Campylobacter agar medium was purchased from Thermo Fisher Scientific. Acetylhydroxamic acid (AHA), jack bean urease (JBU, type III with specific activity 40.3 U/mg solid) and urea were obtained from Sigma Aldrich. Boric acid (BA) and sodium fluoride (NaF) were purchased from Maclin (Shanghai, China). Dithiothreitol (DTT) and L-cysteine (L-cys) were obtained from Solaibao (Beijing, China). Glutathione (GSH) was obtained from Meilun (Dalian, China). HEPES (Amresco >99%) was from BioFroxx. All chemicals and reagents were of analytic purity.

SGR was purchased from Zunyi (Guizhou, China) and authenticated by one of our authors (Qiang Lu). A voucher specimen has been deposited at the Zhuhai Campus of Zunyi Medical University for reference (No. 20240516). The materials were crushed using swing grinder. Medicinal powder was extracted with 70% ethanol in 1:15 (g: mL) ratio using hot reflux method, which was followed by successive repeated twice. The extracting solution was filtrated via a 200-mesh sieve and centrifuged at 8000 rpm for 20 minutes. The resulting supernatant was then concentrated and lyophilized under vacuum conditions. Additionally, dried sample in a loose or powdered state is viewed as the standard and stored at -4 °C.

SGR extraction was dissolved in acetonitrile and filtrated through 0.22 μm microporous membrane. The Acquity UPLC system equipped with the Waters Xevo G2 Q-Tof system integrated with a switchable electrospray ion source interface (ESI) was used to UPLC-MS/MS analysis. Analytical separation was conducted by Acquity UPLC BEH C18 (100 mm×2.1 mm, 1.7 μm). The mobile phase is acetonitrile (eluent A) and formic acid aqueous solution (eluent B), with a linear gradient elution: 0∼2.0 min, 3%A;2.0∼12.0 min, 3%~21%A; 12.0∼17.0 min, 21%~46%A; 17.0∼25.0 min, 46%~70%A; 25.0∼28.0 min, 70%~100%A, at a flowrate of 0.4 mL/min. The injecting volume was 5 μL and the column oven temperature was 40°C. Complete ESI positive ionization scanning from m/z 50-1200Da. Data collection and analysis were performed using TOF-MS^e software and Peak View 1.2 software, and the main active components of SGR were deduced according to the precise molecular weight and secondary fragment information.

H. pylori was inoculated in Campylobacter agar and grown on Columbia agar with appropriate bovine serum albumin at 37°C, 98% humidity, and low aerobic conditions (5% O₂, 10% CO₂, and 85% N₂) for 72 hours. Three days later, H. pylori was collected by scraping and then suspended in phosphate buffered saline (PBS). Besides, the H. pylori concentration was calibrated to 1×10⁸ CFU/mL by turbidimetric method. Standard HPU was extracted from H. pylori strain ATCC 43504 following the approach detailed by Matsubara et al. (2003). The resulting HPU preparation represents a crude enzyme extract, which has been widely adopted for initial inhibitor screening studies (Li et al., 2018; Tan et al., 2017; Yu et al., 2015). All inhibition assays included appropriate controls (enzyme blanks and solvent controls) to ensure specific detection of urease activity.

In this study, three H. pylori strains—ATCC 43504, NCTC 26695, and ICDC 111001—were utilized to evaluate the antimicrobial activity of SGR. Mueller-Hinton blood agar plates were prepared with varying SGR concentrations ranging from 0 to 1.5 mg/mL. A 100 μL suspension of each H. pylori strain was inoculated onto the respective SGR-supplemented plates. Positive controls (metronidazole) and negative controls (solvent water) were included in experiments. The plates were then incubated under microaerophilic conditions for three days. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of SGR or metronidazole at which no bacterial growth was observed compared to negative control wells.

The protein concentration of HPU was tested utilizing BCA protein detection kit. Standard urease test mixture consists of 150 mM urea in HEPES buffer (20 mM). HPU solution of varying concentrations was mixed with 150 mM urea as the substrate in a HEPES buffer (20 mM), and then reacted at 37 °C for 20 min. Urease vitality was assessed based on ammonia levels generated during the reaction. Ultimately, residual urease activity was measured by modified Berthelot (phenol hypochlorite) at 595 nm. This experiment was performed three times in parallel. The result revealed that the HPU activity was determined to be 17.0 U/mg compared to JBU (40.3 U/mg).

Test drug solution with equal volume and different concentration was mixed with urease and incubated in a 96-well plate at 37°C for 20 min. AHA was used as the positive control, while urease with urea (no depressor) served as the negative control. Moreover, urea solution (150 mM) was admixed and coincubated at ambient temperature for 20 min. Residual urease activity was measured via using the modified Berthelot method. The percentage of residual activity (RA%) was calculated as (A^sample - A^blank)/(A^{negative control} - A^blank)×100%, where A^blank represents the background absorbance without urease. Calculating the half-maximal inhibitory concentration (IC₅₀) of the depressor to assess the impact of the test drug towards enzyme activity. Each experiment was conducted in triplicate for validation.

Residual urease activity was determined by pre-incubating test drug, urease mixture with a series of urea concentrations. Michaelis constant (K_M ) and maximum velocity (v_max ) values are obtained from the Lineweaver-Burk plots of 1/v and 1/urea by plotting the reciprocal reaction velocity and substrate concentration. Variation characteristics of urease kinetic parameters K_M and v_max were analyzed by adding different concentrations of test products to determine the type of inhibiting effect on HPU. Each sample was performed in triplicate.

A functional relationship was established by measuring the impact of incubation time on ammonia concentration in the presence or absence of test drug solution. Before the reaction began, test drug mixed with urease was immediately reacted in a non-pre-incubated system. In contrast, test product was mixed with urease and incubated for 20 minutes prior to the addition of urea to the pre-incubation system. Urease vitality was determined according to standard measurements at various time points. Using a curve-fitting computer program, the experimental points are fitted into the following integral equation describing the progression curve:

Where, P_t represents the accumulated product yield at time t and 0, V₀ and V_s denote the initial and steady-state velocities of the reaction, while k_app stands for the apparent velocity constant.

Urease in the mixture was pre-incubated with the SGR solution (2 mg/mL) for 20 minutes. Subsequently, 1.25 mM GSH was introduced and coincubated with the pre-incubator to detect residual urease activity of the mixture at diverse time intervals. Residual enzyme vitality was inspected both before and after the introduction of GSH. Experiment was carried out three times in parallel.

Molecular docking software AutoDock Vina 1.1.2 was utilized to analyze the potential binding sites of astilbin and engeletin to urease. The selection of these two ureases was based on their complementary biological relevance: HPU (PDB ID: 1E9Y, resolution: 3.00 Å) represents the primary therapeutic target for H. pylori infection, while JBU (PDB ID: 3LA4, resolution: 2.05 Å) serves as a well-characterized reference with conserved catalytic domains that facilitates comparative mechanistic analysis. The 3D structure of astilbin and engeletin was obtained from PubChem database. The compound and target protein formats were converted to PDBQT files using AutoDockTools 1.5.6 software. Before docking, all water molecules were eliminated, and hydrogen atoms were placed on the receptors and given an electric charge. A cubic grid box of 60 × 60 × 60 Å with 0.375 Å spacing was centered at the average coordinates of the two Ni²⁺ ions (for HPU X=127.864, Y=126.349, Z=87.546; for JBU X=-39.959, Y=-44.679, Z=-74.986) to cover the entire active site. Docking employed the Lamarckian GA (10 runs, exhaustiveness=8) with Vina’s scoring function. Method validation confirmed reproducibility (RMSD< 2.0 Å for re-docked ligands). The resulting binding poses were analyzed using PyMOL for 3D visualizations, with binding energies compared between targets to elucidate species-specific interactions.

In this study, GraphPad Prism 13.0 software was utilized for data visualization. Data are presented as mean ± standard error (S.E.M). SPSS 29.0 software was employed for statistical analysis. Results were processed applying one-way analysis of variance (ANOVA) to determine statistical differences between groups, followed by the Dunnett test. The significance degree was set at p< 0.05.

As illustrated in Figure 1 and Table 1 , UPLC-ESI-MS/MS test revealed the 40 compounds including astilbin, engeletin, isoengeletin, neoastilbin, isoastilbin, and neoisoastilbin were identified in the SGR extract under positive and negative ion mode analysis. Moreover, the compounds can be roughly classified as organic acids, flavonoids, phenols, sesquiterpenes, etc., according to the precise molecular weight and secondary fragment information.

As illustrated in Table 2 , SGR exhibited varying degrees of growth inhibition against the three H. pylori strains under a culture condition of pH 7.2. Notably, the MIC of SGR against the standard strain ATCC 43504 was determined to be 1.5 mg/mL. In contrast, the MIC values for the strains NCTC 26695 and ICDC 111001 were significantly lower, both at 0.5 mg/mL, indicating greater susceptibility of these strains to SGR’s antimicrobial effects. In comparison, the standard antibacterial agent metronidazole demonstrated significant anti-H. pylori activity against strain ICDC 111001, with an MIC of 2.0 μg/mL.

As shown in Figure 2 , SGR demonstrated a significant inhibitory effect on HPU and JBU, with IC₅₀ values of 1.04 ± 0.01 mg/mL and 1.01 ± 0.01 mg/mL, separately. In addition, the IC₅₀ values of AHA, as standard urease depressor, were 4.93 ± 0.11 μg/mL and 1.56 ± 0.10 μg/mL for inhibiting HPU and JBU, separately.

As illustrated in the Lineweaver-Burk plot, all lines crossed at one location on x-axis, indicating that the kinetic parameter K_M of inhibition of HPU by SGR remained basically unchanged, while the value of V_max gradually decreased after adding various concentrations of SGR ( Figure 3A ). According to Lineweaver-Burk mapping analysis, the inhibitory type of SGR on HPU was non-competitive depressor. Additionally, the equilibrium parameters for binding of SGR to the free enzyme (K_i ) and to the enzyme-substrate complex (K_is ) were 0.10 ± 0.01 mg/mL and 0.12 ± 0.01 mg/mL, respectively, based on the relationship between the slope or intercept of the line in the Lineweaver-Burk diagram and the concentration of the suppressant ( Figures 3a, b ).

As depicted in Figure 3B , the plot of 1/v versus 1/urea consisted of multiple lines intersecting at one location in the second quadrant. K_M gradually elevated and V_max gradually declined following adding various concentrations of SGR. This suggested that SGR was a mixed suppressant for JBU. Moreover, the equilibrium parameters of K_i and K_is were 0.02 ± 0.02 mg/mL and 0.61 ± 0.08 mg/mL, separately ( Figures 3c, d ).

As illustrated in Figure 4 , depressor concentration and incubation time were found to have significant impacts towards the binding rate between SGR and urease. The curve fitting of the reaction process between SGR and HPU in unincubated and incubated system, shows a characteristic concave curve ( Figures 4A, B ), indicating rapid hydrolysis of urease at the initial velocity (V₀). With the influence of SGR on urease, there was a gradual inhibition on urease activity, resulting in a change in the hydrolysis urease from V₀ to steady-state velocity (V_s) based on the first-order velocity constant (K_app). Similarly, the reactive progress of SGR-JBU combination in unincubated and incubated system displayed a typical concave curve ( Figures 4C, D ), demonstrating that the combination had a constant equilibrium rate V₀ from the beginning, with the hydrolysis urease rate decreasing from V₀ to V_s. The reactive progress curves of JBU and HPU were consistent with the slow-binding suppression depicted by Morrison and Walsh (Morrison and Walsh, 1988).

Three thiol-containing substances (DTT, GSH and L-cys) were utilized to probe the possible inactivation sites of SGR-induced urease. As depicted in Figures 5A, B , the thiol-containing compounds exhibited a higher level of activity on urease than in the free of thiol-containing substances. Therefore, the sulfhydryl group of urease may be tightly relevant to the inactivation of urease by SGR.

Numerous researches have demonstrated that the inorganic substances BA and NaF are competitive urease depressors that repress urease activity via binding to nickel ions in the active center of urease (Mazzei et al., 2019). As illustrated in Figures 5C, D , SGR, BA, and NaF exhibit various degrees of inhibition on urease activity. The urease activity in the SGR mixed system containing BA or NaF significantly decreased, even lower than that in the SGR group, suggesting that BA and NaF may synergistically depress the activity of HPU and JBU with SGR. Therefore, sulfhydryl compounds were shown to restore urease activity more effectively than inorganic compounds. This suggests that SGR may bind to the thiol group, which is the active site of urease.

As seen in Figure 6 , thiol-containing substances can alleviate the inactivation of SRG on urease. Enzyme activity was tightly relevant to the coincubation time of urease, sulfhydryl compounds, and SGR ( Figures 6A, B ). In addition, the addition sequence of urease, sulfhydryl compounds, and SGR has no significant effect on enzyme activity ( Figures 6C, D ).

As depicted in Figure 7 , the urease activity decreased by approximately 80% after co-incubating urease and SGR for 20 minutes compared to its initial activity. However, with the addition of 1.25 mM GSH, HPU or JBU activities recovered approximately 40% of initial levels. The results indicated that the SGR-induced HPU or JBU reaction was reversible. The recovery of urease inhibitory activity by GSH further supports that sulfhydryl at the active site of urease exert a crucial function in the inactivation of urease by SGR.

As depicted in Figure 8 , the active ingredients of SGR have a good inhibitory effect on urease. The IC₅₀ of astilbin depressing HPU and JBU was 1.47 ± 0.01 mM, and 2.22 ± 0.02 mM, separately. The IC₅₀ of engeletin repressing HPU and JBU was separately 5.89 ± 0.01 mM and 6.67 ± 0.01 mM, suggesting that the inhibitory effect of engeletin on urease is not as effective as that of astilbin.

As shown in Figure 9 , the K_M value did not greatly change, whereas the v_max value declined with increasing astilbin concentration, implying that the inhibitory type of astilbin on HPU was non-competitive type ( Figure 9A ). The equilibrium inhibition parameters Ki and Kis were 0.87 ± 0.01 mM and 0.87 ± 0.01 mM, separately ( Figures 9a, b ). In contrast, during the binding process between astilbin and JBU, the K_M value was increased while v_max was decreased with increasing astilbin concentration, consistent with the kinetic characteristics of mixed inhibition ( Figure 9B ). The inhibition parameters Ki and Kis were 0.20 ± 0.01 mM and 1.17 ± 0.10 mM, separately ( Figures 9c, d ).

The molecular docking analysis was carried out, and the resulting interactions were visualized utilizing the Pymol software. The most probable binding modes of ligand with urease were depicted by the enzyme surface and cartoon mode. As shown in Figures 10A-D , astilbin exhibited docking scores of -8.0 kcal/mol for HPU and -7.6 kcal/mol for JBU. In HPU, astilbin formed hydrogen bonds with ARG 338, MET 317, HIS 138, HIS 221, GLY 279, and ALA 169 in the mobile flap region, and interacted with CYS 321, HIS 322, MET 366, and ALA 365 via hydrophobic forces, potentially stabilizing the flap in an open conformation and inhibiting catalytic activity. In JBU, astilbin hydrogen-bonded with GLY 638, MET 637, MET 588, and GLN 635, and engaged in hydrophobic interactions with HIS 593, ALA 440, ARG 609, and ARG 639, similarly stabilizing the flap in an open state and inhibiting enzyme activity.

Additionally, as illustrated in Figures 10E–H , engeletin demonstrated notable affinity for urease, with binding docking scores of -7.5 kcal/mol for HPU and -7.3 kcal/mol for JBU. Specifically, engeletin formed hydrogen bonds with key amino acid residues in the mobile flap region of HPU, including ARG 338, HIS 322, VAL 320, and ASN 168. Similarly, engeletin interacted with the amino acid residues HIS 593, MET 588, MET 637, and GLN 635 of JBU through hydrogen bonding, further highlighting its binding potential.