Introduction

Helicobacter pylori (H. pylori), a gram-negative bacterium, is a widespread pathogen that has infected approximately 50% of the global population1. The importance of H. pylori can be attributed to its classification in the first group of carcinogens by the World Health Organization (WHO)2. This bacterium is known to be responsible for various diseases, such as stomach ulcers, changes in the stomach lining, stomach cancer, and mucosa-associated lymphoid tissue (MALT)3,4.

In clinical practice, H. pylori infection is commonly treated with triple regimens (amoxicillin, metronidazole, and proton pump inhibitors (PPI)) initially and quadruple regimens (PPI, bismuth, tetracycline, and metronidazole) for recurrent cases5. However, the effectiveness of these regimens has considerably decreased due to antibiotic resistance6. To overcome these challenges, researchers have investigated alternative strategies7,8. Urease, an enzyme produced by H. pylori, is one of the potential targets.

H. pylori has developed ingenious mechanisms to thrive in the stomach’s acidic environment. One of its crucial survival strategies revolves around the urease enzyme9. H. pylori, upon entering the stomach, employs urease to hydrolyze urea into ammonia and carbon dioxide. The released ammonia and bicarbonate reduce overall stomach acidity, which provides an appropriate condition for H. pylori colonization and survival10. Therefore, urease inhibitors could disrupt the survival and colonization of H. pylori, leading to more effective and well-tolerated treatments8,11.

Quercetin, a flavonoid found in fruits and vegetables, has been extensively studied for its antioxidant, anti-inflammation, anticancer, anti-viral, and antibacterial properties12. Also, it is known as a urease inhibitor and a preventive agent for H. pylori infection13. However, Quercetin’s practical application has been limited by challenges such as low water solubility, low oral bioavailability, and instability in physiological fluids14,15,16.

There are various methods in drug design and delivery to overcome these limitations, including using liposomes17, micelles18, and polymers19, but nanomedicines are proven to have a high potential to fix these restrictions. Due to their small size (typically ranging between 1 and 100 nm), substantial surface area, and distinctive surface characteristics, nanoparticles (NPs) play a crucial role in drug delivery systems (DDSs), medical nano sensors, biochips, insulin pumps, needleless injectors, and other diverse applications. Additionally, NPs overcome challenges related to drug solubility and bioavailability, preserve drugs from unstable environments, pass drugs through biological barriers, and overcome drug resistance20,21,22. Nanomedicines, which involve the incorporation of drugs with nanocarriers, is an evolving branch of pharmaceutics that has transformed the field of medicine. These innovative approaches have shown promise, particularly in the field of antibacterial activity23. However, attention has yet to be paid to increasing the inhibitory effect of quercetin against urease enzyme with this method so far.

To date, a wide range of properties of silver nanoparticles (AgNPs) have been revealed, including antimicrobial, anticancer, pesticidal, catalytic, and their use as absorbents for water treatment24,25. Recently, it has been demonstrated that AgNPs conjugation with drugs, especially antibiotics offers promising outcomes in the treatment of gastrointestinal ulcers and infectious diseases26,27,28,29,30. Therefore, it could have been an appropriate carrier for quercetin to combat H. pylori.

In this particular study, we aimed to enhance the urease inhibitory activity of quercetin by conjugating it with AgNPs. We hypothesized that the conjugation of quercetin with AgNPs would not only improve its solubility and stability but also enhance its inhibitory effect on the urease enzyme. To achieve this, quercetin was loaded onto AgNPs. Subsequently, the efficacy and inhibitory impact of these complex AgNPs were evaluated compared to free quercetin against the urease enzyme. Binding energy and interactions between quercetin and AgNPs were also calculated via Density Functional Theory (DFT) calculations.

Materials and methods

Materials and instruments

Jack-bean urease enzyme (EC 3.5.1.5) and silver nitrate were provided from Sigma Aldrich (St. Louis, Missouri, United States). Sodium chloride, nitric acid, trisodium citrate, quercetin, and other reagents were purchased from Merck (Darmstadt, Germany). Deionized water was used during nanoparticle synthesis, and all chemical compounds were in analytical grade and used without any additional purification steps.

The pH of the solutions was measured using a Corning 220 pH meter (Cole-Palmer, Teddington, UK). The UV-Vis spectra of the samples were recorded with a UV-Vis spectrophotometer (Jenway 6300), and FT-IR spectra were reported in a 1:1 mixture of lyophilized NPs and KBr pellet using an FT-IR spectrometer (Bruker Tensor27) from 400 to 4000 cm− 1. Additionally, the morphology, size, and crystal structure of the NPs were assessed through DLS (Malvern ZEN3600), TEM (PHILIPS EM208s, 100 kV) and XRD (PHILIPS PW1730) instruments, respectively.

Preparation of AgNPs

AgNPs were synthesized using a modified version of the Frens’ method, which is a chemical reduction technique31. In brief, one mM silver nitrate solution was prepared by dissolving approximately 8.5 mg of AgNO3 in 50 mL of distilled water, and this solution was kept in the dark to prevent slow decomposition due to light exposure. Next, 500 µL of a 0.2 M trisodium citrate solution (pH 8) was added dropwise to the silver nitrate solution while stirring, and the mixture was heated to 100 °C. The reaction was stirred for 40 min at this temperature. After 40 min, the reaction was halted, and the synthesized AgNPs were centrifuged at 2040 RCF (10,000 g) for 10 min, discarding the supernatant. The resulting nanoparticle precipitate was dispersed in deionized water, and the NP suspension was used for further analyses.

Preparation of quercetin-loaded silver nanoparticles (Ag@QNPs)

To synthesize quercetin-loaded silver nanoparticles (Ag@QNPs), fresh solutions of AgNO3 (1 mM) and quercetin (1 mM) were prepared in deionized water. The reaction was carried out by mixing the AgNO3 and quercetin solutions. The reaction mixture was stirred vigorously for about 40 min at 100 °C, and then 500 µL of 0.2 M trisodium citrate solution (pH 8) was added dropwise. After 40 min, the final solution was centrifuged at 4000 rpm for 30 min. The obtained colloidal suspension (Ag@QNPs) was re-dispersed in deionized water for further analyses. Additionally, to quantify the loading efficiency of quercetin onto the AgNPs, the supernatant of the final solution was collected and analyzed.

Urease inhibitory assay

The urease inhibition potential of quercetin, AgNPs, and Ag@QNPs was evaluated using an adapted spectrophotometric approach rooted in the Berthelot reaction. Hydroxyurea served as the positive control, with an IC50 value of 7.6 µg/mL. The reaction mixture included 850 µL of urea (30 mM) and 135 µL of varying concentrations of the inhibitors (quercetin, AgNPs, and Ag@QNPs), bringing the total volume to 985 µL. To initiate the enzymatic reaction, 15 µL of urease enzyme solution (1 µg/mL) prepared in phosphate buffer (100 mM, pH 7.4) was added.

The ammonia concentration was quantified by incubating 500 µL of solution A (composed of 0.5 g phenol and 2.5 mg sodium nitroprusside in 50 mL distilled water) and 500 µL of solution B (containing 250 mg sodium hydroxide and 820 µL of 5% sodium hypochlorite in 50 mL distilled water) with 500 µL of the enzymatic assay solution at 37 °C for 30 min. Absorbance measurements were taken at 630 nm. The uninhibited urease activity was designated as the 100% control32.

Urease Inhibition measurement and data processing

The percentage of inhibition was computed using the following formula:

$${\text{I}}\left( \% \right) = {\text{1}}00{-}{\text{1}}00*\left( {{\text{T}}/{\text{C}}} \right)$$
(1)

Here, I (%) represents the percentage inhibition of the urease enzyme, T (test) denotes the absorbance value of the tested samples (quercetin, AgNPs, Ag@QNPs, and positive control) in the presence of the enzyme, and C (control) refers to the absorbance value of the enzymatic solution without any inhibitor. All experiments were conducted in triplicate. The IC50 value, which indicates the concentration of the inhibitor required to achieve 50% enzyme inhibition, was determined by evaluating the urease inhibitory activity of the compounds at different concentrations. Dose-response curves were plotted using GraphPad Prism 9 software to derive the IC50 values.

Computational methods

All calculations in this work were carried out using the ORCA quantum chemistry package33. Avogadro34, an excellent tool for editing molecular geometries, was used as a graphical interface to process and view ORCA input and output files.

The Ag surface was constructed in a one-dimensional planar arrangement using 40 Ag atoms. The Ag–Ag distance was fixed to 2.62 Å35. The quercetin molecule was optimized on the silver surface in both singlet and doublet states in all directions of the drug configuration using the density functional theory method. In all cases, the silver surface was fixed in its coordination position, and the drug was allowed to optimize. Calculations were performed using the Perdew–Burke–Ernzerhof (PBE) exchange correlational function and def2-TZVPP basis set. For the silver atom, the core potential (ECP) was used to reduce the calculation cost. Also, D3 dispersion interaction correction and basis set superposition error (BSSE) were applied to all calculations to correct binding energies.

Binding energies (B.E.) were calculated based on the following equation:

$${\text{B}}.{\text{E}}. = {\text{E}}_{{{\text{Complex}}}} - \left( {{\text{E}}_{{{\text{Quercetin}}}} + {\text{ E}}_{{{\text{Ag sheet}}}} } \right)$$
(2)

Results and discussion

The main objective of this project is to enhance the inhibitory activity of quercetin on the urease enzyme through the synthesis of Ag@QNPs. Therefore, Ag@QNPs have been synthesized using the Frens method as a reduction reaction. To study and identify the synthesized nanoparticles, various analyses such as DLS, FT-IR, XRD, and TEM techniques have been employed, and an enzymatic assay has been conducted using the Berthelot method.

Physicochemical properties and characterization of AgNPs and Ag@QNPs

UV-Visible analysis of NPs

The Ultraviolet-visible (UV-Vis) technique was used to determine the formation and stability of AgNPs and Ag@QNPs. As can be seen in Fig. 1, the UV-Vis spectrum of AgNPs shows significant absorption at 407 nm, which is a well-known feature of AgNPs36,37. While Ag@QNPs exhibited an absorption peak around 445 nm (Fig. 1), its spectrum profile is similar to that of AgNPs. The 38 nm offset observed in the wavelength indicates the effective loading of quercetin on the AgNPs.

Fig. 1
Fig. 1
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UV–Vis spectra of AgNPs and Ag@QNPs.

FT-IR analysis of Nps

The Fourier Transform Infrared (FTIR) spectroscopic analysis was carried out to confirm the successful loading of quercetin onto the AgNPs. The obtained FT-IR spectrum of quercetin, AgNPs, and AgNPs@Q are illustrated in Fig. 2. According to the recorded spectra, quercetin exhibited characteristic peaks at specific wavenumbers: 3438 cm[-1 corresponding to O-H stretching, 1577 cm[-1 representing C = O stretching, and approximately 1490 cm[-1 indicating C = C stretching. Moreover, absorption bands observed in the range of 650 to 1000 cm[-1, are related to the angular deformation of C = CH in aromatic compounds.

As shown in the spectrum of Ag@QNPs, the band at 1620 cm[-1 in AgNPs shifted to 1590 cm[-1 in Ag@QNPs, which may be attributed to involvement in conjugate formation with quercetin. This shift occurs because the interaction between quercetin and the nanoparticles leads to an increase in symmetry and causes a blue shift. Furthermore, both quercetin and AgNPs compounds exhibit a distinct peak corresponding to the stretching OH functional group at approximately 3400 cm[-1. However, this peak is slightly broadened in the case of Ag@QNPs. These changes indicate successful drug loading onto the nanoparticles and demonstrate that the synthesis has been performed properly.

Fig. 2
Fig. 2
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FT-IR spectra of quercetin (blue), AgNPs (black), and Ag@QNPs (red).

XRD analysis of AgNPs

X-ray Diffraction (XRD) can provide detailed information about the crystalline dimensions of the material. Therefore, the crystal structure, purity, and phase composition of the synthesized AgNPs were investigated using XRD analysis at room temperature. As shown in Fig. 3, the presence of the index peaks at 2θ = 38.10°, 44.32°, 64.44°, and 77.41°, which corresponds to AgNPs38,39,40. The XRD pattern is consistent with the standard card 0717-087-01, confirming the synthesis of crystalline AgNPs without any impurities. The XRD spectrum was used to calculate the crystallite size of synthesized AgNPs. Considering the peak appeared at 38.10°, the Debye-Scherrer equation was used for the crystallite size calculation of Ag NPs. (Eq. 3)

$$D = \frac{{\kappa \lambda }}{{\beta \cos \theta }}$$
(3)

where the Scherrer constant κ = 0.94, wavelength of X-ray Cu Kα radiation λ = 1.54˚A, θ is the Braggs angle, and β is the full-width half maximum of intense XRD peaks of AgNPs41. The crystallite size of 0.62 nm was obtained for the AgNPs, which is consistent with previous reports42.

Fig. 3
Fig. 3
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XRD pattern of AgNPs.

DLS analysis of NPs

Dynamic light scattering (DLS) was applied to measure the average size and the size distribution of AgNPs and Ag@QNPs in deionized water at 25 °C without any filtration. As shown in Fig. 4, the average hydrodynamic diameter of the AgNPs and Ag@QNPs were 70.85 ± 9.68 nm and 104.5 ± 8.53 nm, respectively. The increase in size suggests that quercetin molecules have been successfully loaded onto the nanoparticles.

Also, the zeta potential (ZP) value of AgNPs was obtained to be -23.5 ± 3.16 mV that indicating a robust surface charge that prevents particle aggregation. However, upon loading quercetin onto the AgNPs surfaces, the ZP decreased to -15.2 ± 2.3 mV. Despite this reduction in surface charge, Ag@QNPs can exhibit sufficient repulsive force to prevent aggregation during long-term storage (Table 1).

Fig. 4
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Size distribution by intensity of (a) AgNPs, (b) Ag@QNPs, ζ-potential of (c) AgNPs, (d) Ag@QNPs.

Table 1 The stability of AgNPs and Ag@QNPs.
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TEM analysis of NPs

Transmission electron microscopy (TEM) was used to determine the shape, size, and distribution of NPs. The TEM images of (a) AgNPs and (b) Ag@QNPs are shown in Fig. 5.

The TEM scans of AgNPs demonstrated non-spherical heterogeneous shapes (Fig. 5a), with an average size of approximately 59–69 nm. In contrast, the TEM image of Ag@QNPs showed more spherical nanoparticles with decreased angles and an average size of about 57–77 nm. The particles were also dispersed without any observable aggregation. These results suggest that quercetin was successfully loaded onto the AgNPs.

Fig. 5
Fig. 5
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TEM images along with particle size distribution of (a) AgNPs and (b) Ag@QNPs.

Urease enzymatic assay

The urease inhibitory activity of quercetin, AgNPs, and Ag@QNPs was independently evaluated using an enzymatic assay following the Berthelot protocol. As seen in Table 2, the percent inhibition of Ag@QNPs was calculated to be 97% at 167 µg/mL (considering the drug loading percentage of 12%, it contains 20 µg/mL of quercetin), and the IC50 value was 0.473 ± 0.09 µg/mL. However, quercetin showed a percent inhibition of 74% at 20 µg/mL and an IC50 value of 9.581 ± 0.38 µg/mL. It can be concluded that the activity of quercetin was significantly improved after loading it onto the AgNPs (Table 2). In other words, the conjugation of quercetin to AgNPs had a robust inhibitory effect in comparison to pure AgNPs and quercetin.

Table 2 Inhibitory activity of Quercetin, AgNPs, and Ag@QNPs against urease.
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Quercetin has been previously identified as a urease inhibitor, though its effectiveness is often hindered by poor solubility and bioavailability. Studies have reported IC50 values for quercetin in the range of 10–20 µg/mL, which is consistent with the findings in this research, where an IC50 value of 9.581 µg/mL was observed13,14. While AgNPs alone have been reported to possess some antimicrobial43, antiviral44, and enzyme inhibitory properties, their urease inhibitory activity is generally weak. For example, previous studies have shown limited urease inhibition by AgNPs, which is consistent with the 27% inhibition observed in this study26,27.

Other studies have also explored the use of AgNPs as carriers for urease inhibitors, demonstrating improved inhibitory activity compared to free inhibitors. For example, Zia et al. showed that omeprazole conjugated with AgNPs exhibited enhanced urease inhibition, further supporting the effectiveness of nanoparticle-based delivery systems for this purpose29. Similarly, Saddik et al. investigated the use of azithromycin-loaded AgNPs for the treatment of infected wounds, highlighting the broader potential of nanoparticle-based systems in enhancing drug delivery and therapeutic efficacy45. The novel contribution of this research lies in the synergistic effect achieved by combining quercetin with AgNPs, significantly enhancing urease inhibition. This combination has not been extensively explored in prior studies, making it a noteworthy advancement in the field of urease inhibition.

To contextualize the potency of Ag@QNPs, we compared its urease inhibitory activity (IC50 = 0.473 ± 0.09 µg/mL) with other well-known urease inhibitors, including hydroxyurea (IC50 = 7.6 µg/mL), thiourea (IC50 = 1–2 µg/mL), and acetohydroxamic acid (IC50 = 0.001–4.7 µg/mL)46. While Ag@QNPs exhibit slightly lower potency compared to acetohydroxamic acid, they are significantly more potent than free quercetin (IC50 = 9.581 ± 0.38 µg/mL) and hydroxyurea. This highlights the effectiveness of conjugating quercetin with AgNPs in enhancing its urease inhibitory activity. Furthermore, Ag@QNPs offer potential advantages in terms of biocompatibility and targeted delivery, which are critical for therapeutic applications.

While AgNPs exhibit promising drug delivery and antibacterial capabilities, but researchers must thoroughly assess their cytotoxic effects and biocompatibility within biological systems. Studies suggest that AgNPs induce cytotoxicity primarily through the generation of reactive oxygen species (ROS) and damage to cellular membranes47. The toxicity of AgNPs is influenced by three key factors: size, surface coating, and concentration48. Additionally, combining quercetin with AgNPs offers a potential strategy to mitigate toxicity, as quercetin’s antioxidant properties help counteract ROS formation49. Further research is essential to fully evaluate the biocompatibility and toxicology of Ag@QNPs, including in vitro cytotoxicity assays and in vivo toxicity studies using animal models.

DFT studies

Computational techniques have been used extensively to explore the properties of the nanoparticles or analyze the binding processes and develop predictive modeling50,51. In this study, first, the most stable structure of the quercetin drug was selected and redrawn based on previously published articles52. A silver surface consisting of 40 atoms was chosen, with dimensions of 8 × 5 atoms, allowing for the complete placement of a quercetin molecule on the surface while minimizing the number of silver atoms used. After optimizing the structures of both the surface and the drug, eight initial complexes with different orientations of the drug relative to the surface were created, and structure optimization was performed using the ORCA program package. During the optimization process, the silver atoms were kept completely fixed in place, while the atoms of the drug molecule were allowed to move freely. Figure 6 illustrates the structure of quercetin, the silver surface, and their complex.

After conducting structure optimization calculations, all the initial structures converged into a final configuration, as depicted in Fig. 6c. The figure illustrates that during the optimization process, the drug’s structure became completely flat, with the most significant change being the rotation around the carbon-carbon bond between the two benzene rings. This rotation is attributed to the presence of non-stationary electrons in the benzene ring and its strong interaction with the metal atoms. Such strong interactions have also been noted in other research studies53. The distance between the closest atoms of the quercetin molecule and the silver atoms in the optimized complex is illustrated in Fig. 6c.

Fig. 6
Fig. 6
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Structure of (a) quercetin, (b) the silver surface, and (c) the optimized complex.

The interaction energy between the drug and the surface was calculated using Eq. (2). Table 3 presents the absolute energies for the drug, the surface, and the complex, along with the interaction energies in both singlet and doublet states. The interaction energy obtained in the singlet state was − 53.79 kcal.mol[-1, which aligns well with findings in the literature regarding silver complexes and organic compounds35,54,55. Additionally, the basis function superposition error (BSSE) for correcting binding energies was found to be less than 0.2 kcal.mol[-1, a value that is negligible compared to the interaction energy.

Table 3 Absolute energies calculated for Quercetin, the silver surface, and their complex, along with the interaction energies in both singlet and doublet States.
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Previous DFT studies have explored quercetin’s interactions with metal surfaces like silver, gold, and copper. Brovarets’ and Hovorun demonstrated that quercetin forms stable metal complexes via non-covalent interactions, with interaction energies typically ranging from − 50 to -60 kcal.mol-1 52. Our reported interaction energy of -53.79 kcal.mol[-1 aligns with these findings, confirming strong quercetin-silver interactions. Similar energies have been observed for other organic molecules on silver surfaces. For example, Bhunia et al. reported − 50 to -55 kcal.mol[-1 for pyridine, which flattens due to π-metal interactions35, and Biswas et al. found − 50 to -60 kcal.mol[-1 for methimazole on silver nanoparticles54.

The flattening of quercetin on silver, observed in our study, is consistent with prior research. Naderlou et al. noted similar flattening of organic molecules on metal surfaces, driven by π-metal interactions and non-stationary electrons in benzene rings53. Additionally, the rotation around carbon-carbon bonds in quercetin, as seen in our work, has been reported in other DFT studies, such as Brovarets’ and Hovorun, which highlighted conformational changes in quercetin upon metal interaction52.

Non-covalent interactions, particularly π-metal bonding, stabilize the quercetin-Ag complex, as shown by Takenaka et al. for 2,2′-bipyridyl on silver and gold surfaces55. Our findings reinforce the importance of these interactions in metal-organic systems.

The novelty of our work lies in focusing on quercetin’s interaction with AgNPs to enhance urease inhibition, an underexplored application in nanomedicine. By integrating experimental urease inhibition assays with DFT calculations, we provide a comprehensive understanding of how Ag@QNP conjugates improve inhibition, advancing the field significantly.

Conclusion

This research aimed to enhance the urease inhibitory activity of quercetin through its conjugation with AgNPs. Ag@QNPs were successfully synthesized and characterized using various techniques. Ag@QNPs exhibited significantly higher urease inhibitory activity compared to pure quercetin, demonstrating the synergistic effect of combining quercetin with AgNPs. Computational studies using DFT calculations revealed the nature of the interaction between quercetin and the silver surface, suggesting that non-covalent forces, with acceptable interaction energy, stabilize the structure of the complex. This study demonstrates the potential of Ag@QNPs as a promising approach to enhance the urease inhibitory activity of quercetin. The findings contribute to the development of novel nanomaterials for biomedical applications. Future research should focus on elucidating the detailed mechanism of urease inhibition by Ag@QNPs and evaluating their biocompatibility and toxicity.