Introduction

Helicobacter pylori is a spiral, microaerophilic, Gram-negative bacterium that inhabits the stomach lining of approximately 50% of the human population worldwide1. This organism is one of the major pathogens associated with ulcers and gastric atrophy2,3. The World Health Organization (WHO) has also classified H. pylori as a Group I carcinogen due to its established relationship with mucosa-associated lymphoid tissue (MALT) lymphoma and gastric carcinoma4. Rising antimicrobial resistance, particularly towards clarithromycin and metronidazole, with rates of resistance of around 25% and 65%, respectively, has significantly decreased the effectiveness of standard antibiotic treatments5,6. Another significant process that favors bacterial survival and antibiotic resistance is the formation of biofilms. Many genes are associated with virulence and the formation of biofilms. For example, ureA is involved in acid resistance, spoT regulates biofilm formation, hspR affects motility and adhesion, and cagA and vacA encode toxins7,8. Antimicrobial peptides (AMPs) are short, synthetic, or naturally occurring peptides that possess strong antibacterial properties, primarily by disrupting bacterial membranes9,10. These peptides have shown promise as treatments for these problems11. AMPs selectively interact with bacterial membranes while avoiding host cells due to their cationic and amphipathic properties. Furthermore, AMPs exhibit low rates of resistance development and may have immunomodulatory, antiviral, and anticancer effects12. Ib-AMP4, a peptide first isolated from Impatiens balsamina, serves as the template for the synthetic AMP known as Impatiens balsamina-M113. The peptide’s net charge, hydrophobicity, and antimicrobial potency were all enhanced by the addition of tryptophan and arginine residues. Among several designed analogs, Ib-M1 (Type 1) exhibited the strongest activity against Gram-negative pathogens, with minimal cytotoxicity to mammalian cells14. Nanotechnology offers promising strategies to combat antibiotic-resistant bacteria15. Among metal oxide nanoparticles, zinc oxide is particularly well-known for its broad-spectrum antimicrobial activity, which is primarily attributed to membrane disruption and the generation of reactive oxygen species (ROS)16,17. Although Zinc oxide nanoparticles (ZnO NPs) are widely used in biomedical applications and are considered biocompatible, traditional chemical synthesis methods often employ hazardous solvents and require significant energy, thereby raising environmental concerns18.

On the other hand, a cheaper, safer, and somewhat eco-friendly approach, known as green synthesis, utilizes plant extracts instead of heavy chemicals19. In this case, Anvillea garcinii was chosen specifically due to its strong antibacterial and anti-biofilm effects. The extract of this plant, in fact, effectively inhibited the growth of Staphylococcus aureus and even reduced certain biofilm genes, such as the spa gene20. That makes it a relatively natural option for acting as both a reducing and stabilizing agent in the synthesis of ZnO NPs21. In green synthesis, bioactive phytochemicals such as polyphenols, flavonoids, and terpenoids act as reducing and stabilizing agents. Previous studies have reported that plant extracts rich in these compounds, such as Moringa oleifera leaf extract and Schinus molle seed and leaf extracts, exhibit remarkable antioxidant and antibacterial activities22,23. Moreover, other reports have highlighted the anti-biofilm and inhibitory effects of several herbal extracts, including ginger and clove, against H. pylori24,25. These findings support the use of plant-based extracts in green nanoparticle synthesis as an efficient strategy to enhance their antibacterial and anti-biofilm properties. Similarly, recent studies have shown that green-synthesized nanoparticles can modulate microbial gene expression and enhance antimicrobial performance, providing a sustainable and effective alternative to conventional nanomaterials26. Moreover, conjugating nanoparticles with bioactive agents such as antimicrobial peptides has been reported to improve their stability, selectivity, and therapeutic efficacy while reducing cytotoxicity27. Therefore, combining such biologically active green-synthesized nanoparticles with antimicrobial peptides may further enhance their biological efficacy through synergistic mechanisms. It has also been suggested that AMPs and nanoparticles can be combined to increase stability and selectivity, decrease toxicity, and enhance antimicrobial efficacy28. This combined approach may be especially successful against pathogens such as H. pylori, which form biofilms and are resistant to antibiotics.

The primary goal of this study is to investigate the antibacterial and antibiofilm effects of Ib-M1 peptide, green-synthesized ZnO NPs, and their combination against H. pylori. To gain a better understanding of how these treatments function. Furthermore, we investigated their influence on the expression of virulence-associated genes to elucidate potential mechanisms of action.

Results

Characterization of ZnO NPs

ZnO NPs were successfully synthesized using a green approach employing A. garcinii extract under varying drying temperatures (60 °C and 500 °C) and pH levels (8, 10, and 12). UV–Vis spectroscopy revealed characteristic absorption peaks at 230 nm for ZnO-60 and 238 nm for ZnO-500, indicating strong electronic transitions consistent with their semiconductor nature (Figure S1, Supplementary information). Dynamic light scattering (DLS) analysis showed that particle sizes ranged from 16.4 nm (pH 12, oven-dried) to 63.0 nm (pH 8, furnace-dried) (Table S1, Supplementary information). Higher pH values led to increased negative surface charge, enhancing electrostatic repulsion and yielding smaller, more stable nanoparticles. In general, particles dried at 60 °C exhibited smaller sizes compared to those dried at 500 °C, possibly due to reduced agglomeration and absence of high-temperature-induced recrystallization. Field emission scanning electron microscopy (FE-SEM) confirmed the spherical morphology and size distribution consistent with DLS data (Table 1) (Figure S2, Supplementary information). X-ray diffraction (XRD) analysis revealed sharp and distinct diffraction peaks corresponding to the hexagonal wurtzite crystal structure of ZnO NPs (Figure S3, Supplementary information).

Table 1 The size of ZnO NPs at 60 °C and 500 °C based on pH.
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Crystallite sizes calculated using the Scherrer equation were smaller in ZnO-60 compared to ZnO-500, indicating the impact of calcination on particle growth (Table 2). Fourier-transform infrared spectroscopy (FTIR) confirmed the presence of biomolecules from A. garcinii on the nanoparticle surfaces, which facilitated both reduction and stabilization during synthesis (Figure S4, Supplementary information). ZnO NPs synthesized had IC50 values were effective scavengers of DPPH radicals. IC50 values for both of them was higher than the maximum investigated concentration (500 μg/mL) (Fig. 1). The minor difference between them indicates that the calcination temperature had a negligible influence on antioxidant efficacy. Antibacterial assessments against Escherichia coli (E. coli) (Gram-negative) and S. aureus (Gram-positive), as presented in the corresponding tables, demonstrated that ZnO NPs synthesized under different pH levels and drying conditions exhibited comparable inhibitory and bactericidal effects (Table S2 and S3, Supplementary information). Nanoparticles prepared at pH 12 exhibited superior antibacterial activity, with the lowest minimum inhibitory concentration (MIC) (125 μg/mL) and minimum bactericidal concentration (MBC) (250 μg/mL) values. Based on these findings, ZnO NPs synthesized at pH 12 and dried at 60 °C were selected as the optimal formulation due to their smaller size, stability, and superior biological activities.

Table 2 Crystallite size of ZnO NPs calculated from XRD data using the Scherrer equation.
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Fig. 1
figure 1

DPPH scavenging activity of ZnO-60 and ZnO-500.

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Synthesis and characterization of the Ib-M1 peptide

The purity and molecular weight of the synthetic Ib-M1 peptide were provided by the manufacturer, GenScript (Piscataway, NJ, USA). According to high-performance liquid chromatography (HPLC) analysis, a dominant single peak was observed at a retention time of 11.14 min with an area percentage of 97.82%, indicating high purity (Figure S5 and Table S4, Supplementary information). Minor peaks accounted for less than 1.3% of the total area. The electrospray ionization mass spectrometry (ESI–MS) analysis, also conducted by GenScript in positive ion mode, confirmed the molecular weight of the peptide. Multiple charged peaks were observed at m/z 606.1 \[M + 4H]^4 + , 530.7 \[M + 5H]^5 + , 442.5 \[M + 6H]^6 + , and 883.8 \[M + 3H]^3 + , with the molecular weight calculated from the triply charged ion (m/z 883.8) being approximately 2658.4 Da, closely matching the theoretical mass of 2658.18 Da.

Antibacterial activity of ZnO NPs and Ib-M1 against H. pylori

As shown in Table 3, MIC values ranged from 16 to 32 µg/mL for ZnO NPs and from 500 to 1000 µg/mL for Ib-M1. The MBC values of ZnO NPs were between 32 and 64 μg/mL, whereas Ib-M1 showed MBC values from 1000 to 2000 μg/mL. These results showed that ZnO NPs had stronger antibacterial activity compared to the Ib-M1 peptide. The combination exhibited an additive effect against strain BY-1 (FICI = 0.98), while indifferent effects were observed for strains OC-824 (FICI = 1.51) and MZ-1 (FICI = 1.47). These data suggest that the ZnO NPs and Ib-M1 combination does not produce a strong synergistic effect in most strains but may improve antibacterial potency in specific cases. Further mechanistic investigations are warranted to explore the underlying interactions and potential clinical applications.

Table 3 The FICI values indicating the interaction between ZnO NPs and Ib-M1 peptide against three H. pylori strains.
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Evaluation of bacterial biofilm formation

The biofilm-forming ability of three H. pylori strains was quantitatively assessed using the crystal violet staining method. The MZ-1 strain exhibited strong biofilm production (OD570 = 0.243 ± 0.006), while strains BY-1 and OC-824 demonstrated moderate levels (OD570 = 0.140 ± 0.010 and 0.180 ± 0.009, respectively), indicating strain-specific variability. Statistical analysis revealed a significant difference in biofilm formation among the strains, with MZ-1 producing significantly more biofilm than BY-1 and OC-824 (p < 0.001) (Fig. 2).

Fig. 2
figure 2

Comparison of biofilm biomass among H. pylori strains.

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Assessment of antibiofilm activity

The minimum biofilm eradication concentration (MBEC) of ZnO NPs, the Ib-M1 peptide, and their combination were determined against three H. pylori strains (Table 4). The combination of ZnO NPs and Ib-M1 peptide significantly reduced MBEC values across all strains compared to either agent alone, indicating enhanced anti-biofilm activity (Fig. 3). These findings demonstrate that the combinatorial treatment is more effective in disrupting mature biofilms, supporting the possibility of a synergistic or additive effect.

Table 4 The MBEC values of ZnO NPs, Ib-M1 peptide, and their combination against H. pylori strains.
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Fig. 3
figure 3

Percentage reduction in MBEC of H. pylori strains when treated with the combination of ZnO NPs and Ib-M1 peptide compared to their individual treatments.

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SEM analysis of biofilm disruption

To evaluate the biofilm disruption by the tested compounds, the MZ-1 strain was selected due to its superior ability to form biofilms. Biofilms formed by this strain were treated with ZnO NPs, the Ib-M1 peptide, and their combination at concentrations corresponding to 4 × MIC. The 4 × MIC concentrations were 64 µg/mL for ZnO NPs, 2000 µg/mL for the peptide, and 32.25 µg/mL of nanoparticles combined with 250 µg/mL of peptide in the combination group. Images were acquired at magnifications ranging from ×5000 to ×20,000 to visualize detailed structural features of the biofilms (Fig. 4). Images shown are representative of at least three independent experiments, with multiple fields imaged per condition to ensure reproducibility. SEM analysis revealed that control samples exhibited dense and well-organized biofilms composed of healthy, tightly adherent bacterial cells (Fig. 4A). In contrast, treatment with 4 × MIC of the compounds showed a marked reduction in cell density and disruption of the biofilm matrix (Fig. 4B,C). Cells appeared scattered, with evident morphological alterations, including membrane wrinkling, shrinkage, and structural damage. The combined treatment exhibited the most pronounced biofilm disruption (Fig. 4D), leading to extensive destruction of the biofilm and a substantial decline in bacterial adherence.

Fig. 4
figure 4

SEM images of H. pylori MZ-1 strain biofilms under different treatments. (A) The untreated control shows dense, compact biofilm structure. (B) Treatment with ZnO NPs (64 µg/mL) resulted in a disrupted biofilm and reduced cell density. (C) Treatment with the Ib-M1 peptide (2000 µg/mL) causes disruption of the biofilm and scattered cells. (D) Combination treatment (32.25 µg/mL ZnO + 250 µg/mL peptide) showing extensive biofilm destruction and severe morphological damage. Images were captured at different magnifications (×10,000), with scale bars representing 2–10 µm.

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Gene expression of H. pylori virulence factors following treatment

Specific primers targeting the virulence- and stress-related genes (cagA, vacA, ureA, hspR, and spoT) in H. pylori were successfully designed. All primers generated specific amplicons suitable for qRT-PCR analysis. Detailed information regarding primer sequences, product sizes, and melting temperatures is provided in Table 5.

Table 5 Nucleotide sequences of primers used for qRT-PCR analysis of H. pylori virulence genes.
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To identify the optimal time point for RNA extraction, growth curves of the H. pylori strains were generated by monitoring optical density at 600 nm (OD600) at 0, 6, 12, 24, and 36 h post-inoculation. All strains demonstrated typical bacterial growth patterns, transitioning from the lag phase to the logarithmic (exponential) phase. The MZ-1 strain exhibited maximum growth at 24 h (OD600 ≈ 0.55), whereas BY-1 and OC-824 peaked at approximately 22 h (OD600 ≈ 0.70) and 20 h (OD600 ≈ 0.60), respectively. These points, representing the exponential phase of each strain, were chosen for RNA extraction and subsequent gene expression analysis (Fig. 5). The relative expression levels of the five target genes were analyzed following treatment with ZnO NPs, the Ib-M1 peptide, and their combination across all three H. pylori strains (Fig. 6). Expression of cagA was significantly downregulated in response to all treatments in all strains (p < 0.0001). In particular, ZnO NPs had a stronger suppressive effect than Ib-M1 in the BY-1 and OC-824 strains, whereas in the MZ-1 strain, the combination therapy resulted in the most pronounced inhibition (6A). Similarly, expression of vacA was markedly reduced following treatment (p < 0.0001). Among the tested conditions, the combination of ZnO and Ib-M1 consistently produced the greatest inhibitory effect, especially in the MZ-1 strain, highlighting a possible synergistic interaction (6B). The ureA gene also exhibited significant downregulation in all strains (p < 0.0001). In BY-1, the most substantial reduction was observed with ZnO NPs alone, whereas the combination therapy had a stronger effect in the MZ-1 strain. This suggests a differential response to treatments depending on the strain background (6C). Analysis of hspR expression showed that the combination of ZnO NPs and Ib-M1 led to more prominent downregulation than either agent alone, particularly in strains OC-824 and MZ-1 (p < 0.0001). This indicates that stress-related pathways may also be significantly impacted by the combined treatment (6D). Lastly, expression of the spoT gene was notably reduced in all strains following treatment. Once again, the combination therapy exerted the strongest suppressive effect in the MZ-1 strain, despite some variability in response across strains (p < 0.0001) (6E).

Fig. 5
figure 5

Growth curves of H. pylori strains based on optical density at 600 nm (OD600) measurements. Peak growth was observed at 21 h (BY-1), 24 h (OC-824), and 22 h (MZ-1), which were used as optimal time points for RNA extraction.

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Fig. 6
figure 6

The impact of ZnO NPs, Ib-M1 peptide, and their combination on gene expression was examined: cagA (A), vacA (B), ureA (C), hspR (D), and spoT (E).

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Flow cytometry of FITC-labeled H. pylori in AGS cells

According to Fig. 7, flow cytometry analysis was conducted to assess the internalization of FITC-labeled H. pylori into AGS gastric epithelial cells. In untreated infected cells (positive control), 21.9 ± 5.63% of AGS cells were FITC⁺, indicating a high degree of bacterial invasion (7A). Treatment with ZnO NPs at 1/2 MIC reduced the FITC⁺ population to 10.57 ± 2.37% (7 B), while the Ib-M1 peptide (1/2 MIC) further decreased the invasion rate to 8.06 ± 1.75% (7 C). Notably, the combination of ZnO NPs and the Ib-M1 peptide (each at 1/2 MIC) resulted in a synergistic effect, significantly reducing the percentage of FITC⁺ AGS cells to 3.6 ± 0.28% (7 D). For each sample, at least 10,000 events (cells) were collected and analyzed using flow cytometry, a standard protocol for quantifying bacterial invasion in host cells. Results are reported as mean ± SD from three separate experimental replicates. Statistical analysis revealed that all treatment groups significantly differed from the control (p < 0.01), and the combination treatment was significantly more effective than either treatment alone (p < 0.05).

Fig. 7
figure 7

Flow cytometric analysis of AGS cells infected with FITC-labeled H. pylori following various antibacterial treatments. (A) Positive control; (B) ZnO NPs; (C) Ib-M1 peptide; (D) Combination treatment. The percentage of intracellular H. pylori invasion is significantly reduced in group D compared to control.

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Cellular invasion of H. pylori to AGS cells

The ability of H. pylori to invade AGS gastric epithelial cells was assessed under different treatment conditions. When AGS cells were treated with ZnO NPs at 1/2 MIC, bacterial invasion was reduced by 25 ± 3.2% compared to the untreated control. In contrast, treatment with peptide Ib-M1 at 1/2 MIC achieved a 70 ± 4.5% reduction in bacterial invasion. Most notably, the combined treatment of ZnO NPs and peptide Ib-M1 (each at 1/2 MIC) resulted in the highest inhibitory effect, with an 85 ± 2.8% reduction in bacterial invasion compared to the control. All experiments were performed in triplicate, and results are reported as mean ± standard deviation. Statistical analysis showed that each treatment significantly reduced bacterial invasion compared to the control (p < 0.01). Moreover, the combined effect of the treatments was significantly stronger than that of either individual treatment alone (p < 0.05).

Cell viability assessment

The cytotoxic effects of ZnO NPs, Ib-M1 peptide, and their combination on AGS gastric carcinoma cells were assessed using the MTT assay with three independent replicates (Fig. 8). IC50 values were estimated to be approximately 48.19 µg/mL for ZnO NPs (Fig. 8A), 1362 µg/mL for Ib-M1 peptide (Fig. 8B), and 145.6 µg/mL (ZnO NPs)/250 µg/mL (Ib-M1) for their combination (Fig. 8C). Data are presented as mean ± standard deviation (SD). Statistical analysis revealed significant differences between groups (p < 0.0001).

Fig. 8
figure 8

MTT assay results showing the cytotoxic effects on AGS cells. (A) Effect of Ib-M1 peptide on cell viability at different concentrations. (B) Effect of zinc oxide ZnO NPs on cell viability. (C) Combined effect of Ib-M1 peptide and ZnO NPs on cell viability. Statistical analysis was performed using one-way ANOVA comparing each concentration to the control group (p < 0.0001).

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Discussion

The ZnO NPs synthesized in this study exhibited strong antibacterial activity against H. pylori, with MIC values ranging from 16 to 32 µg/mL. Notably, these MIC values are lower than those previously reported, where ZnO NPs showed MICs of 25–50 µg/mL29. These values are much lower than those reported elsewhere, where complete H. pylori inhibition required up to 4000 µg/mL ZnO NPs, highlighting the efficiency of our green-synthesized ZnO NPs. The enhanced antibacterial activity probably can be attributed to the optimized synthesis at pH 12 and 60 °C, which produced small (~ 18 nm), highly crystalline nanoparticles with a high surface area, facilitating Zn2⁺ release and ROS generation30. Moreover, as A. garcinii extract has shown antibacterial activity against S. aureus, it likely enhances the effects of our green-synthesized ZnO NPs20.

Compared with other antimicrobial peptides described as being significantly effective against H. pylori, such as bicarinalin (MIC50 ≈ 0.99 µg/mL) and Epi-1 (8–12 µg/mL), the MIC of Ib M1 (500–1000 µg/mL) appears relatively high31,32. In a previous study, the antimicrobial peptide Ib-M1 showed significant antibacterial activity against E. coli O157:H7 strains, with a reported MIC as low as 4.7 μmol/L (approximately 12.4 μg/mL)14. In this study, MIC values against H. pylori were higher (500–1000 μg/mL, ~ 188–376 μmol/L), likely due to physiological differences and antibiotic resistance mechanisms compared to E. coli. The unique outer membrane and surface charge of H. pylori may reduce the binding and membrane-disrupting effects of cationic peptides like Ib-M133,34. Moreover, species-specific mechanisms of resistance and disparities in membrane lipid composition can also suppress the antibacterial action of the peptide35. These factors, taken together, can justify the high MIC values observed against H. pylori. The combination of Ib-M1 peptide with ZnO NPs significantly reduced the MIC values of the peptide and enhanced its antibacterial activity. This effect was additive in some strains but indifferent in others, which may be explained by differences in efflux pumps activity among H. pylori strains.These results indicate that combining peptides and nanoparticles can enhance antibacterial efficacy, depending on the strain and experimental conditions36.

In the antibiofilm assay, both ZnO NPs and Ib-M1 effectively disrupted mature H. pylori biofilms at 4 × MIC, consistent with previous studies. Similarly, Medina-Ramírez IE37 showed the antibiofilm efficacy of ZnO NPs in comparable models. Comparable effects have also been reported for antimicrobial peptides38,39. Interestingly, combining ZnO NPs and Ib-M1 at lower concentrations than when used alone significantly reduced biofilm biomass and bacterial viability, suggesting that agents with different mechanisms may better target biofilms even without observable synergy in planktonic conditions40. Altogether, these results highlight the potential of combining nanomaterials and antimicrobial peptides as a promising strategy to combat biofilm-associated H. pylori infections. Flow cytometry analysis showed that ZnO NPs and Ib-M1 independently inhibit H. pylori invasion into AGS cells (FITC⁺ cells: 10.57 ± 2.37% and 8.06 ± 1.75%, respectively). Colony-forming unit (CFU) assays revealed 25% and 70% reduction in invasion by ZnO NPs and Ib-M1, respectively, while their combination reduced invasion by 85%, consistent with flow cytometry results and reflecting disruption of bacterial adhesion and entry mechanisms41 . Notably, combining treatments at sub-inhibitory concentrations (1/2 MIC each) markedly suppressed bacterial internalization (3.6 ± 0.28%), significantly more than either treatment alone (p < 0.05). This synergy may result from complementary mechanisms, as ZnO NPs generate reactive oxygen species and membrane stress to prevent H. pylori invasion42, the Ib-M1 peptide may directly bind to bacterial surfaces or interfere with virulence factor-mediated uptake43,44. Previous studies have also reported similar synergistic effects. For example, the Ib-M1 peptide, structurally resembling host defense peptides like β-defensins, may disrupt bacterial membranes through its cationic and hydrophobic properties and bind to adhesins such as BabA and SabA, key players in bacterial adherence to host receptors45. Collectively, these findings show that ZnO NPs and Ib-M1 not only have significant anti-invasive activity, but their combination at sub-lethal doses synergistically inhibits H. pylori invasion into gastric epithelial cells. This strategy may provide a novel and effective approach to target early pathogenic interactions, especially amid rising antibiotic resistance.

This study demonstrated that ZnO NPs and Ib-M1 significantly inhibit the expression of key H. pylori virulence and regulatory genes (cagA, vacA, ureA, hspR, and spoT). These findings are consistent with previous reports showing that metal-based nanoparticles can reduce CagA activity and peptide treatments can modulate bacterial gene expression46,47, including green-synthesized copper nanoparticles that inhibit MexA efflux pump expression48, highlighting their potential to suppress bacterial pathogenicity at the gene expression level. Notably, the extent of gene expression reduction varied among strains49,50. While cagA and ureA were consistently downregulated across all three strains, vacA, hspR, and spoT showed more variable responses, likely due to intrinsic genetic differences and variations in regulatory pathways and membrane composition affecting peptide and nanoparticle action51. The genes investigated in this study play a critical role in the adhesion, motility, biofilm formation, and virulence of H. pylori. Therefore, the downregulation of these genes by ZnO NPs and Ib-M1 may indicate their potential inhibitory effects on biofilm development as well as the reduction of bacterial virulence, which should be examined in future studies52,53. Strain-dependent variations in response, likely due to differences in surface charge, membrane lipid composition, or stress response systems, impact the susceptibility of different strains to antimicrobial agents. Furthermore, the efficiency of cellular uptake and biophysical interactions—such as electrostatic binding—between nanoparticles or peptides and the bacterial membrane plays a crucial role in explaining these differential responses54,55.

Cytotoxicity profiles, assessed by MTT assay (three replicates) showed IC50 values of ~ 48 µg/mL for ZnO NPs and 1356 µg/mL for Ib-M1, indicating higher safety for the peptide The relatively high cytotoxicity of ZnO NPs is inherent to Zn2⁺ ion release and ROS generation; however, the green synthesis using A. garcinii extract may partially mitigate these effects by providing phytochemical capping, which limits excessive oxidative stress and enhances biocompatibility. Notably, the IC₅₀ value obtained for ZnO NPs in this study is consistent with previously reported ranges for biogenic ZnO nanoparticles and generally lower than that of chemically synthesized ZnO NPs, suggesting improved safety of the green-synthesized form56,57. In the combination treatment, concentrations above and below the MIC were tested. The peptide concentration was kept constant due to its high safety margin, while ZnO NPs were varied. The combination showed a higher IC50 for ZnO than ZnO alone, suggesting the peptide may reduce oxidative or membrane-disruptive effects, improving safety while maintaining antibacterial efficacy. Furthermore, to better evaluate the therapeutic window, the selectivity index (SI = IC₅₀/MIC) was calculated for each treatment. ZnO NPs and Ib-M1 peptide alone exhibited SI values of 1.5 and 2.7, respectively, indicating moderate selectivity. Interestingly, the ZnO NPs in the combination treatment showed a markedly higher SI (≈9.3), suggesting an improved safety margin and reduced cytotoxicity while maintaining strong antibacterial potency. These results further support the potential of combining ZnO NPs with Ib-M1 as a safer and more effective therapeutic approach against H. pylori. Strain-dependent variations were observed, with some strains showing additive or even antagonistic interactions. Such context-specific outcomes may result from overlapping mechanisms, functional interference, or differences in cellular uptake58,59. Therefore, further molecular investigations—such as transcriptomic analyses or real-time imaging are warranted to elucidate these complex interactions and to guide the rational design of more effective combination therapies.

Conclusion

This study demonstrates that the integration of green-synthesized ZnO NPs with the antimicrobial peptide Ib-M1 exerts significant antibacterial and antibiofilm effects against H. pylori. While ZnO NPs displayed potent antimicrobial activity with relatively low MIC values, Ib-M1 showed higher MICs but excellent biocompatibility, and their combined use enhanced overall efficacy. The combination treatment effectively downregulated key virulence and regulatory genes, thereby limiting bacterial invasion into gastric epithelial cells. Cytotoxicity assessments confirmed a favorable safety profile, highlighting the potential of integrating nanomaterials with antimicrobial peptides as a promising strategy to overcome antibiotic resistance and improve therapeutic outcomes against H. pylori infections. Despite promising in vitro results, this study has limitations. The experiments were conducted under controlled laboratory conditions, which may not fully replicate the complex in vivo gastric environment. Future studies should investigate the pharmacokinetics, biodistribution, and long-term safety of these agents in vivo using approporiate animal models. Additionally, exploring the molecular mechanisms underlying strain-dependent responses and the interaction dynamics between nanoparticles and peptides could provide insights to optimize combination therapies. Addressing these aspects will be crucial for translating these findings into clinical applications against H. pylori infection.

Ethical approval

This study was approved by the Ethics Committee of Isfahan University of Medical Sciences (Ethics code: IR.MUI.MED.REC.1402.292), and all procedures were conducted in accordance with national and international ethical guidelines, including the Declaration of Helsinki. Informed consent was obtained from all subjects or their legal guardians prior to sample collection.

Materials and methods

H. pylori culture

Three H. pylori strains were used in this study, including two clinical isolates resistant to clarithromycin and metronidazole (H. pylori strains BY-1 and OC-824) obtained from the Helicobacter Research Labortaory, Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran, and one clinical strain resistant to metronidazole but sensitive to clarithromycin (H. pylori strain MZ-1), which was provided by the Department of Bacteriology and Virology, Isfahan University of Medical Sciences, Isfahan, Iran. The identity of all three strains was confirmed by PCR and sequencing.

H. pylori was cultured on Columbia agar base (Oxoid, Basingstoke, UK) supplemented with 10% fetal calf serum (FCS; Sigma, USA), 7% defibrinated sheep blood, and the antibiotics amphotericin B (Sigma, USA; 2 mg/L), polymyxin B (Sigma, USA; 2500 IU/L), and vancomycin (Sigma, USA; 6 mg/L). Plates were incubated at 37 °C in a microaerophilic environment (5% O2, 10% CO2, 85% N2) using a CO2 incubator (Innova CO-170, New Brunswick Scientific, USA). Broth cultures in Brucella broth (QUELAB, Canada) with 10% fetal bovine serum (FBS, Gibco, USA) were incubated under the same conditions.

Preparation of plant extract

The A. garcinii plant belongs to the Asteraceae family and is native to the southern regions of Iran. It was taxonomically confirmed by Dr. Mokhtar Zolfi Bavaryani, a plant taxonomist at the Research and Education Center for Agriculture and Natural Resources of Bushehr Province, Iran, in March 2021. The plant material was collected from the Tangestan district, Bushehr Province, Iran, and a voucher specimen has been deposited in the herbarium of this institution under the voucher number 3365, which is publicly accessible for reference. The collection of plant material was conducted in accordance with institutional and national guidelines, and permission for collection was obtained from the local environmental authority.

The leaves were washed thoroughly to remove any dust and debris, dried in an oven at 40 °C for 48 h, and then ground into fine powder. To prepare the aqueous extract, 5 g of A. garcinii leaf powder was added to 50 mL of double-distilled water (DDW) and boiled at 80 °C for 30 min using a magnetic stirrer. After cooling, the extract was filtered using Whatman No. 1 filter paper and stored at 4 °C until use.

Synthesis of ZnO NPs

In this study, the aqueous extract of A. garcinii was used to create ZnO NPs in an environmentally friendly manner. The plant extract served as both a stabilizing and reducing agent during nanoparticle formation.

To analyze the structural and physicochemical properties of the synthesized nanoparticles, several techniques were employed. The surface morphology and particle size were investigated using FE-SEM (Carl Zeiss Sigma 500 VP, Osterode, Germany). Functional groups were detected using FTIR (Jasco, Tokyo, Japan). DLS (Zetasizer Nano ZS90, Malvern Instruments, UK) was used to measure the hydrodynamic size and zeta potential. The crystalline structure was identified by XRD (ASENWARE AW-XDm300, Shenzhen, China). UV–Vis spectroscopy (V670 series UV/Vis spectrophotometer, Jasco, Tokyo, Japan) was used to evaluate optical properties and determine the band gap energy. Antioxidant activity was evaluated using the DPPH radical scavenging assay, and IC₅₀ values were calculated. The standard broth microdilution method (CLSI guidelines) determined the MIC and MBC against E. coli and S. aureus to evaluate antibacterial activity60,61.

Chemical synthesis and structural analysis of the Ib-M1

The synthetic antimicrobial peptide Ib-M1 (sequence: EWGRRMMGRGPGRRMMRWWR-NH2), consisting of 20 amino acids, was synthesized by GenScript (Piscataway, NJ, USA). The identity and purity of the peptide were confirmed by HPLC and ESI–MS as provided by the manufacturer. The peptide was dissolved in ultrapure water and stored at − 20 °C until use.

Evaluation of the antibacterial activity

The antimicrobial activities of the Ib-M1 peptide and ZnO NPs against three H. pylori strains were evaluated using the microbroth dilution method. Two-fold serial dilutions of green-synthesized ZnO NPs (8–500 µg/mL) and Ib-M1 peptide (31–2000 µg/mL; GenScript, USA) were prepared in Brucella broth (QUELAB, Canada) supplemented with 10% FCS (Sigma, USA). Subsequently, a bacterial suspension was added to each well of a 96-well microtiter plate (SPL Life Sciences, Korea) to achieve a final inoculum of approximately 1 × 105 CFU/mL. Negative control wells consisted of bacterial suspensions without any treatment. The plates were incubated at 37 °C under microaerophilic conditions for 48 h. MICs was defined as the lowest concentration at which no visible bacterial growth was observed and determined by measuring optical density at 600 nm (OD600) using a UV–Vis spectrophotometer (V-670, Jasco, Japan). For MBC determination, 10 µL from wells showing no visible growth were plated onto Columbia agar base (Oxoid, UK) supplemented with 5% sheep blood and incubated for 72 h. The MBC value was defined as the lowest concentration at which no bacterial colonies were observed62.

Checkerboard assay for synergistic activity

To assess the synergistic antibacterial activity of ZnO NPs in combination with the Ib-M1 peptide against H. pylori, a checkerboard microdilution assay was performed in 96-well microtiter plates (SPL Life Sciences, Korea). Two-fold serial dilutions of each compound were prepared and arranged in a checkerboard format, with ZnO NPs along the X-axis and Ib-M1 peptide (GenScript, USA) along the Y-axis, to generate a matrix of concentration combinations. The tested concentrations ranged from 8 to 128 μg/mL for ZnO NPs and 125 to 2000 μg/mL for Ib-M1. A bacterial suspension of H. pylori was added to each well to achieve a final concentration of approximately 1 × 105 CFU/mL. The plates were incubated at 37 °C for 72 h under microaerophilic conditions without shaking. After incubation, bacterial growth was visually assessed based on turbidity, and the MIC of each agent alone and in combination was recorded. The Fractional Inhibitory Concentration Index (FICI) for each ZnO NPs and Ib-M1 peptide combination was computed using a standard formula to assess the nature of their interaction.

FICI = (MIC of A in combination/MIC of A alone) + (MIC of B in combination/MIC of B alone). Based on the calculated FIC values, the interaction between the two antimicrobial agents was classified as63:

  • FICI ≤ 0.5: synergistic

  • 0.5 < FICI ≤ 1: additive

  • 1 < FICI < 4: indifferent

  • FICI ≥ 4: antagonistic

Evaluation of bacterial biofilm formation

Bacterial cells grown on Columbia agar plates (Oxoid, UK) were harvested and resuspended in brucella broth (QUELAB, Canada) supplemented with 2% FCS (Sigma, USA) to achieve a standardized optical density (OD600 ≈ 0.1). Then, 200 μL of the bacterial suspension was transferred into each well of a sterile 96-well microtiter plate (SPL Life Sciences, Korea). The plates were incubated at 37 °C under microaerophilic conditions for 48 h to allow biofilm formation. After incubation, non-adherent cells were gently removed by washing each well three times with 200 μL of phosphate-buffered saline (PBS; Gibco, USA). The attached biofilms were fixed with 200 μL of 99% methanol (Merck, Germany) and allowed to air-dry. The wells were then stained with 0.1% crystal violet solution (Sigma, USA) for 15 min. Excess dye was removed by washing with distilled water, and the bound stain was solubilized with 200 μL of 96% ethanol (Merck, Germany). Finally, absorbance was measured at 570 nm using a microplate reader (BioTek ELx800, USA) to quantify the biofilm biomass. Positive and negative controls were included. as follow: the positive control consisted of bacterial cultures without any treatment, representing maximal biofilm formation. The negative control consisted of wells containing only the medium without bacteria, representing background absorbance64,65.

Assessment of the MBEC

For determining the MBEC of ZnO NPs, the Ib-M1 peptide, and their combination against H. pylori, mature biofilms were first established. Overnight bacterial cultures were adjusted to an OD600 of 0.1 (~ 106 CFU/mL) and inoculated into 96-well flat-bottom polystyrene microtiter plates (SPL Life Sciences, Korea). The plates were incubated for 48 h at 37 °C under microaerophilic conditions to allow biofilm maturation. Then, non-adherent cells were gently removed by washing with phosphate-buffered saline (PBS; Gibco, USA), and fresh brucella broth (QUELAB, Canada) containing various concentrations of the antimicrobial agents was added to the established biofilms. The tested concentrations were 4 × MIC for ZnO NPs, 4 × MIC for the Ib-M1 peptide (GenScript, USA), and a fixed combination of 4 × MIC ZnO NPs + 4 × MIC Ib-M1 peptide. Prior to MBEC assays, a preliminary screening was performed using serial multiples of the MIC (1×, 2×, 4× and 8×). The preliminary results showed that 4 × MIC was the minimal concentration achieving ≥ 99.9% reduction in CFU for mature biofilms of all tested strains; therefore, 4 × MIC was selected for the MBEC experiments. Control groups included: (i) untreated biofilms, (ii) brucella broth with 2% FCS and containing antimicrobial agents without bacteria, and (iii) brucella broth with 2% FCS alone. The plates were further incubated for 24 h under the same conditions. Biofilm biomass was quantified by crystal violet staining (0.1% w/v; Sigma, USA) and measured at OD570 using a microplate reader (BioTek ELx800, USA). Bacterial viability was assessed by counting CFUs on Columbia agar (Oxoid, UK). The MBEC was defined as the lowest concentration capable of reducing viable bacterial counts by at least 99.9% compared to the untreated control29,66. The percentage reduction in MBEC for the combination treatment compared to individual treatments was calculated using the below formula:

$${\text{Percentage}}\;{\text{reduction}} = {\text{ MBEC}}_{{{\text{combination}}}} - {\text{MBEC}}_{{{\text{individual}}}} {\text{/MBEC}}_{{{\text{individual}}}} \times {1}00$$

SEM analysis of H. pylori biofilm

H. pylori biofilm was developed on sterile glass coverslips (Thermo Fisher Scientific, USA) placed in 6-well polystyrene microtiter plates (SPL Life Sciences, Korea) containing brucella broth (QUELAB, Canada) supplemented with 2% FCS (Sigma-Aldrich, USA) under microaerophilic conditions at 37 °C for 72 h. Following biofilm development, the coverslips were treated for 24 h with ZnO NPs, the Ib-M1 peptide (GenScript, USA), and their combination, each applied at a concentration of 2 × MIC. To remove planktonic cells, the coverslips were gently rinsed three times with phosphate-buffered saline (PBS; Gibco, USA).

The remaining biofilms were fixed overnight at 4 °C with 2.5% glutaraldehyde (Merck, Germany), then washed with cacodylate buffer (Electron Microscopy Sciences, USA). Dehydration was performed by immersing the samples sequentially in a graded ethanol series (25%, 50%, 75%, 95%, and 100%; Merck, Germany). Afterward, the samples were freeze-dried (Christ Alpha 1–2 LDplus, Germany) to eliminate residual moisture. Finally, a thin layer of gold was sputter-coated (EMITECH K550X, UK) onto the samples, and surface morphology was observed using SEM (Quanta 200, FEI, USA) equipped with an EDAX silicon drift detector (SDD, 2017)67.

Gene expression analysis of H. pylori virulence factors

Primers specific to the target genes were designed based on sequences retrieved from the NCBI database, using AlleleID 6.7 software (Premier Biosoft, USA). Primer specificity was confirmed using NCBI Primer-BLAST, and the validated primers were synthesized by Pishgam Biotech Co. (Tehran, Iran). The 16S rRNA gene was used as the internal control. To evaluate the effects of ZnO NPs, the peptide Ib-M1, and their combination on the expression of H. pylori virulence genes (cagA, vacA, ureA, hspR, and spoT), the bacteria were cultured in brucella broth (QUELAB, Canada) supplemented with 10% FBS (Gibco, USA) under microaerophilic conditions at 37 °C for 24 h. To determine the optimal timepoint for RNA extraction, growth curves were generated for each strain by measuring the optical density at 600 nm (OD600) using a UV–Vis spectrophotometer (V670, JASCO, Japan) at 0, 6, 12, 24, and 36 h post-inoculation. The initial inoculum was adjusted to an optical density at 600 nm (OD600) of 0.05. The exponential phase, as identified through the growth curves, was selected for subsequent gene expression analysis. Bacterial cultures were treated with ZnO NPs, Ib-M1 peptide, and their combination at sub-inhibitory concentrations (1/2 MIC) for durations determined by the growth kinetics. Total RNA was extracted using the FavorPrep™ Total RNA Mini Kit (Favorgen Biotech Corp., Taiwan), following the manufacturer’s instructions. The quality and concentration of extracted RNA were evaluated using agarose gel electrophoresis and quantified with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA). Complementary DNA (cDNA) was synthesized from total RNA using the Pars Tous™ cDNA synthesis kit (Pars Tous Biotechnology, Iran) with random hexamer primers. Quantitative real-time PCR (qRT-PCR) was carried out using gene-specific primers and the Amplicon™ 2X RealQ Plus Master Mix (Amplicon, Denmark) on a Rotor-Gene Q real-time PCR system (QIAGEN, Germany), following the manufacturer’s protocol. The 16S rRNA gene was used as the internal reference, and relative gene expression (fold change) was calculated using the 2^−ΔΔCt method, where ΔCt represents the difference between the Ct of the target gene and the Ct of 16S rRNA, and ΔΔCt represents the difference between ΔCt of the treated sample and ΔCt of the control68.

Flow cytometric analysis of H. pylori invasion to AGS gastric epithelial cells

To label H. pylori with fluorescein isothiocyanate (FITC), the bacteria were first cultured in Brucella broth (QUELAB, Canada) for 24 h under microaerophilic conditions. Following incubation, bacterial cells were harvested by centrifugation (10,000 × g, 5 min, 4 °C) and washed twice with sterile normal saline. The resulting pellet was resuspended in 0.1 M sodium bicarbonate buffer (NaHCO2, pH 9.0) to adjust the bacterial concentration to 0.5 McFarland standard (~ 1 × 108 CFU/mL). Fluorescein isothiocyanate (FITC; Sigma-Aldrich, USA) was added at a final concentration of 100 µg/mL, and the mixture was incubated in the dark at room temperature with gentle shaking for 30 min. After labeling, the bacteria were washed three times with phosphate-buffered saline (PBS; Gibco, USA) by centrifugation at 12,000 × g for 3 min to remove unbound dye. The FITC-labeled H. pylori were resuspended in PBS. For invasion assays, AGS cells (ATCC CRL-1739) were seeded into 24-well plates and co-incubated with FITC-labeled H. pylori at an MOI of 50 bacteria per host cell. During infection, tested antibacterial agents were added, including ZnO NPs (1/2 MIC), the synthetic peptide Ib-M1 (1/2 MIC), and their combination (each at 1/2 MIC). The co-culture was maintained at 37 °C under 5% CO2 for 4 h. To eliminate extracellular (non-internalized) bacteria, gentamicin (100 µg/mL; Bio Basic, Canada) was added to the medium during the final hour of incubation. After treatment, the human gastric adenocarcinoma cell line (AGS) cells were washed with PBS, detached using 0.25% trypsin–EDTA (Gibco), and analyzed by flow cytometry (e.g., FACSCalibur, BD Biosciences, USA) to quantify bacterial internalization based on FITC fluorescence69,70.

Cellular invasion assay using CFU counting

To investigate the ability of H. pylori to invade host cells, AGS cells were used. These cells were obtained from the Pasteur Institute (Tehran, Iran) and cultured in RPMI-1640 medium (Gibco, Invitrogen, Carlsbad, CA, USA). The medium was enriched with 2% FBS (Gibco, USA) and 1% penicillin–streptomycin (Gibco). Cells were plated in 12-well plates at a density of 1 × 105 cells per well. They were incubated for 24 h at 37 °C in an atmosphere with 5% CO2 to allow proper adhesion and growth. After incubation, the culture medium was replaced with antibiotic-free medium. Treatments included ZnO NPs (1.2 MIC), Ib-M1 peptide (1.2 MIC), or both together. Treatments were added simultaneously with H. pylori bacteria at a multiplicity of infection (MOI) of 50. After 5 h of incubation, gentamicin (100 μg/mL) was added for 1 h to eliminate extracellular bacteria. The wells were emptied and washed three times with PBS. Next, the cells were lysed with 150 μL of saponin. Then, 10 μl from each well was transferred onto Columbia blood agar. After 24 to 48 h of incubation, the plates were examined, and colonies were counted to determine the number of intracellular bacteria. The untreated control group consisted of AGS cells infected with H. pylori but without any ZnO NPs or Ib-M1 peptide treatment, serving as the baseline for bacterial invasion. The percentage of invasion for each treatment was calculated relative to the untreated control using the following formula71:

$$\% {\text{Invasion}} = {\text{CFU}}\;{\text{of}}\;{\text{treatment}}/{\text{CFU}}\;{\text{of}}\;{\text{control}} \times {1}00$$

Each experiment was performed in triplicate, and results are reported as mean ± SD.

Cell viability assessment using MTT assay

The cytotoxic effects of ZnO NPs, the Ib-M1 peptide, and their combination on AGS cells were evaluated using the MTT assay. AGS cells sourced from the Pasteur Institute (Tehran, Iran) were cultured in RPMI-1640 medium (Gibco, Invitrogen, Carlsbad, CA, USA) enriched with 2% FBS (Gibco, USA) and 1% penicillin–streptomycin (Gibco). Cells were seeded into 96-well tissue culture plates at a density of 1 × 105 cells/mL (100 μL per well) and incubated at 37 °C in a humidified atmosphere with 5% CO2 for 24 h to allow for adherence. Following incubation, the growth medium was replaced with 100 μL of fresh RPMI medium containing 2% FBS and various concentrations of ZnO NPs (16, 32, 64, 128, and 256 µg/mL), Ib-M1 peptide (200, 500, 1000, and 2000 µg/mL), or their combination (each at 1/2 MIC). Control wells received medium with 2% FBS only (no treatment). The cells were then incubated under the same conditions for an additional 24 h. After treatment, 20 μL of MTT solution (5 mg/mL in PBS; Sigma-Aldrich, USA) was added to each well, and the plates were gently shaken for 5 min before incubation at 37 °C for 4 h to allow for the formation of formazan crystals. Subsequently, the supernatant was carefully removed, and 200 µL of dimethyl sulfoxide (DMSO; Merck, Germany) was added to each well to dissolve the crystals. The plates were shaken for another 5 min, and the absorbance was measured at 560 nm using a microplate reader (BioTek ELx800, USA). All experiments were performed in triplicate. The IC50 values (concentration causing 50% inhibition of cell viability) were determined using a dose–response curve generated in GraphPad Prism software (version 8.0.2 GraphPad Software, San Diego, CA, USA)72.

Statistical analysis

Data were statistically analyzed using GraphPad Prism software version 8.0.2 (GraphPad Software Inc., San Diego, CA, USA), following standard analytical protocols. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to evaluate differences among groups. A p-value of < 0.05 was considered statistically significant. The IC50 values were obtained by fitting dose–response curves using nonlinear regression in GraphPad Prism. All major experiments, including MIC/MBC determination, MBEC assays, qRT-PCR, and cytotoxicity tests, were performed with at least three independent biological replicates. Each biological replicate was measured in triplicate as technical replicates to ensure accuracy and reproducibility of the results.