Component characterization of Smilax glabra Roxb., and its inhibitory activity against Helicobacter pylori through targeted suppression of its secreted urease

Abstract

Background: Smilax glabra Roxb. (SGR), known as "tufuling" in China, is a medical and edible plant, which has anti-inflammatory, antibacterial and antineoplastic activity. SGR is extensively utilized in the remedy of gastroenteric disorders associated with H. pylori infection. However, the precise mechanism underlying the anti-H. pylori function of SGR remains to be elucidated.

Aim: The inhibitory impact of SGR on the growth of H. pylori was examined. Subsequently, SGR against H. pylori urease (HPU) and jack bean urease (JBU) was investigated to illuminate the inhibitory effects, kinetic types, sites of inhibition, and potential mechanisms of action.

Methods: UPLC-ESI-MS/MS was applied to identify the components of SGR. The anti-H. pylori effect of SGR was conducted by agar dilution method. The enzyme inhibitory activities of SGR and its primary constituents were assessed through a modified spectrophotometric Berthelot (phenol-hypochlorite) assay. The kinetics of urease inhibition were analyzed using Lineweaver-Burk plots. To explore the underlying mechanisms, sulfhydryl group reagents and Ni²⁺ binding depressors were employed. Additionally, molecular docking simulations were conducted to examine the binding interactions between the main compounds of SGR and urease.

Results: A total of 34 compounds including astilbin, engeletin, isoengeletin, neoastilbin, isoastilbin and neoisoastilbin are identified in SGR. SGR was observed to inhibit the growth of three H. pylori strains (ATCC 43504, NCTC 26695, and ICDC 111001) with minimum inhibitory concentration (MIC) values spanning a range of 0.5 to 1.5 mg/mL. Moreover, SGR exerted 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. Enzyme kinetics analysis showed that SGR was a slow binding, non-competitive depressor to HPU, and a slow binding, mixed depressor to JBU. In-depth mechanism exploration showed that thiol compounds had better protective effect on HPU or JBU than inorganic substances, implying that the active site of SGR repressing urease may be the sulfhydryl group. Furthermore, glutathione reactivated SGR-inhibited urease, demonstrating that the inhibition was reversible. Additionally, astilbin and engeletin exhibited a certain inhibitory role towards urease activity, with astilbin inhibiting urease more than three times as strongly as engelitin. Enzyme kinetics analysis established that the inhibitory role of astilbin on enzymes was consistent with that of SGR. Molecular docking study indicated that astilbin and engeletin interacts with sulfhydryl groups at the active site of urease.

Conclusion: These results indicated that SGR could prominently inhibit H. pylori growth through targeted suppression of its secreted urease. This investigation provides substantial experimental evidence supporting the consideration of SGR as a safe and promising natural treatment for H. pylori-associated gastrointestinal diseases.

Keywords: Helicobacter pylori; Smilax glabra Roxb.; astilbin; molecular docking; thiol; urease.

Figure 1

Chemical composition analysis of Smilax glabra Roxb. (SGR). (A) The plants, rhizomes, and 70% ethanol extracts of SGR. Total ion chromatogram of SGR in positive (B) and negative ion mode (C). (D) Flavonoids including astilbin, engeletin, isoengeletin, neoastilbin, isoastilbin, neoisoastilbin was identified as the main chemical component of SGR.

Figure 2

Inhibitory effects of various concentrations of SGR on HPU (A) and JBU (B). Inhibitory role of AHA towards HPU (C) and JBU (D). The experimental data are exhibited as means ± SEM (n = 3).

Figure 3

Enzymatic kinetics analysis of SGR against urease. Lineweaver-Burk plots were constructed to illustrate the inverse of reaction velocities against the reciprocal of urea concentration for HPU (A) and JBU (B). These plots were generated in the presence of SGR at dosages of 0.0, 0.50, 1.0, and 2.0 mg/mL. (a, c) The inhibitive constant K_i was gained by plotting the slope of the Lineweaver Burk plot against the dosages of SGR. (b, d) The inhibitive constant K_is was gained by plotting the intercept of the Lineweaver Burk plot against the dosages of SGR. The experimental data are emerged as means ± SEM (n = 3).

Figure 4

Reactive progress curves of SGR against HPU and JBU. Reaction progress curve system can be divided into non-preincubated system [HPU (A), JBU (C)] and preincubated system [HPU (B), JBU (D)]. Curves were generated by evaluating the correlation between ammonia amount and incubating time (0–45 minutes) in the presence of SGR at dosages of 0.0, 0.5, 1.0, and 2.0 mg/mL. Experimental data are presented as means ± SEM (n = 3).

Figure 5

Impacts of sulfhydryl compounds on SGR-induced HPU (A) and JBU (B) inactivation. Impacts of inorganic substances on SGR-induced HPU (C) and JBU (D) inactivation. The dosages of SGR, sulfhydryl compounds (comprising DTT, GSH, and L-cys) and inorganic compounds (including NaF and BA) were 1.5 mg/mL, 1.25 mM and 1.25 mM, separately. Experimental data are emerged as means ± SEM (n = 3). *p< 0.05, **p< 0.01 vs. urease; #p< 0.05, ## p<0.01 vs. SGR group.

Figure 6

Effects of the incubation time and addition sequence of sulfhydryl reagents SGR-modified HPU (A, C) and JBU (B, D). Enzymic activity was assessed following co-incubations for 5, 10, 20 and 40 minutes. The compound enclosed in brackets was preincubated for 20 minutes, after which the final compound (outside brackets) was introduced and incubated for another 20 minutes. The concentrations of sulfhydryl compounds and SGR were 1.25 mM and 1.5 mg/mL, separately. Data are emerged as means ± SEM (n = 3). *p< 0.05, **p< 0.01 vs. the first column of each group.

Figure 7

Reactivation of SGR-inactivated HPU (A) and JBU (B) with 1.25 mM GSH. The dosage of SGR repressing HPU and JBU was 1.5 mg/mL. Enzyme viability was inhibited by SGR (•) and partially recovered after GSH addition (▴).

Figure 8

Inhibitory effect of the main active ingredients of SGR on urease. Astilbin-induced enzyme inactivation on HPU (A) and JBU (B). Inhibitory action of engeletin towards HPU (C) and JBU (D). Data are presented as means ± SEM (n = 3).

Figure 9

Kinetic investigation of urease inhibition by astilbin. Lineweaver-Burk plots of HPU (A) and JBU (B) were described in the non-existence and existence of diverse astilbin dosages. (a, c) The inhibitory parameter Ki was gained by plotting the slopes of Lineweaver-Burk plots versus astilbin dosages. (b, d) The inhibitory parameter Kis was gained from the plot of the intercepts of Lineweaver-Burk plots versus astilbin dosages.

Figure 10

Molecular docking analysis of astilbin and engeletin with urease. Enzyme surface and cartoon mode of the interplay between astilbin and HPU (A, B)/JBU (C, D). Enzyme surface and cartoon representations of the interaction between engeletin and HPU (E, F)/JBU (G, H). The yellow dashed line represents hydrogen bonding interactions.