Elsevier

Journal of Ethnopharmacology

Volume 162, 13 March 2015, Pages 69-78
Journal of Ethnopharmacology

Biological evaluation and molecular docking of baicalin and scutellarin as Helicobacter pylori urease inhibitors

https://doi.org/10.1016/j.jep.2014.12.041Get rights and content

Abstract

Ethnopharmacological relevance

Baicalin and scutellarin are the principal bioactive components of Scutellaria baicalensis Georgi which has extensively been incorporated into heat-clearing and detoxification formulas for the treatment of Helicobacter pylori-related gastrointestinal disorders in traditional Chinese medicine. However, the mechanism of action remained to be defined.

Aim of the study

To explore the inhibitory effect, kinetics and mechanism of Helicobacter pylori urease (the vital pathogenetic factor for Helicobacter pylori infection) inhibition by baicalin and scutellarin, for their therapeutic potential.

Materials and methods

The ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol–hypochlorite) method. The inhibitory effect of baicalin and scutellarin was characterized with IC50 values, compared to acetohydroxamic acid (AHA), a well known Helicobacter pylori urease inhibitor. Lineweaver–Burk and Dixon plots for the Helicobacter pylori urease inhibition of baicalin and scutellarin was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode. Moreover, cytotoxicity experiment using Gastric Epithelial Cells (GES-1) was evaluated.

Results

Baicalin and scutellarin effectively suppressed Helicobacter pylori urease in dose-dependent and time-independent manner with IC50 of 0.82±0.07 mM and 0.47±0.04 mM, respectively, compared to AHA (IC50=0.14±0.05 mM). Structure-activity relationship disclosed 4′-hydroxyl gave flavones an advantage to binding with Helicobacter pylori urease. Kinetic analysis revealed that the types of inhibition were non-competitive and reversible with inhibition constant Ki of 0.14±0.01 mM and 0.18±0.02 mM for baicalin and scutellarin, respectively. The mechanism of urease inhibition was considered to be blockage of the SH groups of Helicobacter pylori urease, since thiol reagents (l,d-dithiothreitol, l-cysteine and glutathione) abolished the inhibitory action and competitive active site Ni2+ binding inhibitors (boric acid and sodium fluoride) carried invalid effect. Molecular docking study further supported the structure-activity analysis and indicated that baicalin and scutellarin interacted with the key residues Cys321 located on the mobile flap through Ssingle bondπ interaction, but did not interact with active site Ni2+. Moreover, Baicalin (at 0.59–1.05 mM concentrations) and scutellarin (at 0.23–0.71 mM concentrations) did not exhibit significant cytotoxicity to GES-1.

Conclusions

Baicalin and scutellarin were non-competitive inhibitors targeting sulfhydryl groups especially Cys321 around the active site of Helicobacter pylori urease, representing potential to be good candidate for future research as urease inhibitor for treatment of Helicobacter pylori infection. Furthermore, our work gave additional scientific support to the use of Scutellaria baicalensis in traditional Chinese medicine (TCM) to treat gastrointestinal disorders.

Introduction

Helicobacter pylori infection is the main cause of various gastric diseases, including chronic gastritis, gastric lymphoma, peptic ulcers, and stomach cancer (Parsonnet et al., 1994). At least half of the world׳s population is estimated to be infected by this bacterium (Conteduca et al., 2013). One of the main hallmarks of Helicobacter pylori is its constitutive urease production that generates a protective ammonium cloud from urea (Clyne et al., 1995), allowing it to survive in a hostile acidic environment. Moreover, this enzyme plays an important role in Helicobacter pylori adhesion, and urease-negative Helicobacter pylori mutants failed to colonize the gastric mucosa (Mobley et al., 1995).

Structural studies of Helicobacter pylori urease (HPU) have revealed a dinuclear Ni active site with a carbamylated lysine residue that bridges the deeply buried metal atoms (Ha et al., 2001). Around the active site, the hydrophilicity of the amino acids is characterized by highly flexible flap that undergoes an induced fit (Amtul et al., 2002). Interestingly, it has been revealed that the urease catalytic activity strongly depends on its multiple cysteinyl residues bearing sulfhydryl groups, especially those that is located on the mobile flap closing the active site (Ha et al., 2001, Zaborska et al., 2007).

Noticeably, urease was widely distributed in nature among plants, fungi, and bacteria (Mobley and Hausinger, 1989). Biochemically, the best-characterized plant urease was that from jack bean (Canavalia ensiformis) which was widely employed as a model of urease for inhibitory studies. But only some of the latter having profound medical implications like the one we studied currently. Since urease is the major colonization and virulence factor for Helicobacter pylori (Covacci et al., 1999), strategies based on urease inhibition are now essential lines of treatment for Helicobacter pylori infection. Hitherto, hydroxamic acids, phosphoramidates, urea derivatives, and quinones have been used as specific urease inhibitors (Kosikowska and Berlicki, 2011). However, the effective application of urease inhibitors directed to treat Helicobacter pylori infection has been limited either by unsatisfied bioavailability, or by toxicity with some of them. (Dominguez et al., 2008, von Kreybig et al., 1968). Consequently, it is worthwhile to discover and comprehensively study alternative urease inhibitors.

Scutellaria baicalensis Georgi, commonly known as “Huang-Qin” in Chinese, has extensively been incorporated into heat-clearing and detoxification formulas for the treatment of gastrointestinal disorders such as dyspepsia, gastritis, and diarrhea (Martin and Dusek, 2002). Research established that extractions of it have multiple bioactivities such as antimicrobial, anti-inflammatory and anti-oxidative activities (Yoon et al., 2009, Jeong et al., 2011, Lu et al., 2011). Baicalin (BA) and scutellarin (SL) (Fig. 1), the principal bioactive components of the root and the aerial part of this medicinal plant (Zhang et al., 2009), shared pharmacological activities including antioxidant (Shieh et al., 2000, Hong and Liu, 2004), anti-inflammatory (Yoon et al., 2009, Chen et al., 2013), and neuroprotective (Chai et al., 2013, Xu et al., 2013). Although it has been reported that BA and extractions of Scutellaria baicalensis exhibited anti-Helicobacter pylori activity (Shih et al., 2007, Wu et al., 2008) as well as inhibition on Helicobacter pylori–induced gastric inflammation (Shih et al., 2007), little has been done to elucidate the mechanism underlying their anti-Helicobacter pylori activity. While extensive studies have been carried out to investigate wide range of enzymes inhibitory activity of BA and SL (Chen et al., 2001, Tao et al., 2008, Deng et al., 2012, Jian et al., 2012, Wu et al., 2013b). However, with regard to HPU, pharmacological and biological evaluation of BA and SL toward this enzyme is unclear.

In our previous study, we have discovered that BA and SL ablated jack bean urease (JBU) activity effectively (Tan et al., 2013, Wu et al., 2013a). On the basis of the similarities in sequence and common structure for pivotal catalytic characteristic, it has been assumed that ureases from different sources have similar catalytic mechanism (Mobley et al., 1995). However, it was found that significant kinetic differences existed between the HPU and JBU (Cesareo and Langton, 1992). Therefore, it is necessary to explore the inhibitory potency, kinetics and mechanism of BA and SL on HPU activity for its therapeutic potential, as a continuation of our previous work whereas fresh insights into clinical implications. Meanwhile, the different structure-activity relations were observed, which should provide some information for developing novel urease inhibitor for treatment of Helicobacter pylori infection. Furthermore, our work should give additional scientific support to the use of Scutellaria baicalensis on traditional Chinese medicine (TCM) to treat gastrointestinal disorders.

Section snippets

Materials

Baicalin (CAS number: 21967-41-9) and scutellarin (CAS number: 27740-01-8) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity evaluated by high performance liquid chromatography (HPLC) was over 98%. Jack bean urease (type III with specific activity 40.3 U/mg protein), and other reagents were purchased from Sigma Aldrich (Steineheim, Germany). Bradford Protein Assay Kit was purchased from Beyotime Institute of

Effect of BA and SL on Helicobacter pylori urease activity

Both BA and SL inhibited HPU catalytic activity in dose-dependent manner, with SL being the superior inhibitor, compared to acetohydroxamic acid (AHA), a well-known HPU inhibitor (Fig. 2). At 0.42 mM, AHA reduced HPU catalytic activity by 54%, much greater than the inhibitory action that either BA or SL carried on HPU. This trend was also showed at the following two higher doses (0.56 and 0.75 mM). However, at 1 mM, such tendency was broken since SL reduced HPU catalytic activity by 88% which was

Discussion

In the present study, we have demonstrated that BA and SL inhibited HPU activity in a dose-dependent manner. (Fig. 2, Fig. 3), from which the data illustrated the following implications: (i) BA and SL suppressed HPU effectively. IC50 of BA and SL were approximately six and three times larger than AHA (Table 1), respectively; (ii) IC50 of BA was approximately two times larger than SL, suggesting 4′-hydroxyl group played a positive role in urease inhibition which gave accordance to the point that

Conclusion

Our findings suggested that BA and SL effectively inhibited the activity of HPU in a non-competitive and reversible manner. Structure-activity relationship disclosed 4′-hydroxyl gave flavones an advantage to binding with HPU. The sulfhydryl groups around the active site especially Cys321 were the targeted residue responsible for HPU inhibition. Although further investigation is required to study the physiological relevance of these results, these data and the relatively safe profile of BA and

Acknowledgments

This work was supported by Grant from National Natural Science Foundation of China (No.81374043), Specialized Research Fund for the Doctoral Program of Higher Education, Guangdong International Cooperation Project (No. 2012B050300002), The Special Funds from Central Finance of China in Support of the Development of Local Colleges and University, Educational finance Grant No.276(2014), Doctoral Fund of Ministry of Education of the People׳s Republic of China (No.20134425110009) and College

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