title: Biological evaluation and molecular docking of baicalin and scutellarin as Helicobacter pylori urease inhibitors.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
  
  
  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 Ni(2+) 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 Ni(2+) 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 S-H·π interaction, but did not interact with active site Ni(2+). 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.
authors.affiliation: College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, P.R. China.
authors.forename: Xiao-Dan
authors.initials: XD
authors.lastname: Yu
authors.affiliation: Guangzhou Wanglaoji Pharmaceutical Company Limited, Guangzhou, Guangdong 510450, P.R. China.
authors.forename: Rong-Bo
authors.initials: RB
authors.lastname: Zheng
authors.affiliation: College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, P.R. China; Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, The Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510120, P.R. China.
authors.forename: Jian-Hui
authors.initials: JH
authors.lastname: Xie
authors.affiliation: College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, P.R. China.
authors.forename: Ji-Yan
authors.initials: JY
authors.lastname: Su
authors.affiliation: The First Affiliated Hospital of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510405, P.R. China.
authors.forename: Xiao-Qi
authors.initials: XQ
authors.lastname: Huang
authors.affiliation: Guangdong Institute of Microbiology, Guangzhou 510006, P.R. China.
authors.forename: Yong-Hong
authors.initials: YH
authors.lastname: Wang
authors.affiliation: College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, P.R. China.
authors.forename: Yi-Feng
authors.initials: YF
authors.lastname: Zheng
authors.affiliation: College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, P.R. China.
authors.forename: Zhi-Zhun
authors.initials: ZZ
authors.lastname: Mo
authors.affiliation: College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, P.R. China.
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Wu
authors.affiliation: College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, P.R. China.
authors.forename: Dian-Wei
authors.initials: DW
authors.lastname: Wu
authors.affiliation: College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, P.R. China.
authors.forename: Ye-er
authors.initials: YE
authors.lastname: Liang
authors.affiliation: The First Affiliated Hospital of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510405, P.R. China. Electronic address: gancaozhf@126.com.
authors.forename: Hui-Fang
authors.initials: HF
authors.lastname: Zeng
authors.affiliation: College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, P.R. China; Dongguan Mathematical Engineering Academy of Chinese Medicine, Guangzhou, University of Chinese Medicine, Dongguan 523000, P.R. China.
authors.forename: Zi-Ren
authors.initials: ZR
authors.lastname: Su
authors.affiliation: College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, P.R. China. Electronic address: hping331@126.com.
authors.forename: Ping
authors.initials: P
authors.lastname: Huang
mesh_terms: D047310:Apigenin / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005419:Flavonoids / Q000737:chemistry / Q000494:pharmacology*; D005965:Glucuronates / Q000737:chemistry / Q000494:pharmacology*; D016480:Helicobacter pylori / Q000201:enzymology*; D006801:Humans; D062105:Molecular Docking Simulation; D014510:Urease / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D005419:Flavonoids; D005965:Glucuronates; C484876:scutellarin; C038044:baicalin; D047310:Apigenin; D014510:Urease
keywords: Baicalin; Helicobacter pylori; Molecular docking; Non-competitive inhibitior; Scutellarin; Urease