Green Synthesis: An Eco-Friendly Approach for the Synthesis of Silver Nanoparticles Functionalized with Operculina turpethum and It’s In vitro and in vivo Biological Activities

Introduction

Nanoparticles (NPs) are incredibly small particles with dimensions typically ranging from 1 to 100 nanometers. They exhibit unique physical, chemical, and biological properties that are distinct from their bulk counterparts. NPs have garnered significant attention in various fields due to their potential applications in various sectors like drugs, electronics, energy production, and environmental remediation.^1–4 Their small size allows for a large surface-to-volume ratio, enabling enhanced reactivity, increased surface area for interactions, and improved functionality.^5,6 NPs can be synthesized using different methods, including chemical, physical, and biological approaches, offering versatility in their size, shape, and composition.⁷ However, the unique properties of NPs also warrant careful consideration of their potential environmental and health impacts, necessitating comprehensive studies on their synthesis, characterization, and safe handling.⁸

Silver nanoparticles (Ag NPs) have appeared as a promising candidate for various applications including wastewater treatment due to their unique characteristics such as high surface area, small size, and unique physiochemical properties.^9–12 The small size and high surface area of Ag NPs make them highly reactive and effective in inhibiting the growth of microorganisms.¹³ The antibacterial activity of Ag NPs is attributed to several mechanisms such as the release of silver ions, the disruption of the bacterial cell membrane, and the inhibition of bacterial enzymes. Hence, they have garnered significant attention in recent years as a candidate for the development of novel antibacterial, antifungal, and antiviral agents.¹⁴

Ag NPs have shown promise as antibacterial agents due to their broad-spectrum activity against a range of microorganisms.¹⁵ The efficacy of Ag NPs in inhibiting bacterial growth has been demonstrated in various studies.¹⁶ The synthesis of NPs including Ag NPs using plant extracts has emerged as a sustainable and eco-friendly approach for nanoparticle synthesis.^17,18 Plant extracts possess several secondary metabolites, such as flavonoids, alkaloids, and terpenoids, which is responsible for the reduction and stabilizing in the synthesis of Ag NPs. This method is cost-effective, non-toxic, and eco-friendly compared to other chemical methods, making it an attractive approach for the large-scale production of Ag NPs^19,20.

O. turpethum is a medicinal plant that belongs to the Convolvulaceae family. It is commonly found in India, Sri Lanka, and other tropical regions.²¹ The plant is known by various names such as Indian jalap, turpeth, and nisoth. The roots of the plant are used in traditional remedies to treat various ailments such as constipation, fever, and skin infections.²² The plant comprises many bioactive compounds such as terpenoids, flavonoids, and alkaloids, which have been shown to possess antioxidant, enzyme inhibitory, anti-inflammatory, and antimicrobial properties.^23–26 Therefore, the use of O. turpethum extract for the synthesis of Ag NPs can provide a sustainable and eco-friendly approach for the development of novel antibacterial and enzyme inhibitory agents.

The use of plant extracts for the synthesis of Ag NPs has many advantages over other methods.^27,28 First, plant extracts are readily available and can be obtained from a variety of sources. Second, plant extracts are non-toxic and eco-friendly, making them suitable for biomedical and environmental applications. Third, plant extract-mediated synthesis of silver NPs is cost-effective and requires less energy compared to other chemical methods.^29–32 Therefore, the use of plant extracts for synthesizing of Ag NPs can provide a sustainable and eco-friendly approach for the development of novel antibacterial^19,20,33 and enzyme-inhibitory therapeutic agents.^34–36

Numerous studies have been conducted about the antibacterial and enzyme inhibitory properties of Ag NPs synthesized using plant extracts. For example, Ag NPs synthesized using Azadirachta indica extract exhibited potent antibacterial properties and significant enzyme inhibition due to the interaction of bioactive compounds of the plant with the biological targets, highlighting their applicability in treating microbial infections and enzyme-related disorders.^37,38 Similarly, Moringa oleifera-mediated Ag NPs showed remarkable antimicrobial effects, attributed to the phytochemicals present in the plant extract.³⁹ The synthesis of Ag NPs using Aloe vera and Ocimum sanctum extracts further demonstrated the ability of plant-based secondary metabolites, such as flavonoids and terpenoids, to act as reducing and stabilizing agents, making this approach both eco-friendly and effective.^40,41 These studies validate the role of plant-mediated green synthesis of Ag NPs and provide a foundation for exploring diverse plant species rich in bioactive compounds for nanoparticle production. Building on this rich foundation, the current study leverages the bioactive potential of O. turpethum to synthesize Ag NPs and evaluates their in vitro and in vivo biological activities, thus contributing to the growing body of knowledge in green nanotechnology.

Despite these advances, there remains a significant gap in identifying and exploring new medicinal plant species with potent bioactive compounds for Ag NP synthesis. O. turpethum, a medicinal plant has been traditionally used for its anti-inflammatory, antimicrobial, and enzyme-inhibitory properties. However, its potential as a reducing and stabilizing agent in Ag NPs synthesis has not been extensively investigated. Moreover, while previous studies have focused primarily on the antibacterial and enzyme-inhibitory activities of plant-mediated Ag NPs, their in vivo pharmacological effects, such as sedative and analgesic properties, remain largely unexplored.

To address this gap, the present study aims to:

1. Utilize the methanolic extract of O. turpethum to synthesize Ag NPs through a green and sustainable approach.
2. Characterize the synthesized Ag NPs to confirm their physicochemical properties.
3. Evaluate their in vitro antibacterial and enzyme-inhibitory activities.
4. Investigate their in vivo sedative and analgesic effects, providing novel insights into their pharmacological potential.

By integrating in vitro and in vivo assessments, this study not only expands the scope of plant-mediated Ag NP research but also provides a comprehensive understanding of their therapeutic applications. Our findings contribute to the advancement of green nanotechnology and highlight the potential of O. turpethum-mediated Ag NPs as multifunctional bioactive agents for biomedical applications.

Materials and Methods

The dried powder of O. turpethum was procured from the local market in Peshawar, Pakistan. Analytical-grade methanol (99.8% pure) was obtained from Merck, and silver nitrate (99.99% pure) was purchased from Sigma-Aldrich. Thiourea (99% pure), acarbose (99% pure), and acetazolamide (≥99% pure), Diclofenac sodium (98–102% pure) and diazepam (98% pure), acetic acid (≥99.7% pure) and saline solution (0.9% NaCl) and all the other chemical used was of analytical grade. All the chemicals and reagents were sourced from China.

Plant Collection and Extraction

In this study, the dried powder of O. turpethum was purchased from the local market in Peshawar, Pakistan in March 2023 and then subjected to methanol extraction. Methanol is a common solvent used in plant extraction due to its ability to extract various phytochemicals effectively. To extract the bioactive compounds from the plant material, 10 grams of the dried powder were added to 100 mL of methanol in a closed conical flask. The mixture was periodically shaken at 40 degrees Celsius for 24 hours to ensure the complete extraction of phytochemicals.⁴² The temperature and duration of the extraction were optimized to obtain a high yield of bioactive compounds. This method of extraction is commonly used to obtain phytochemicals from plant material and is effective in extracting a wide range of compounds.⁴³ After the extraction was completed, the mixture was filtered using filter paper to remove any insoluble residue. The resulting filtrate was then used as a reducing and stabilizing agent in the synthesis of Ag NPs.

Synthesis of Ag NPs

Silver nanoparticles were synthesized by mixing 10 mL of 5 mm AgNO₃ solution with 2.5 mL of methanolic extract of O. turpethum. The reaction mixture was stirred at 40°C for 4 hours. The successful synthesis of Ag NPs was confirmed by a color change to brown and a UV-Vis (Model-UV2601) absorption peak at 400 nm.

Characterization of Ag NPs

Comprehensive characterization of the synthesized Ag NPs was conducted using multiple techniques. FTIR (Model FTIR-990) analysis was used to validate the reduction of metal ions and to identify the functional groups involved in the reaction. By analyzing the infrared absorption spectra, we gained insights into the chemical interactions between the plant extract and the silver ions, providing valuable information about the synthesis mechanism. To further investigate the morphology and size of the Ag NPs, field-emission scanning electron microscopy (JEM 2100, Jeol CRL) was utilized. FESEM allowed for high-resolution imaging of the nanoparticles, revealing their surface morphology and confirming their size and shape. This analysis provided a visual representation of the synthesized Ag NPs, aiding in the understanding of their structural properties. Furthermore, energy-dispersive X-ray spectroscopy (ADX-8000 minI) was used for elemental composition of the synthesized Ag NPs. By detecting and analyzing the characteristic X-ray emissions, EDS provided insights into the elemental composition of the nanoparticles. This analysis not only confirmed the presence of silver but also provided information about the potential presence of other elements or contaminants, shedding light on the overall composition of the synthesized Ag NPs.

In vitro Activities

Antibacterial Activity

Using a standardized protocol, the antibacterial properties of the crude methanol (MeOH) extract from O. turpethum plant and the synthesized AgNPs (silver nanoparticles) were evaluated against Staphylococcus aureus, a Gram-positive bacterium. The bacterial strains were stored in Mueller-Hinton agar at 4°C in a refrigerator. To assess the antibacterial activity, the modified agar well diffusion method was employed, with Mueller-Hinton agar serving as the growth medium. The culture was inoculated onto petri dishes and incubated at 37°C for 24–72 hours. Before use, the petri dishes were sterilized. Next, 0.6 mL of the prepared bacterial broth culture was mixed with 20 mL of sterilized molten Mueller-Hinton agar and poured into each petri dish. Wells with a diameter of 6 mm were created in the agar medium using a sterilized borer. The crude MeOH extract (50 µL) from the plant and the synthesized AgNPs (50 µL) were separately added to the wells using a micropipette. A standard drug, Linezolid, was included as a control by using a disc soaked in 50 µL of the drug. To ensure proper diffusion, the petri dishes were placed in a laminar flow hood for one hour. Subsequently, the plates were incubated for 24 hours in incubator at 37°C. The zone of inhibition, representing the area where bacterial growth was inhibited, was measured.

Enzyme Inhibition Evaluation

The potential of enzyme inhibitory of the crude methanol (MeOH) extract from O. turpethum plant and the green synthesized AgNPs was evaluated against various enzymes. For the evaluation of the urease inhibitory activity, the crude methanolic extract and Ag NPs were tested at concentrations of 0.2 µg.mL⁻¹ and 0.25 µg.mL⁻¹, respectively. Thiourea, a known urease inhibitor, was used as the standard inhibitor at a 0.2 µM concentration. Similarly, the α-glucosidase inhibitory capability, the crude extract (0.2 µg.mL⁻¹) and Ag NPs (0.25 µg.mL⁻¹) were investigated respectively. Acarbose, was used as a standard drug. To check carbonic anhydrase II enzyme inhibition, the crude MeOH extract and AgNPs were tested at concentrations of 0.2 µg.mL⁻¹ and 0.25 µg.mL⁻¹, respectively. A well-known inhibitor acetazolamide, was used as the standard drug at a 0.2 µM concentration. The xanthine oxidase inhibitory activity of the MeOH extract and Ag NPs were evaluated at same concentrations as used for carbonic anhydrase II enzyme inhibition. The enzyme inhibitory assays were performed by incubating the enzymes with the respective test samples and substrates under specific conditions. The activities of the enzymes were measured, and the percentage inhibition was calculated by comparing the activity observed in the control samples. Additionally, IC₅₀ values, representing the concentration required to inhibit 50% of the enzyme activity, were determined. The data obtained from the experiments were subjected to statistical analysis to assess the significance of the inhibitory effects exhibited by the crude MeOH extract and AgNPs against the tested enzymes.

In vivo Screening

For vivo activities, albino mice (20–25 g) were used. The animals were maintained under standard laboratory conditions (temperature: 22 ± 2°C, 12-hour light/dark cycle) with access to food, water, and libitum. The study was conducted following ethical guidelines and approved by the institutional animal ethics committee.

In vivo Analgesic Activity

The analgesic activity was assessed using the acetic acid-induced writhing test. Mice were divided into groups (n = 6 per group) and treated as follows: saline (10 mL/kg, control), methanolic extract (25, 50, and 100 mg/kg), silver nanoparticles (2.5, 5, and 10 mg/kg), and diclofenac sodium (1.5 mg/kg, reference drug). One hour after oral administration of the respective treatments, 0.6% acetic acid solution was injected intraperitoneally to induce writhing. The number of writhes was counted for 20 minutes post-acetic acid injection, and the percentage inhibition of writhing was calculated for each treatment group.⁴⁴

In vivo Sedative Activity

The sedative activity was evaluated using the open-field test. Mice were divided into groups (n = 6 per group) and treated with saline (10 mL/kg, control), methanolic extract (25, 50, and 100 mg/kg), silver nanoparticles (2.5, 5, and 10 mg/kg), and diazepam (0.25 mg/kg, reference drug). One hour after administration, each mouse was placed individually in an open field apparatus, and the number of square crossings was recorded for 5 minutes as a measure of locomotor activity. Reduction in locomotor activity was considered indicative of sedative effects, and the percentage effect was calculated for each group.⁴⁵

Statistical Analysis

The data were expressed as mean ± standard error of the mean (SEM). A p-value of less than 0.05 was considered statistically significant, with notations *p < 0.05, **p < 0.01, and ***p < 0.001 indicating varying levels of significance.

Results

The Ag NPs were successfully synthesized using a methanolic extract of O. turpethum and were visually checked by observing a distinct color change in the reaction mixture (Figure 1), which turned from its original color to a characteristic brown hue. This change in color is often associated with the reduction of Ag ions and the subsequent production of Ag NPs as confirmed by the UV-Vis spectrophotometry.⁴⁶ The phytochemicals present in the extract act as a reducing and stabilizing agent thus avoiding the use of toxic chemicals. The green synthesis of Ag NPs using O. turpethum is primarily facilitated by its rich phytochemical composition which plays a crucial role in the reduction and stabilization of Ag NPs. Flavonoids and phenolics act as strong reducing agents, donating electrons to Ag⁺ ions, leading to their reduction into Ag^o nanoparticles. Meanwhile, terpenoids and alkaloids contribute to the stabilization of the synthesized Ag NPs by capping their surface, preventing aggregation and enhancing stability.

Figure 1 MeOH plant extract (a) and synthesized Ag NPs (b).

Characterization

UV-Vis Spectroscopy

UV-Vis spectroscopy is a commonly used analytical technique for the characterization of nanoparticles. In the present study, the formation of silver nanoparticles (Ag NPs) was validated by observing a change in the color of the solution from pale yellow to brown, as well as the appearance of a characteristic absorption peak at 400 nm⁴⁷ in the UV-Vis spectrum as shown in Figure 2a. This peak is a result of the surface plasmon resonance (SPR) of the nanoparticles, which is a phenomenon where the conduction electrons in the metal nanoparticles oscillate collectively in response to the incident electromagnetic radiation. The intensity and position of the SPR peak depend on the size, shape, and composition of the nanoparticles, as well as the dielectric environment. A peak within the range of 400–450 nm suggests the formation of small, spherical Ag NPs, while a redshift (higher wavelength) may indicate larger particle size or slight aggregation. The sharp and well-defined nature of the peak in our spectra confirms the uniformity and stability of the synthesized Ag NPs, further supported by their extended stability over time without significant changes in absorbance.

Figure 2 UV-Vis spectrum (a), FTIR spectrum (b) of plant extract and synthesized Ag NPs.

FTIR

FTIR spectroscopy was used to investigate the different functional groups present in the plant extract and the synthesized Ag NPs.⁴² The FTIR spectra of the crude methanolic extract and green-synthesized Ag NPs reveal the presence of several functional groups. The broad peak observed between 3500–3300 cm⁻¹ in the crude extract is typically associated with O-H stretching vibrations from alcohols, phenols, or carboxylic acids, with the broadening indicating hydrogen bonding interactions. The sharp peaks at 3200 cm⁻¹ and 3000 cm⁻¹ in the crude extract are likely due to N-H stretching vibrations of amines or C-H stretching vibrations of aliphatic hydrocarbons. A weak peak at 2500 cm⁻¹ could be attributed to O-H stretching of carboxylic acids, while the sharp peak at 1652 cm⁻¹ observed in both the crude extract and Ag NPs is likely related to the C=O stretching of amide bonds. The sharp peaks at 1425 cm⁻¹ and 2509 cm⁻¹ in the crude extract may be due to C-H bending vibrations of alkyl groups or C=O stretching in carboxylic acids and C≡C stretching in alkynes, respectively. The high-intensity peak at 1009 cm⁻¹ in both the crude extract and Ag NPs corresponds to C-O stretching vibrations from phenolic groups. The shift in the broad peak at 3500–3000 cm⁻¹ in the synthesized Ag NPs, which becomes narrower, indicates the reduction and capping of Ag ions by the functional groups in the plant extract. Additionally, the weakening of the peaks at 3200 and 3000 cm⁻¹ in the Ag NPs suggests that amines and hydrocarbons in the extract interact with the silver ions during nanoparticle synthesis. These findings highlight the involvement of specific functional groups in the reduction and stabilization of Ag ions during the green synthesis of Ag NPs. The FTIR Spectrum of the crude methanolic extract and Ag NPs is shown in the Figure 2b

FTIR spectroscopy analysis of the crude extract and synthesize nanoparticles Ag NPs indicated the contribution of several functional groups in the green synthesis of Ag NPs. The existence of diverse functional groups presents in the plant extract played a crucial role in the reduction and capping of Ag ions to form Ag NPs, and the disappearance of certain peaks in the FTIR spectrum of the synthesized Ag NPs indicated their involvement in the nanoparticle’s synthesis.

FESEM

FESEM (Field-Emission Scanning Electron Microscopy) is an invaluable tool for studying the surface morphology and size of nanomaterials. In our study, we utilized FESEM to analyze the synthesized Ag NPs (silver nanoparticles) derived from O. turpethum. The FESEM analysis revealed that these nanoparticles exhibit a spherical shape during their growth process. To gain a comprehensive understanding of the synthesized nanoparticles, we captured FESEM images at both low and high resolutions. These images effectively portray the intricate texture of the extract in which the nanoparticles are grown. By examining the FESEM images, we can delve deeper into the structural details and characteristics of the nanoparticles and their surrounding environment. The FESEM image is shown in Figure 3.

Figure 3 Low resolution (a) and High resolution (b) FESEM images of the synthesized Ag NPs.

EDS

In the EDS (Energy-Dispersive X-ray Spectroscopy) analysis of the synthesized Ag NPs (silver nanoparticles), elemental signals were observed at specific energy levels. The carbon (C) signal appeared at 0.06 keV, the oxygen (O) signal at 0.13 keV, and the silver (Ag) signal at 0.04 keV.

Quantitatively, the relative percentages of these elements were determined from the EDS spectrum. The carbon signal accounted for approximately 29.32% of the detected elements, followed by oxygen at 32.97%, and silver at 0.42%. These findings offer valuable insights into the elemental makeup of the synthesized Ag NPs. The EDS spectrum of the synthesized Ag NPs is given in the inset of Figure 4.

Figure 4 The EDS spectrum of the green synthesized Ag NPs.

In vitro Activities

Antibacterial Activity

The bactericidal activity of (Ag NPs) and the crude methanol (MeOH) extract from the O. turpethum plant was evaluated against the bacteria S. aureus. As controls, distilled water was used as a negative control, and Linezolid, a standard drug, was used as a positive control. The results said that distilled water had no inhibitory effect on the bacterial strain, indicating its lack of antibacterial activity. In contrast, Linezolid exhibited the highest inhibitory value of 25 mm against S. aureus, confirming its potent antibacterial activity. Ag NPs demonstrated significant inhibitory activity with a mean value of 14 mm against S. aureus, indicating their antimicrobial potential. The crude plant extract exhibited the lowest mean value of 12 mm for inhibitory activity against the bacterial strain. The results also revealed that various solutions containing Ag NPs displayed inhibitory effects against tested gram-positive bacteria, as shown in Table 1 and Figure 5.

Table 1 Antibacterial Effect of Plant Ext., Ag NPs (Zone of Inhibition)

Figure 5 Antibacterial activities of MeOH plant extract and synthesized Ag NPs.

Urease Inhibitory Activity

O. turpethum’s methanolic extract significantly reduced urease activity, with a percentage inhibition of 72.65%. The proportion of inhibition for the green synthesized silver nanoparticles, however, was 96.09%, indicating even inhibitory efficacy. The IC₅₀ value for the nanoparticles was found to be 25.65±0.97 µg.mL⁻¹, whereas the IC₅₀ value for the methanolic extract was found to be 68.02±1.22 µg.mL⁻¹ as shown in Table 2 According to these findings, the crude methanolic extract has a less inhibitory effect on urease activity than the green synthetic silver nanoparticles made from O. turpethum.

Table 2 Enzyme Inhibitory Potential of Crude Extract Silver Nanoparticles Using Methanolic Extract of O. turpethum

α-Glucosidase Activity

An enzyme involved in the breakdown of carbohydrates, -glucosidase, was inhibited by the MeOH extract and greenly synthesized AgNPs. Although the inhibitory effects were present in both samples, the nanoparticles showed a higher level of inhibition. These findings show the potential of NPs from O. turpethum as -glucosidase inhibitors, which may be investigated for treating disorders involving glucose metabolism. The nanoparticles were evaluated at a concentration of 0.25 µg.mL⁻¹, whilst the methanolic extract was examined at a concentration of 0.2 µg.mL⁻¹. The table displays the % inhibition. With a percentage inhibition of 30.76%, the methanolic extract showed a moderate inhibitory action against -glucosidase. The greenly synthesized AgNPs, on the other hand, had improved inhibitory activity, with a percentage inhibition of 48.09% as indicated in Table 2.

Carbonic Anhydrase II Enzyme Activity

The carbonic anhydrase II enzyme was moderately inhibited by the methanolic extract of O. turpethum, with a percentage inhibition of 45.09%. The proportion of inhibition for the green synthesized silver nanoparticles was 85.09%, in comparison, which showed much stronger inhibitory activity. The nanoparticles’ IC₅₀ value was determined to be 0.66±0.80 µg.mL⁻¹ (Table 2). These results indicate that as compared to the crude methanolic extract, the green synthesized silver nanoparticles had a more effective inhibitory impact on the carbonic anhydrase II enzyme. The information shows that, in comparison to the methanolic extract, the nanoparticles have a substantially stronger inhibitory impact. The nanoparticles’ lower IC₅₀ values indicate that they are more effective in inhibiting carbonic anhydrase II enzyme activity.

Xanthine Oxidase Activity

The xanthine oxidase enzyme was significantly inhibited by the methanolic extract of O. turpethum, with a percentage inhibition of 66.98%. With a percentage inhibition of 90.97%, the green synthesized silver nanoparticles, however, demonstrated an even stronger inhibitory efficacy. The IC₅₀ value for the nanoparticles was 14.23±1.01 µg.mL⁻¹, whereas the IC₅₀ value for the methanolic extract was determined to be 92.09±1.98 µg.mL⁻¹. According to the data given in Table 2, when compared to the crude methanolic extract, green synthesized silver nanoparticles from O. turpethum have a stronger inhibitory effect on xanthine oxidase activity.

In vivo Biological Screening

Analgesic Effect

For the analgesic activity, the methanolic extract showed increased efficacy with higher doses, achieving percentage effects of 47.89% ± 2.50 at 25 mg/kg, 59.34% ± 2.26* at 50 mg/kg, and 65.87% ± 2.01** at 100 mg/kg. In comparison, the silver nanoparticles demonstrated a notably higher analgesic activity even at lower doses. A 2.5 mg/kg dose of nanoparticles yielded an effect of 61.45% ± 1.63**which was comparable to the 100 mg/kg dose of the methanolic extract. The analgesic effect increased further with the nanoparticles, reaching 75.09% ± 1.95*** at 5 mg/kg and 81.98% ± 1.85*** at 10 mg/kg. Diclofenac sodium at 1.5 mg/kg served as the standard analgesic and exhibited the highest effect at 86.02% ± 0.24*** as shown in the Table 3

Table 3 Analgesic Activity of Crude Extract Ag NPs Using Methanolic Extract of O. turpethum

Sedative Activities

The methanolic extract displayed a dose-dependent sedative effect, with 25, 50, and 100 mg/kg doses producing sedative effects of 30.98% ± 2.00, 39.12% ± 1.98, and 50.09% ± 1.87**respectively. While the silver nanoparticles also showed a same trend in their sedative activity, with higher doses resulting in higher effects. At a dose of 2.5 mg/kg, the nanoparticles produced a sedative effect of 14.98±1.20***which increased to 25.09% ± 1.23** at 5 mg/kg and further to 35.09±1.65** at 10 mg/kg. Diazepam at 0.25 mg/kg, used as the reference sedative, showed a minimal effect of 1.14% ± 1.00*** in this model as shown in the Table 4.

Table 4 Sedative Activity of Crude Extract Silver Nanoparticles Using Methanolic Extract of O. turpethum

Discussion

Plants serve as rich sources of phytochemicals with diverse therapeutic applications, and these compounds possess inherent capabilities for reducing and stabilizing processes conducive to nanoparticle formation.^48,49 In this study, we utilized the methanolic extract of O. turpethum to synthesize silver nanoparticles (AgNPs) with the aim of enhancing their biological applications. The nanoparticle synthesis followed a standardized protocol documented in the literature.⁴² The successful synthesis of AgNPs was substantiated through UV spectroscopy, revealing an absorption peak around 400 nm in the spectrum a characteristic feature commonly associated with silver nanoparticles.⁵⁰ The confluence of observed color changes and the specific absorption peak in the UV-Vis spectrum collectively provided robust confirmation of the effective synthesis of silver nanoparticles. Mechanistically, When an AgNO₃ solution is mixed with the plant extract, bioactive compounds such as flavonoids, terpenoids, and phenolics reduce Ag⁺ to Ag^o. This reduction is marked by a color change, typically to yellowish-brown, due to surface plasmon resonance. The reduced silver atoms aggregate to form nuclei, which grow into nanoparticles as more silver is reduced. Simultaneously, the phytochemicals stabilize the nanoparticles by capping their surfaces, preventing aggregation and ensuring uniform size. The synthesized nanoparticles underwent comprehensive characterization employing FTIR, SEM, and EDS. In the FTIR spectrum, alterations in the intensity of peaks in both the crude extract and AgNPs indicated the involvement of functional groups in the reduction process. Additionally, the disappearance of the sharp peak at 1425 cm⁻¹ in the NPs suggested its role in the synthesis process SEM analysis demonstrated the uniform growth of the synthesized nanoparticles, providing insight into their structural characteristics. Furthermore, EDS analysis confirmed the presence of Ag ions in the reaction mixture, underscoring the purity of the synthesized nanoparticles. The biological activities of the synthesized NPs were also evaluated. The significant inhibitory activity of Ag NPs against S. aureus underscores their antimicrobial potential. These results are consistent with previous studies reported in the literature, reinforcing the reliable antimicrobial properties of Ag NPs,^42,51 and validating the reliable antibacterial properties of silver nanoparticles. The antibacterial activity of AgNPs synthesized from O. turpethum was evaluated against Staphylococcus aureus, with the nanoparticles exhibiting a zone of inhibition of 14 mm. The antibacterial activities of the green synthesized NPs are due to the production of ROS and the interaction of the metal ions with the cell membrane, which disrupts the cell membrane.^52–54 This result is comparable to the findings reported by Shahid et al (2024), where AgNPs synthesized from Callistemon viminalis also showed a similar zone of inhibition (14.5 mm) against S. aureus.⁵⁵ Abdussalam-Mohammed et al, 2025 also reported the antibacterial activities of green synthesized Ag NPs, and comparable results have been observed.¹¹ The inhibition of enzymes is also due to the various effective interactions including hydrogen bonding, dipole-dipole interaction and Van der Waals forces of the NPs functionalized with secondary metabolites with the active sites of the enzyme. The enzyme inhibition activity of the AgNPs in this study was promising. Specifically, the AgNPs exhibited 96.09% inhibition of urease at 0.25 µg/mL, with an IC₅₀ value of 25.65±0.97 µg/mL. For xanthine oxidase, the AgNPs showed a 90.97% inhibition at the same concentration, with an IC₅₀ value of 14.23±1.01 µg/mL. Gul et al (2021), reported the inhibition of enzymes where Ricinus communis-mediated AgNPs from roots (R-AgNPs) showed 94.2% inhibition against urease with an IC₅₀ value of 36.81±0.05 µg/mL and 83.6% inhibition against xanthine oxidase (IC₅₀ value 3.60±0.04 µg/mL). The R. communis leaf-mediated AgNPs (L-AgNPs) showed a similar trend in inhibition, with 92.1% urease inhibition and 83% inhibition against xanthine oxidase.⁵⁶ The enzyme inhibition results from O. turpethum AgNPs in this study are in line with previous reports, highlighting the potential of plant-mediated AgNPs for therapeutic applications. The results indicate a promising avenue for the development of antibacterial agents derived from O. turpethum. Moreover, the significant inhibitory effects on various enzymes, evident in urease, α-glucosidase, carbonic anhydrase II, and xanthine oxidase assays were also observed. In the context of urease inhibitory activity, the superior efficacy of Ag NPs compared to the crude methanolic extract suggests enhanced potential for urease inhibition, offering a promising avenue for the development of therapeutic urease inhibitors This shows that the urease inhibitory potential of silver nanoparticles made using O. turpethum has increased, which may be related to the unique features and capabilities of the nanoparticles.⁵⁷ Additionally, the heightened inhibitory activity of AgNPs against α-glucosidase, in comparison to the methanolic extract, suggests potential applications in managing disorders related to glucose metabolism. The lower IC₅₀ values, indicative of a stronger inhibitory impact on carbonic anhydrase II enzyme activity, further underline the potential effectiveness of green-synthesized AgNPs as inhibitors, encouraging further exploration into their therapeutic implications in physiological processes. The substantial inhibitory effect on xanthine oxidase activity by these nanoparticles suggests their potential in therapeutic applications for disorders associated with purine metabolism. The enhanced physicochemical characteristics and improved interactions of the nanoparticles with the enzyme may be responsible for their increased inhibitory activity, which results in a more dramatic inhibition of xanthine oxidase. The in vivo results demonstrate that the silver nanoparticles of O. turpethum exhibit enhanced analgesic effects compared to the methanolic extract. The nanoparticles achieved significant analgesic activity at much lower doses, with a 2.5 mg/kg dose producing an effect like that of the highest dose of the methanolic extract (100 mg/kg). This suggests that the nanoparticle formulation increases the bioavailability and systemic absorption of active compounds, allowing for more effective pain relief at lower concentrations. Furthermore, the highest analgesic efficacy observed at 10 mg/kg of nanoparticles (81.98% ± 1.85***) was close to the standard diclofenac sodium (86.02% ± 0.24***), indicating the potential of these nanoparticles as a potent alternative analgesic. Conversely, the sedative effects of the silver nanoparticles showed a same trend with increasing doses. While the methanolic extract displayed a straightforward dose-dependent increase in sedative activity, the nanoparticles’ highest sedative effect was recorded at 10 mg/kg. The low sedative effect of diazepam in this particular study model, despite being a standard reference drug, suggests that the parameters used to assess sedation may differ from conventional models. These findings highlight the promising analgesic potential of silver nanoparticles derived from O. turpethum. The observed sedative effects suggest that these AgNPs could be further explored as therapeutic agents for managing conditions related to anxiety, stress, and sleep disorders. Similarly, the analgesic properties could provide a new avenue for pain management, offering a more eco-friendly alternative to conventional synthetic drugs. However, further research is needed to clarify the mechanisms behind the reduced sedative effects of the nanoparticles and to optimize their formulation for potential therapeutic use. More research is needed to delve deeper into the active ingredients, optimize nanoparticle production, and ensure their safety and efficacy in humans. But these initial findings offer a compelling case for further exploration, potentially leading to exciting new medicines rooted in this ancient medicinal plant.

While this work successfully demonstrates the green synthesis of AgNPs and evaluates their biological activities, there are a few limitations. First, the synthesis process, although effective on a small scale, may require optimization for large-scale production, particularly for industrial or clinical applications. Additionally, while the in vitro results are promising, further investigation into the in vivo toxicity, biocompatibility, and long-term stability of the synthesized AgNPs is necessary for validating their clinical potential. Lastly, the exact mechanisms underlying the biological activities of the AgNPs, including their interactions with cellular components, remain to be fully explored.

Conclusion

This study highlights the potential of green-synthesized AgNPs from O. turpethum for biomedical applications. The AgNPs demonstrated significant antibacterial and inhibition of enzymes, confirming their effectiveness as antimicrobial agents. Additionally, their dose-dependent sedative and analgesic effects suggest therapeutic potential for pain and neurological disorders. This eco-friendly and cost-effective synthesis method offers a sustainable approach to developing novel therapeutic agents, reducing reliance on traditional chemical methods. Future studies could further explore the clinical applications and long-term stability and in-depth toxicity studies of these AgNPs.