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Assessment of antimycobacterial activity and DFT studies of green-mediated CdO-doped Ag nanoparticles using Diethyl phthalate enriched Sauropus androgynus leaf extract
* Corresponding author: E-mail address: nathan.amalphysics@gmail.com (M. Amalanathan)
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Received: ,
Accepted: ,
Abstract
Tuberculosis (TB) is a potentially fatal disease. The need for novel anti-tubercular drugs has arisen due to the emergence of a high degree of treatment resistance and the predominance of Mycobacteria other than TB (MOTT). Accordingly, the current study reports the facile production of silver and silver-cadmium oxide nanomaterials utilizing leaf extract from Sauropus androgynus. The synthesized nanoparticles (NPs) were characterized using an ultraviolet-visible spectrophotometer (UV-Vis), X-Ray Diffraction analysis (XRD), Scanning Electron Microscope (SEM), Energy Dispersive X-Ray analysis (EDAX), High Resolution Transmission Electron Microscope analysis (HR-TEM), and fourier transform infrared (FTIR) studies. An aggregated spherical form with Ag, Cd, and O signals was observed in the SEM-EDAX analysis, and the XRD shows distinctive crystallinity. For the Ag-CdO nanocomposite and bare AgNPs, the average particle size was 17 nm and 23 nm, respectively, as determined by HR-TEM analysis. Ag and Ag-CdO NPs demonstrated distinct inhibition zones ranging from 13 to 16 mm, demonstrating their inhibitory efficacy, when the antibacterial activity was tested against Staphylococcus and Pseudomonas at various concentrations. A promising in vitro antimycobacterial activity against M. smegmatis was also demonstrated by the Ag-CdO nanocomposite. According to Density Functional Theory (DFT) studies, diethyl phthalate (DP) found in Sauropus androgynus leaf extract is what causes the extract to function as a reducing agent when Ag+ ions are reduced to Ag0. Good ligand interaction was shown in molecular docking studies of PDB ID: 5D6N, which correlated with the in vitro antimycobacterial activity. As a result, the synthesized NPs may be useful as antibacterial and antitubercular agents.
Keywords
Antimycobacterial
DFT
Green synthesis
Nanoparticles
M. smegmatis
1. Introduction
Tuberculosis (TB) is a chronic bacterial infection, and a large proportion of individuals infected with Mycobacterium tuberculosis (MTB) have latent TB. However, some individuals may progress to active TB. Based on the review by the World Health Organization (WHO), about two billion individuals were infected with MTB. Of these two billion people, eight million had active TB, and about two million people died due to active TB every year [1]. MTB affects about a third of the world’s population and is expected to infect about 30 million people in the next two decades, according to the WHO (WHO, 2008). Ten million people were diagnosed with TB in 2017, and more than 1.6 million died. It also continues to be a global health emergency due to the rapid spread of aggressive drug-resistant strains, as well as latent infections [2].
An indigenous plant from Southeast Asia, Sauropus androgynus (L.) is widely grown for traditional medicinal uses. The leaves of this plant have the ability to function as antioxidants and are useful in stimulating human milk production [3]. Because of its substantially higher vitamin content and nourishment than other vegetables, it is known as the “multigreen” vegetable [4]. The leaves of S. androgynus contain approximately 6-10% protein. In Asian countries, this herb has long been utilized as a medicine. In traditional medicine, S. androgynus is administered to relieve fever [5]. The existence of bioactive components in the leaf extract, such as alkaloids, tannins, saponins, and flavonoids, indicates its potential antibacterial activity [6]. Several medical ailments, including cholestasis, cough, ophthalmia, pain, coryza, and erythrina, have been known to be treated with various portions of this plant [7].
In the present era, nanotechnology has become a major focus of research and development to address body ailment issues. Metal nanomaterials such as Au, Ag, Se, etc., and oxides of nanomaterials like CuO, ZnO, NiO, MnO, etc., have been used as antibacterial, targeted drug delivery systems, antimycotics, antioxidants, anticancer agents, and so on [8]. Among them, Silver nanoparticles (AgNPs) have been found to function as anti-bacterial agents through multiple mechanisms such as rupturing bacterial membranes and cell walls to induce cell leakage, and lowering antioxidant levels to cause redox imbalance and oxidative damage to bacterial DNA [9]. AgNPs may make drug-resistant MTB strains more susceptible to treatment, as they are resistant to most organic antibiotics that are currently in use. This makes them a promising tool in anti-TB therapy [10]. AgNPs have been the most popular among several types of biosynthesized metal NPs because of their distinct chemical, biological, and physical characteristics [11]. When AgNPs are synthesized by the green approach, the plant extract containing proteins, flavonoids, ketones, aldehydes, tannins, carboxylic acids, phenolic acids, and aldehydes oxidizes Ag+ to Ag0 [12]. New-age bio-nanoformulations involve combining nanotechnology and traditional medicine.
Cadmium oxide is an inorganic compound. Recent oxide studies have focused considerable interest on cadmium oxide because of its strong conductivity and low band gap energy [13]. CdO NPPs have a vital function in biomedical applications because of their antibacterial characteristics. The generated microstructures have demonstrated exceptional resilience against fungal and bacterial infections. This is due to the existence of reactive oxygen species (ROS), the release of cadmium Cd2+ ions, and the distinctive size and form of the material NPs [14].
Doping CdO with metallic Ag will possess the ability to change its physical characteristics, and as a dopant, it increases electrical conductivity, optical band gap, and n-type conductivity. However, the effectiveness of Sauropus androgynus leaf extract in enhancing antimycobacterial activity has not been investigated in earlier investigations. The optical and microbial characterizations were carried out by the experimental studies and to identify the interaction of biomolecules present in the leaf extract with the target proteins that inhibit the growth of TB is resolved by computational techniques for drug designing such as molecular docking and Density Functional Theory (DFT), which are important steps in the drug discovery process.
2. Materials and Methods
2.1. Materials
Chemicals required for synthesis, such as cadmium acetate and silver nitrate, were purchased from Merck India Ltd., and utilized without extra purification.
2.2. Preparation of leaf extract
Sauropus androgynus leaves were washed with distilled and running water to remove dust and pollutants. Following that, for 7 days, the leaves were allowed to air dry at room temperature. Dried leaves were ground into powder using a mortar and pestle. A 100 mL of distilled water was added to 5 g of dried leaf powder, and the mixture was allowed to boil for 30 mins. The resulting solution was allowed to cool at room temperature and filtered using Whatman No. 1 filter paper. The filtrate was stored for subsequent experimental analysis.
2.3. Synthesis of AgNPs
For the synthesis of AgNPs, 0.1 M AgNO3 is dissolved in 10 mL of water. The solution is kept at continuous stirring until the salt gets completely dissolved. To this solution, 10 mL of the Sauropus androgynus leaf extract was added. After 15 mins of stirring, the solution changes to a brownish color, which indicates the formation of AgNPs (i.e., Ag+ to Ag0). The solution was heated at 70°C for 2 to 3 h under stirring conditions. The solution was then centrifuged twice to obtain the pure settled NPs. The settled down particles were cleaned using deionized water and ethanol. The obtained by-product was then dried at 80°C for 24 h in a hot air oven [7].
2.4. Synthesis of Ag-CdO NPs
For the synthesize of Ag-CdO NPs, 2% silver nitrate (AgNO3) is mixed and diluted with 25 mL DI water. Cadmium acetate was also diluted in 100 mL DI water. The two diluted salt solutions were mixed together. To this mixture, 20 mL of the Sauropus androgynus leaf extract was added dropwise. After the solution changed from colorless to brown, the mixture was incubated at 37°C for a period of 24 h. The solution was then centrifuged twice to obtain the pure settled NPs. These particles were cleaned using deionized water and ethanol. To obtain the Ag-CdO NPs, the precipitate was calcined at 500°C for 3 h.
2.5. Characterization techniques
Various analytical approaches were used to acquire a better understanding of the properties of the synthesized NPs. The optical properties were initially studied with a UV-Visible Spectrophotometer (Jasco V630 UV-Visible spectrophotometer). The observations were carried out between 200 and 800 nm to analyze the NPs’ absorption and transmission properties. The crystallinity and properties of the structure were explored by X-ray diffraction analysis (XRD). The XRD patterns were acquired using a Bruker D8 diffractometer with CuKα (λ=1.54056 Å) radiation at 40 kV/30 mA. The crystalline structure and phases of the synthesized NPs were determined using an XRD pattern measured at 20 to 80 degrees in 2θ. Shimadzu Prestige 20IR spectrometer was used to record the Fourier transform infrared (FT-IR) spectra between 400-4500 cm−1 for identifying functional groups. Surface morphology and NP size were obtained using SEM (Carl Zeiss) and energy dispersive X-ray analysis (EDAX) (Quanta FEG 250), respectively, while elemental composition was determined by EDAX. To investigate HR-TEM (High Resolution Transmission Electron Microscopy) micrographs, a copper grid was prepared by sonicating a fully dispersed material in an ethanolic solution for 10 mins. At an ambient room temperature, the grid was cooled before undergoing the analysis. The GC-MS (QP 2010 series, Shimadzu, Tokyo, Japan) apparatus was utilized to analyze the extracts.
2.6. Antibacterial activity
The zone of inhibition was found using the agar disc diffusion method to conduct the antibacterial screening of the samples. The samples were assessed against pathogenic Gram-positive and Gram-negative bacteria, Staphylococcus sp., and Pseudomonas sp., respectively. To make the medium, 38 g of Mueller Hinton Agar (Hi Media) was dispersed in 1000 mL of distilled water. The medium was autoclaved for 15 mins at 121°C and 15 lbs of pressure (pH 7.3). Following cooling and pouring the autoclaved medium into the Petri plates (25 mL per plate), the plates were swabbed with Staphylococcus and Pseudomonas spp. to allow for solidification. Subsequently, samples weighing 50µL (50 µg) were put onto a sterile disc and applied to the swabbed plate surface. The plates were kept at 37°C for a full day of incubation. After subsequent incubation, inhibitory zones were investigated and measured in millimeters with a transparent ruler (containing the disc).
2.7. Anti-mycobacterial activity
The agar well diffusion method is commonly used to assess the antibacterial activity of a test material. Mueller-Hinton agar (15-20 mL) was put onto glass petri plates of equal size and allowed to harden. A standardized inoculum of the test organism was evenly distributed on the plates using a sterile cotton swab. Each plate was poked using a sterile cork borer to create four 8 mm wells (20 mm apart). The test sample (50 and 100 µL) was introduced to wells T1 and T2 from a 10 mg/mL stock. Ciprofloxacin (40µL from 4 mg/mL stock) and the sample dilution solvent (DMSO) behaved as positive and negative controls. After 24 h of incubation at 36°C the plates were examined, and the area surrounding the wells where the bacterial growth was inhibited was quantified in millimeters.
2.8. Quantum chemical calculations
Diethyl Phthalate (DP) was found to be the main phytoconstituent in the Sauropus androgynus leaf extract after gas chromatography-mass spectrometry (GCMS) analysis. It was evident that this phytoconstituent was significantly present in the extract as it exhibited a retention-peak area of 59.71%. Furthermore, other components that contribute to the overall composition of the Sauropus androgynus leaf extract were also discovered by the analysis. The Gaussian 09 W program for the basis set of B3LYP/6-311++g(d,p) is used to do theoretical calculations using the DFT for all of the atoms in the DP molecule (optimized molecular structure), as illustrated in Figure 1. The DFT study will provide a more detailed insight into DP’s electronic structure and characteristics, allowing for a better understanding of its possible pharmacological effects and interactions within the synthesized nanoparticles (NPs). The quantum chemical parameters for the optimized molecular structure of DP were found, including the energy of the highest occupied molecular orbital (HOMO), the energy of the lowest unoccupied molecular orbital (LUMO), the dipole moment (D), and the energy gap (∆E).
- Optimized structure of DP.
3. Results and Discussion
3.1. UV-Vis analysis
The importance of light absorption properties is essential for understanding the behavior of NPs. To investigate the optical characteristics of the synthesized NPs, UV-Vis was employed. The UV-Vis absorption spectrum is a useful tool for examining the formation of metal NPs, and the absorption spectra of AgNPs are significantly impacted by their size. The position and intensity of the localized surface plasmon resonance (LSPR) peak in the UV-Vis region can vary when the particle size of the synthesized Ag NPs varies. The collective oscillation of conduction electrons in metals possesses absorption and scattering of light at a particular wavelength causing LSPR. This is due to the excitation of AgNPs by incident light. LSPR peak positions and intensity are determined by several parameters such as particle size and particle shape [15]. The LSPR peak generally shifts to shorter wavelengths (blue shift) as the size of Ag NPs diminishes. This behavior is ascribed to quantum confinement effects, which develop as an outcome of electron confinement to a smaller volume.
The absorbance spectrum for green synthesized NPs of pure Ag and Ag-CdO has been shown in Figure 2. The absorption band corresponding to the Ag NP was found at 415 nm. It shows that the peak was blue-shifted. The absorption peak in this range was generated by the presence of Ag+ ions in the solution [16] and may possibly be attributable to the smaller size of AgNPs. After the Ag-CdO nanocomposite synthesis, an absorption maximum was observed at 455 nm. The redshift was attributed to the transmission of electrons from CdO to AgNPs. In simpler terms, when materials interact with each other, they can change the surface by creating new chemical bonds and vacant spaces, like the vacancy of oxygen due to CdO [17].
- Absorbance UV spectra of Ag and Ag-CdO NPs.
Additionally, the amount of light absorbed decreased when CdO was added to AgNPs. This is because the concentration of the sample of Ag-CdO nanocomposite is lower than that of AgNPs. The Tauc relation was utilized to calculate the optical band gap from the absorption spectrum [18](Eq. 1).
where constant is denoted as C, the Planck’s constant as h, the molar extinction coefficient as α, the band gap energy of the material is Eg, and n determines the type of transition. Eg represents the direct permitted band gap for n=1/2. As seen in Figure 3, the value of Eg was calculated from the intercept of the linear section of the (αhν)2 versus hν plot on the hν axis. When comparing Ag (3.3 eV) to Ag-CdO (3.2 eV), it was discovered that the band gap value decreased. It was evident that Eg decreased when the NPs formed, and the low band gap was achieved when CdO was added to the sample simultaneously. The introduction of the semiconductor effect caused a shift in carrier concentration, which in turn caused the optical bandgap to drop. When the band gap energy decreases, the electron-hole interaction increases on the CdO surface’s active sites. This leads to a better oxidation process and enhances the sample’s optical property.
- Tauc plot indicating the energy gap of the synthesized NPs.
3.2. Structural XRD analysis
The size and structure of the synthesized NPs were determined by X-ray diffraction. As seen in Figure 4, the XRD patterns of the Ag and Ag-CdO NPs revealed peaks of diffraction that appeared at specific 2θ values. Both samples’ crystalline phases were identified by comparing their XRD to standard powder diffraction patterns (JCPDS card). The grain size was calculated using the apparent peaks, and the line broadening of the diffraction lines corrected for instrumental broadening was used to quantify the particle size of each sample using Scherrer’s Eq. (2).
- XRD Intensity peak of Ag and Ag-CdO NPs.
Where λ is the wavelength (λ=1.542Å) (CuKα), β is the full width at half maximum (FWHM) of the line, and θ is the diffraction angle.
The XRD study indicated that the crystallite size of synthesized Ag was 30 nm. However, the size of the crystallite decreased to 28 nm upon the addition of CdO. This considerable decrease could be attributable to the decrease in Ag concentration caused by the addition of CdO.
The presence of both Ag and CdO diffraction peaks suggests that the CdO phase has been introduced into the Ag phase. These findings were also confirmed by the EDAX and HRTEM analyses. Moreover, the inclusion of doping resulted in an increase in the primary peak’s small broadness. Figure 4 illustrates the diffractogram that displays the Bragg reflections with 2θ values at 38.11, 44.27, 64.50, 77.42, and 81.45, which correspond to the planes and, accordingly, the green synthesized Ag NPs.
This affirms the face centered cubic structure (FCC) structure of Ag-NPs, which is in accordance with the standard unit cell of the structure (JCPDS: 01-087-0717). Ag-doped CdO NP XRD pattern showed clear peaks at 2θ values of exactly 33.12, 38.43, 55.26, and 66.11. Comparing the Ag-CdO nanocomposite to its constituent parts, analysis of Figure 4 showed slight variations in diffraction angles and peak intensities in the XRD pattern. These changes can be attributed to variations in crystallite sizes and arrangements of the Ag-CdO NPs. The broadening of peaks in the patterns was caused by the reduction in particle size, which was ascribed to the experimental circumstances of nucleation and crystal nuclei growth.
The dislocation density of the system can be used to determine the number of imperfections and defects in the crystal lattice and is determined using the formula (Eq. 3).
In addition, the displacement of atoms relative to their reference lattice position resulted in the development of microstrain (ε), which can be calculated by the following relation [5,19] (Eq. 4).
The number of unit cells and particle size of Ag and Ag-CdO NPs have an inverse correlation with their dislocation densities. Table 1 shows the calculated structural properties of the produced NPs, including crystallite size, microstrain, and dislocation density. It indicates that the dislocation density and microstrain of the NPs increase while the grain size decreases. The effectiveness of biological capping agents in extract and functional biological applications is notably demonstrated by the crystallinity value of plasmonic AgNPs.
| Nanoparticle | Crystalline size nm | Dislocation density δ × 10-3 (nm-2) | Microstrain ε × 10-3 |
|---|---|---|---|
| Ag | 30 | 0.754 | 3.425 |
| Ag-CdO | 23 | 15.992 | 13.419 |
In AgNPs and Ag-CdO NPs, the impact of CdO incorporation on crystallite size and lattice strain was evaluated using the Williamson-Hall (W-H) approach. From the W-H analysis the microstrain for AgNPs was found to be more negative compared to Ag-CdO NPs. This implies that the undoped AgNPs undergo increased lattice strain, perhaps because of internal defects and structural defects; however, the slope became less negative when CdO is added, suggesting that stress is redistributed throughout the crystal lattice and that total strain is decreased. This reduction is due to the higher dislocation density and strain effects generated by CdO, which limit crystal growth and refine NPs. The overall W-H analysis demonstrated that stress redistribution, increased lattice defects, and restricted grain expansion were the causes of the crystallite size drop during CdO doping, which results in structural stabilization. These findings emphasized the importance of CdO in changing the crystallization dynamics of AgNPs, which may affect their functional capabilities in future applications.
The lattice parameter study found a modest decrease after CdO was included in AgNPs. The conventional FCC silver structure agreed with the calculated lattice value of 4.086 Å for pure AgNPs. The value decreased to 4.080 Å, indicating lattice contraction, after Ag was doped with CdO. This reduction is due to compressive stress, atomic interactions, and the probable substitution of CdO into the Ag lattice. This contraction was further supported by the slight structural distortions and peak shifts shown in XRD, which were in line with higher strain and dislocation density, as verified by Williamson-Hall analysis.
3.3. Fourier transform infrared analysis
Functional groups are basically known to stabilize the NPs. To analyze those functional groups fourier transform infrared (FTIR) analysis is a significant instrument. The FTIR graph is generated by plotting the oscillations and their intensity against the wavenumber of light (cm-1) to which the specimen has been subjected. Each material has a distinct frequency at which it absorbs light, and every functional group has distinct absorption bands. Figure 5 shows the functional groups of the synthesized NPs. Ag and Ag-CdO NPs were shown to have potentially active functional groups extending from 4000 cm-1 to 500 cm-1 by FTIR analysis. Due to the -OH stretching vibration of the -COOH group and the C-H stretching vibration of the alkyl group, the resulting graph displays a large peak in the range of 3600 cm-1 to 3001 cm-1.
- Functional group vibrations of the synthesized NPs.
The mode of stretching in the hydroxyl group in Cd (OH)2 gave rise to the bands at 3454 cm-1 and 3471 cm-1. The peak at 2933 cm-1 at higher wavenumbers (lower frequencies) was caused by the antisymmetric stretch of CH2 methyl groups, primarily from lipids. The phytoconstituents of the plant, mostly proteins, interact with the silver protein, as confirmed by the bands found in the spectra between 1700 and 1400 cm-1 that correspond to the amide groups, the most significant band in the protein infrared spectrum. The medium transmittance at 1645 cm-1 was induced by the C=O carbonyl stretching vibration, which confirmed the presence of amide. This characteristic of the β-sheet protein structure led to the stabilization of the NPs. Alkynes, esters, and carboxylic groups generated silver NPs, and the amide group stabilized those particles, as revealed by the peak at 1389 cm-1 [20].
Further study of the FTIR spectra revealed that the peak at 1023 cm-1 could portray the methoxy group. Additionally, the peak analysis indicated that the carboxyl and methoxy groups were mainly responsible for Ag and Ag-CdO NP capping. Peaks below 1000 cm-1 show metal-oxygen interaction, indicating the role of these functional groups in NP capping [21]. The absorbance at 630 cm-1 reveals the formation of the Cd-O band, which confirms the presence of Cadmium oxide NPs. This result is consistent with the results of the FTIR analysis, demonstrating that the Sauropus androgynus leaf extract can decrease and stabilize silver and silver-doped Cadmium oxide NPs.
3.4. EDAX analysis
The EDAX spectrum in Figure 6 (a,b) shows the elemental analysis of the synthesized nanomaterials. Based on the bio-reduction method, EDAX was used to perform the elemental analysis of Ag and Ag-CdO NPs. Pure sample confirmed the presence of pure Ag and the doped sample confirmed the presence of Ag, Cd, and O. The exact peak at 3 keV corresponds to the origination of pure silver NPs. The crystalline nature of the NPs is indicated by the sharp peak. In the case of Ag-CdO NPs, the purity of the sample was shown by the 44.43% weight percentage of Cd metal, 26.42% of Ag, 19.49% of oxygen, and 9.66% of sodium. Weak peak of Na in the doped sample may be due to the phytochemical contained in the plant extract, which was added as the reducing agent.
- EDAX spectrum of (a) Ag and (b) Ag-CdO.
3.5. SEM analysis
A high energy electron beam was used to examine a sample in a scanning electron microscope (SEM) to generate images of the sample. The signals produced by the electrons’ interactions with the sample’s component atoms provide information concerning its composition, surface topography, and other characteristics, including electron conductivity.
Figure 7 (a,b) displays a typical SEM representation of pure and Ag-doped CdO NPs. AgNPs showed a spherical shape, while the CdO-doped AgNPs changed their shape from sphere, which was notably different due to the addition of the new compound CdO. Consequently, there was minimal impact on the structure when some cadmium oxide is substituted with silver. Generally, the Morphology of Ag-CdO is spherical. But the obtained morphology was aggregated with a spherical shape. The morphology may be aggregated due to the opposite properties and their force of attraction due to ionic bonds.
- Morphological SEM representation of (a) Ag (b) Ag-CdO NPs..
3.6. HR-TEM analysis
HR-TEM was used to investigate the interior morphology and size of both the Ag and Ag-CdO nanoparticles. The average particle size of the synthesised nanoparticles was also measured. The silver nanoparticles were capped by biomolecules, most likely proteins, as evidenced by the intriguing finding that the borders of the particles seemed lighter than the centres. With the usage of Image J software, the particle size has been calculated. Moreover, the nanoparticles’ surface morphology showed that they were uniformly distributed over the surface, with some aggregating. This indicates that the powder particles are slightly agglomerated, and a closer look reveals the existence of spherical nanoparticles. Here, the particle size distribution is skewed to the long side. In comparison with Ag, the mean size of the particles of Ag-CdO was found to be smaller, indicating the strong efficacy of the synthesised sample. Figures 8 (a-d) denote the HRTEM morphology, Interplanar spacing, SAED pattern and histogram of the synthesised Ag nanoparticles. Vice versa Figures 9 (a-d) denote the HRTEM morphology, Interplanar spacing, SAED pattern and histogram of the synthesised Ag-CdO nanoparticles.
- (a) HR-TEM Morphology of Ag (b) interplanar spacing of Ag (c) SAED pattern of Ag (d) Histogram of AgNPs.
- (a) HR-TEM Morphology of Ag-CdO (b) interplanar spacing of Ag-CdO (c) SAED pattern of Ag-CdO (d) Histogram of Ag-CdO NPs.
Phase purity (spinel) of the synthesised materials was revealed by the relatively good match between the inter-planar distances obtained from the SAED rings and the corresponding indexing sequence determined from the XRD data. As a result of the presence of well-ordered edges, the lattice planes were formed. Moreover, the Ag and Ag-CdO have a polycrystalline structure composed of numerous lattices. The well-defined inter-planar spacing (d) of the Ag and Ag-CdO was determined from the peak, and it was found to be d = 0.24 nm and 0.25 nm in the inset of the interplanar spacing figures. As a result, it had been anticipated that Ag-CdO would have a far larger effect on many biomedical applications than AgNPs.
The histogram figure depicts the particle size distribution of synthesised samples, which ranges from 24 to 31 nm with an average size of 26.94 nm for Ag and 12 to 48 nm with an average size of 17 for Ag-CdO NPs.
3.7. Antibacterial activity
A great deal of emphasis has been placed on the high rate of resistance of different microbes to the majority of antimicrobial drugs. The health of humans and animals is seriously threatened by pathogenic microbes that are resistant to antibiotics. As therapeutic antimicrobial agents are used extensively, resistance to these compounds keeps growing at an alarming rate. Mueller–Hinton agar is most commonly used to test for antibiotic susceptibility [22]. Nanomaterials combined with antibiotics can render the material harmless and further enhance its antibacterial activity [23].
In the present study, the synthesized NPs were analyzed for their antibacterial activity against Staphylococcus spp, which is a Gram-positive bacterium that causes suppurative disorders in animals and mankind, and Pseudomonas, a gram-negative bacterium that is commonly found in surroundings such as water and soil (Centre for Disease Control and Prevention, 2019). The peptidoglycan layer is encircled by an outer membrane in Gram-negative bacteria. Gram-negative bacteria are more resistant to certain drugs and immunological responses because of the outer membrane, which contains lipopolysaccharides and functions as a barrier to the entry of certain substances. But in the case of Gram-positive bacteria, this outer membrane is absent [3]. Streptomycin was employed in this technique as a reference standard to compare the outcomes of Ag and Ag-doped CdO NPs. The zone of inhibition that is visible on the agar plate indicates the extent to which the NPs have inhibited the effect. When the sample’s inhibitory region is wider than 6 mm, it is generally regarded as possessing greater antibacterial activity.
Ag and Ag-doped CdO NPs exhibited exceptional antibacterial activity against the Gram-positive and Gram-negative bacteria, in accordance with results of their antibacterial activity. In comparison, the maximum inhibitory region for Staphylococcus spp and Pseudomonas spp in AgNPs was 0 and 10 mm, respectively, while the maximum inhibitory region for Ag-CdO NPs was 13 mm and 16 mm, respectively. The results showed a relationship between cell capsular disintegration and the variations in bactericidal activity between Ag and Ag–CdO NPs. While the study’s results indicate that sample NPs significantly inhibited both Gram-positive and Gram-negative bacteria, the antibacterial activity of the synthesized NPs against Gram-negative bacteria (Pseudomonas spp) was found to be superior to that of Gram-positive bacteria (Staphylococcus spp). This might be because gram-positive and gram-negative bacteria have different compositions in their cell walls. It indicates that the thin-walled Gram-negative strain of Pseudomonas spp is more vulnerable to cell wall damage than the thick-walled Gram-positive strains of Staphylococcus spp. The cell membrane of gram-negative bacteria is ruptured by CdO NPs. Concerning an extensive range of pathogens, Ag has exceptional antibacterial activity. The growth of Pseudomonas is therefore inhibited by the Ag+ ions and CdO NPs in combination. When metal is doped on the surface of the metal oxide NPs, it increases the charge separation of e− and h+, which in turn lowers the band gap energy and prolongs the time for recombination, and increases the activity of antibacterial agents.
As a result of the production of ROS and the release of Cd2+ ions, Ag-CdO NPs exhibit strong antibacterial activity. ROS that form on the surface of the NPs cause oxidative stress to the bacterial cell wall. The bacterial cell eventually dies as a result of this stress. The hydroxyl radical (OH+) and superoxide anion radical (O3-) found in ROS have the potential to cause damage to cell membranes and DNA. Due to their shared electrostatic attraction, the bacteria and NPs become attached [24]. This interaction prevents the growth of bacteria, and the generated ROS destroys the cell. This inhibitory effect could additionally be explained by the interaction between the ions released from the NPs and the thiol groups of proteins on the bacterial cell membrane. The cell membrane is then opened by these cellular proteins to let the nutrients through. Degradation of the cell capsular membrane was associated with the variation in the antibacterial activity of Ag and Ag-CdO NPs. Figure 10 (a,b) shows the response of the antibacterial agent against Ag and Ag-doped CdO NPs, and Table 2 lists the region of inhibition. This study provides evidence that Ag-CdO nanocomposites, which release Ag+ and Cd2+ ions are a powerful source of antibacterial biocides that inhibit the growth of harmful bacteria. These compounds can also be developed further to be used as antibacterial agents in biomedical fields.
- Inhibition of the antibacterial agent against (a) Ag and (b) Ag-doped CdO NPs.
| Bacteria name | Zone of inhibition (mm in diameter) sample and concentration (50 µI) | |||
|---|---|---|---|---|
| Ag | Ag - Cdo | PC(25mg) | NC | |
| Staphylococcus sps (G+ve) | NIL | 13mm | 15mm | NIL |
| Pseudomonas sps (G-ve) | 10mm | 16mm | 15 mm | NIL |
3.8. In vitro anti-mycobacterial activity
In the present study, the antimycobacterial activity of nanosynthesized Ag and Ag-CdO was investigated against M. smegmatis. It was detected that M. smegmatis was resistant to Ag-CdO NPs, possessing inhibition zones of 19 mm and 21 mm at two different concentrations of the sample (500 μg and 1000 μg, respectively). Pure AgNPs showed no inhibition at any concentration, ensuring the absence of activity against bacterial growth. This exemplified the capability of the Ag-CdO NPs as a functional antimycobacterial agent. The zone of inhibition has been depicted in Figure 11 (a,b).
- Inhibition zone against M. smegmatis by (a) AgNPs and (b) Ag-CdO NPs.
The color changes in the dye confirmed the interdictory effect of the NPs, which has been depicted in Figure 12. The blue color of the dye indicated the inhibition of M. smegmatis growth, while a transition to pink color signified the growth of the bacteria, implying the absence of inhibition. The color of AgNPs changed from blue to pink. It shows that medium control showed better reduction of alamar blue than the synthesized AgNPs, resulting in its inability to resist the growth of M. smegmatis. But in the case of Ag-CdO NPs, every concentration showed blue color, and particularly at the concentration of 31.25 µg blue color of the dye remained the same with enhanced activity, attributing its potential to resist the growth of M. smegmatis. It also proved the complementary inhibitory activity of the doped NP. According to these results, Ag-CdO may have promising antimycobacterial properties. New strategies for battling mycobacterial infections, such as TB, a worldwide health concern, may be made possible by more investigation and study of these agents.
- Inhibitory activity of Mycobacteria by alamar blue assay.
3.9. Quantum chemical studies
To analyze the reduction behavior of DP, identified using GCMS analysis, which is the highest biomolecule present in the Sauropus androgynus leaf extract, DFT analysis was carried out. The Sauropus androgynus leaf extract contains a number of additional chemical compounds. However, we can infer that DP is responsible for the reduction of metal nitrate precursors because it makes up a larger portion of the extract. The quantum chemical indices like EHOMO, ELUMO, ∆E, the dipole moment (μ), absolute electronegativity(χ), hardness (η), softness(σ), and from DFT calculations, as have been presented in Table 3.
| Molecular properties | Energy(eV) |
|---|---|
| EHOMO | -9.5292 |
| ELUMO | -5.0533 |
| Energy gap | 4.4759 |
| Ionization potential (IP) | 9.5292 |
| Electron affinity (EA) | 5.0533 |
| Electronegativity (x) | 9.8179 |
| Chemical potential (p) | -9.8179 |
| Global hardness(n) | 2.2379 |
| Softness (S) | 0.2234 |
| Electrophilicity index (ro) | 21.533 |
| Dipole moment | 3.5168 |
Frontier orbital theory was employed to determine the adsorption sites of the compounds in the extract and their interactions with the surface of AgNO3. The disparity between a neutral system’s LUMO and HOMO is the excitation energy of a molecule [25]. LUMO indicates the electron-accepting capacity (empty state) and HOMO represents the electron-donating capacity of the molecule (filled state). These concepts can be utilized to explain molecular electronic transitions, intramolecular charge transfers, and molecular charge [26].
The energy difference results from a significant degree of interaction charge transfer (ICT) through the π-conjugated pathway that connects the efficient electron acceptor groups to the electron donor groups at the end. The reactivity of the molecule is enhanced by the binding nature of DP to the surface of the metal halide thereby increasing the ability to deplete silver nitrate (i.e., AgNO3 to metal). The stability of the complex, which is formed on the surface of the metal halide is analyzed by the energy gap ∆E. The generated complex will also be more stable when the value of ∆E is low.
The results of GCMS analysis showed that the extract contained the compound DP, which was found to be the most abundant. The EHOMO value of −9.5292 eV in the present study indicated that DP adsorbed on the surface of AgNO3 and caused the reduction of Ag+ to Ag0. The HOMO orbitals (Figure 13) were situated on both the oxygen atoms and the aromatic ring of the molecule, suggesting that the lone pair of electrons on the oxygen atom enhanced the electron donating ability. The LUMO existsing the aromatic carbon atoms of the compound and also extended slightly to O8, O12, O20, and O25 atoms. The electronic charge delocalization on those atoms and the ring is indicated by the lowest LUMO value. The Sauropus androgynus leaf extract has a higher capacity to reduce the metal ions and a higher binding ability to the surface of the metal halide, as evidenced by its ELUMO value of 5.0533 eV and the LUMO orbitals of DP’s deficient center. As a result of the lower value of ∆E (4.4759 eV), there is an increased ability to convert Ag+ to Ag0 since minimal energy will be required to remove the electron from an oxygen atom.
- HOMO-LUMO electron density cloud of DP.
An additional significant measure derived from quantum chemistry calculations is the dipole moment (μ), which represents the arrangement of electrons within a molecule. The polarity of a polar covalent bond can also be determined through measurement [27]. When it comes to the reaction between the Sauropus androgynus leaf extract and AgNO3, the values of dipole moment play a crucial role.
The higher the dipole moment, the more effectively the extract accumulates on the surface of AgNO3, allowing the constituent compounds to proceed with the reaction. In this case, the Sauropus androgynus leaf extract exhibits a high dipole moment of 3.5168 Debye. This high dipole moment causes the extract to predominantly bind to the surface of AgNO3 and react with it. Specifically, the adsorption on the surface of AgNO3 occurs when the part of the molecule has the lowest hardness (η) and highest softness (σ), further facilitating the reaction process. Diphenylamine plays a significant role in the synthesis of metal NPs, particularly in the reduction of Ag+ to Ag0 NPs. It is expected that DP has a least hardness of 2.23 eV and the highest softness on oxygen atoms with a value of 0.223. Due to this property, DP can easily bind to the surface of AgNO3, enhancing the reduction of Ag+ to Ag0 NPs. Table 3 provides evidence that DP is strongly bound to the metal halide, thereby increasing the reducing ability of the metal halide to form metal NPs. The dipole moment, ionization potential, and global hardness values further support the strong binding of DP to the metal halide, highlighting its crucial role in the NP synthesis process.
3.9.1. Natural bonding orbital analysis
In the realm of computational chemistry, the natural bonding orbitals play a crucial role in understanding the donor-acceptor interactions between different molecular orbitals. These interactions provide invaluable insight into the bonding and antibonding behavior of electrons within molecules. NBOs are especially helpful in providing more understanding of the filled bonding or lone pair Lewis type NBOs, which are essential for interacting with Rydberg non-Lewis or empty antibonding NBOs. This interaction provides insight into molecular behavior and electrical structure. The donor-acceptor interaction energy in the NBOs is often estimated through second-order perturbation theory analysis of the Fock matrix, shedding light on the stability and reactivity of the molecular system.
The relationship between the stabilization energy E (2) and NBO interaction intensities is crucial in understanding the electron delocalization phenomenon. Essentially, as the E value increases, the donor orbital exhibits a stronger tenor to contribute an electron to the acceptor orbital. This results in a greater degree of electron delocalization, signifying the enhanced stability of the molecular system. Understanding these principles can significantly impact the study and analysis of molecular structures and their electronic properties. The stabilization energy and interactions of the molecule are provided in the Table 4.
| Donor NBO (i) | ED | Acceptor NBO (j) | ED | E (2) Kcal/mol | E(j) – E(i) a. u | E(i, j) a. u |
|---|---|---|---|---|---|---|
| n (C1-C2) | 1.9725 | π* (C1-C2) | 0.320 | 18.99 | 0.28 | 0.066 |
| π* (C5-C6) | 0.3236 | 20.77 | 0.29 | 0.070 | ||
| π* (C11-O25) | 0.2441 | 17.89 | 0.28 | 0.065 | ||
| n (C3-C4) | 1.9796 | π* (C1-C2) | 0.3524 | 21.90 | 0.28 | 0.070 |
| π* (C5-C6) | 0.3236 | 19.60 | 0.29 | 0.068 | ||
| n (C5-C6) | 1.9696 | π* (C1-C2) | 0.3524 | 20.07 | 0.28 | 0.067 |
| π* (C3-C4) | 0.3205 | 20.63 | 0.28 | 0.069 | ||
| π* (C7-O20) | 0.2235 | 9.01 | 0.29 | 0.047 | ||
| LP1O8 | 1.9640 | σ*(C7-O20) | 0.0256 | 7.58 | 1.17 | 0.084 |
| LP2O8 | 1.7870 | π* (C7-O20) | 0.2235 | 43.57 | 0.35 | 0.111 |
| LP1O12 | 1.9657 | σ*(C11-O25) | 0.0207 | 7.17 | 1.17 | 0.082 |
| LP2O12 | 1.7928 | π* (C11-O25) | 0.0207 | 44.75 | 0.35 | 0.112 |
| LP2O20 | 1.8444 | σ*(C6-C7) | 0.0671 | 18.25 | 0.67 | 0.101 |
| σ*(C7-O8) | 0.1006 | 31.60 | 0.64 | 0.129 | ||
| LP2O25 | 1.8444 | σ*(C1-C11) | 0.0647 | 17.48 | 0.68 | 0.100 |
| σ*(C1-O12) | 0.0979 | 32.15 | 0.63 | 0.129 |
The molecule stabilizes as a result of the strong intramolecular hyper conjugative interaction between the π electrons of the C-C bond and the anti-C-C bond of the ring. The conjugation of the corresponding π-bonds in benzene rings is caused by the interactions π (C–C) and their antibonding π* interactions. Strong π-electron delocalization within the ring results in a wide range of stability with 9.01-21.90 kcal/mol, as indicated by the electron density at the conjugated π bonds (0.22–0.35) and π* bonds (0.28–0.29) of benzene rings. In the ring, the resonance effect is caused by the largest reported delocalization. Bonding (π) and antibonding (π*) orbitals overlap one another causing charge transfer interactions, or steric interactions charge transfer (ICT), which stabilizes the system. The NBO analysis anticipated a strong n→π* interaction due to the lone pairs of oxygen atoms LP2O12 → π*(C11-O25), which had a stabilization energy of 44.75 kcal/mol. The weakening of the bond associated with antibonding orbitals is also facilitated by interactions that involve lone pairs and vacant antibonding orbitals, such as LP1O8 → σ*(C7-O20), LP2O20 → σ* (C6-C7) and σ*(C6-C7), and LP2O20 → σ*(C1-C11) and σ*(C11-C12), which have high E(2) values of 43.57 kcal/mol, 18.25 kcal/mol, 31.60 kcal/mol, 17.48 kcal/mol, and 32.15 kcal/mol, respectively. The compound’s structure is stabilized in part by these identified interactions within the molecule.
Table 5 shows the Mulliken charges of DP. The following atoms have been found to have the greatest potential emphasis for interaction with the AgNO3 surface: C2, C3, C4, C5, C7, C10, C11, C14, O20, and O25. Due to their greater negative charge, these atoms can function as nucleophilic reagents. Heteroatoms with higher negative values are generally better at donating electrons and have an increased ability to adsorb on AgNO3 surfaces and initiate the reduction process. Therefore, while preparing AgNPs with Sauropus androgynus leaf extract, DP in the extract is a better compound that triggers more reduction of AgNO3 and functions as a good stabilizing agent.
| Atoms | Mulliken charge | Natural charge |
|---|---|---|
| C1 | 0.7515 | -0.1250 |
| C2 | -0.3138 | -0.1602 |
| C3 | -0.2832 | -0.1945 |
| C4 | -0.3114 | -0.1786 |
| C5 | -0.3973 | -0.l693 |
| C6 | l.l053 | -0.0847 |
| C7 | -0.9322 | 0.8l50 |
| O8 | 0.0674 | -0.5578 |
| C9 | -0.0897 | -0.0342 |
| Cl0 | -0.564l | -0.5889 |
| Cll | -0.4377 | 0.8067 |
| Ol2 | -0.0043 | -0.5657 |
| Cl3 | 0.0257 | -0.03l9 |
| Cl4 | -0.58ll | -0.5899 |
| Hl5 | 0.l442 | 0.2059 |
| Hl6 | 0.l538 | 0.2035 |
| Hl7 | 0.l466 | 0.2048 |
| Hl8 | 0.2028 | 0.l892 |
| Hl9 | 0.l7l0 | 0.l832 |
| O20 | -0.l978 | -0.587l |
| H2l | 0.l882 | 0.22l6 |
| H22 | 0.l683 | 0.2095 |
| H23 | 0.l788 | 0.2090 |
| H24 | 0.l755 | 0.2240 |
| O25 | -0.l879 | -0.5904 |
| H26 | 0.l935 | 0.l846 |
| H27 | 0.l702 | 0.l834 |
| H28 | 0.l593 | 0.2060 |
| H29 | 0.1491 | 0.2079 |
| H30 | 0.1490 | 0.2037 |
3.9.2. Molecular docking
The utilization of molecular docking enables us to investigate the ligand-binding relationship between small molecules and proteins, which is of paramount importance for the screening of virtual libraries of drug-like molecules for the development of new drugs [28]. Using in silico molecular docking experiment, the current bioactive component DP of Sauropus androgynus leaf extract is docked to assess its antimycobacterial efficacy. Any species with a higher negative binding energy indicates that it has a better docking capability to bind to the targeted protein.
The anti-tubercular activity of DP molecule was examined using the protein PDB: 5D6N is chosen. The target protein structure was extracted from the Research Collaboratory for Structural Bioinformatics (RCSB) using Protein Data Bank (PDB) format (RCSB Protein Data Bank) [29]. The Ligand PDB file has been obtained from the optimized molecule structure. Ligand and water molecules were removed from the target proteins using the AutoDock Tools [16] user interface, and polar hydrogen bonds were added to the target proteins using AutoDock Tools. The preparation of the receptor was also undertaken. Table 6 illustrates the docking parameters. The protein-ligand interactions have been illustrated in Figure 14.
| Protein (PDB:ID) | Binding residues | Bond distances (A) | Binding energy (kcal/mol) |
|---|---|---|---|
| 5D6N | THR463 | 3.18 | -5.3 |
| THR463 | 3.44 | ||
| GLY424 | 3.26 | ||
| GLY463 | 2.91 | ||
| THR463 | 3.33 | ||
| THR463 | 3.02 | ||
| GLY424 | 2.93 |
- Protein-Ligand interaction of 5D6N.
The DP molecule showed an increased binding of −5.3 kcal/mol. Seven hydrogen bonds of the amino acids THR463, GLY424, and GLY463 were formed by the molecule, which is docked deeply into the binding pockets of 5D6N.3.18 A˚ and 3.26 A˚ are the bonding range of THR463 and GLY424 residues of nitrite oxygen atoms. 2.91 A˚ and 2.93 A˚ are the bond distances of two hydrophobic interactions (GLY463 and GLY424) of carboxyl group. The stability of the compound is also due to the hydrophobic interactions. The outcomes showed activity of DP as a potential compound that targets protein 5D6N to possess antimycobacterial effectiveness against M. smegmatis. The results of the experimental data were also fairly similar to the in vitro antimycobacterial activity.
4. Conclusions
In conclusion, it has been shown that the Sauropus androgynus leaf extract is capable of bio-reducing aqueous Ag+ and Ag-CdO ions. Strong indications of Ag and Ag-CdO components were observed in the NPs, according to EDAX analysis. FTIR spectra reveal the presence of organic components in the synthesized NPs, showing that the organic components of the leaf extract of Sauropus androgynus were involved in the synthesis process as capping and reducing agents. Structural and optical enhancements confirmed the existence of CdO on the Ag surface. The results of HR-TEM investigation showed that the NPs were spherically agglomerated and ranged in size from 12 to 48 nm. The XRD pattern corroborated the crystalline nature of the synthesized AgNPs and Ag-CdO NPs. Based on DFT investigations, DP present in the Sauropus androgynus exhibits strong antimycobacterial activity against Mycobacterium smegmatis and plays a crucial role in decreasing AgNO3. Additionally, it functions as a superior stabilizing agent. Also, when tested against pathogenic microbes, the biosynthesized Ag-CdO NPs showed greater antibacterial activity than Ag NPs. The antimycobacterial activity results demonstrated that M. smegmatis was effectively inhibited by the Ag-CdO nanocomposite. As a result, it has a strong potential for use as an anti-mycobacterial medication. This study introduces a new method for efficiently synthesizing Ag-CdO NPs from natural materials, which could be useful in a variety of applications. These findings could be applied to future biological and drug formulation applications, among other exciting prospective uses.
Acknowledgment
This work was funded by the Researchers supporting project number (RSP-2025R7) King Saud University, Riyadh, Saudi Arabia. The authors also have gratefully acknowledged UGC-DAE Consortium for scientific Research for providing the partial financial support through the CRS project proposal no. CRS/2022-23/662 UGC India.
CRediT authorship contribution statement
All authors of this study contributed equally to data collection, data analysis and manuscript writing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Declaration of Generative AI and AI-assisted technologies in the writing process
The authors confirm that no artificial intelligence (AI)-assisted technologies were used in the writing, editing, or formatting of this manuscript. Furthermore, no images or figures included in the manuscript were generated or manipulated using AI tools.
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