally, the presence of additional carbon (C) peaks, likely originating from the plant extract, was detected by the EDX (Fig. 3B). Figure 3C depicts the morphological attributes of AgNPs synthesized via green synthesis using the aqueous extract of A. racemosus Willd aerial parts. The characterization reveals that AgNPs-AR exhibits a face-centered cubic shape, spherical characteristics, and size distribution. The results from EDX and DLS analyses cooperatively contribute to the refinement of TEM parameters.
Antioxidant activity
Green synthesized AgNPs-AR’s antioxidant potency was investigated by DPPH free radical scavenging assay and reducing power assay (Fig. 4). DPPH free radical scavenging assay was represented in Fig. 4A with compared to standard ascorbic acid. Better DPPH radical scavenging activity was shown by AgNPs-AR (IC50:81.96 µg/mL) as compared to standard (IC50:41.01 µg/mL) (Fig. 4A).
The reducing property of AgNPs-AR at different concentrations ranging from 150 to 550 µg/mL was employed where reducing power properties of nanoparticles were of in a dose-dependent manner (150 < 250 < 350 < 450 < 550 µg/mL) (Fig. 4B).
α-Amylase inhibition activity of AgNPs-AR
To evaluate the in vitro antidiabetic efficacy, the α-amylase inhibition assay of AgNPs-AR was performed where acarbose was used as a standard. The results (Fig. 4C) demonstrated a significant α-amylase inhibitory effect of AgNPs-AR (IC50: 535.76 µg/mL) compared to acarbose (IC50: 105.77 µg/mL). As the concentration of AgNPs-AR increased, more significant inhibition was observed.
Cytotoxicity potential of AgNPs-AR
ACHN cells line with a dose-dependent manner of AgNPs-AR were studied to evaluate cytotoxicity activity by MTT assay. Cancer cell line viability decreased after treatment with AgNPs-AR made from A. racemosus Willd aerial extract. The result showed that biosynthesized AgNPs-AR reduced cell viability in a dose-dependent manner. The AgNPs-AR showed inhibition of cell viability of 50% (IC50) at the concentration of 52.15 µg/ml (Table 2).
Table 2
Cytotoxicity activity of AgNPs-AR.
Sample | Concertation (µg/ml) | % of Cytotoxicity | IC50 (µg/ml) |
AgNPs-AR | 50 | 47.93 | 52.25 |
100 | 67.39 |
150 | 76.54 |
200 | 77.22 |
250 | 81.41 |
Antibacterial activity of AgNPs-AR
A. racemosus-mediated green synthesized AgNPs showed antibacterial activity against - Gram-positive (E. coli) and Gram-negative (S. aureus) bacteria (Fig. 5). The AgNPs-AR exhibited more extraordinary inhibitory ability against the Gram-negative E. coli than the Gram-positive S. aureus. At the lowest AgNPs-AR dose of 0.031 mg/mL, there was a 9 ± 0.56 mm zone of inhibition for S. aureus, with the MBC observed at 0.125 mg/mL (Fig. 5A, Table 3, and Table 4). Conversely, for Gram-negative E. coli, AgNPs-AR demonstrated a 9.00 ± 1.00 mm zone of inhibition at the same lowest dose of 0.031 mg/mL (Fig. 5B, Table 3, and Table 4). AgNPs-AR demonstrated E. coli, MIC, and MBC values of 0.062 and 0.25 mg/mL, respectively. However, the crude extract showed no such excellent anti-microbial activities.
Table 3
Zone of inhibition (mm) of AgNPs-AR against different tested microorganisms
(mg/mL) | E. coli | S. aureus |
1 | 14.33 ± 0.57 | 13.33 ± 0.57 |
0.5 | 13.66 ± 1.15 | 11.66 ± 0.57 |
0.25 | 13.33 ± 0.57 | 11.33 ± 0.57 |
0.125 | 11.66 ± 0.57 | 11.00 ± 1.00 |
0.062 | 11.33 ± 0.57 | 10.66 ± 0.57 |
0.031 | 9.00 ± 1.00 | 9.00 ± 0.56 |
Table 4
MIC and MBC of AgNPs-AR against tested microorganisms
| E. coli | S. aureus |
MIC | 0.062 mg/mL | 0.031 mg/mL |
MBC | 0.25 mg/mL | 0.125 mg/mL |