Anti- Helicobacter pylori, anti-biofilm activity, and molecular docking study of citropten, bergapten, and its positional isomer isolated from Citrus sinensis L. leaves

Abstract

Introduction: Citrus sinensis L. is a candidate plant with promising antimicrobial potential. In the current study, the phytochemical investigation of C. sinensis leaf extract led to the isolation of three coumarins, namely bergapten, xanthotoxin, and citropten.

Methods: The chemical structures of the isolated coumarins were elucidated using NMR and ESI-MS techniques. The total aqueous ethanol leaf extract and the isolated coumarins were evaluated for their antimicrobial effects against Helicobacter pylori using the MTT-micro-well dilution method and its anti-biofilm activity using MBEC assay, as compared to clarithromycin.

Results: The results showed that citropten scored the lowest MIC value at 3.9 μg/mL and completely inhibited the planktonic growth of H. pylori. In addition, it completely suppressed H. pylori biofilm at 31.25 μg/mL. These findings have been supported by molecular docking studies on the active sites of the H. pylori inosine 5'-monophosphate dehydrogenase (HpIMPDH) model and the urease enzyme, showing a strong binding affinity of citropten to HpIMPDH with seven hydrogen bonds and a binding energy of -6.9 kcal/mol. Xanthotoxin and bergapten showed good docking scores, both at -6.5 kcal/mol for HpIMPDH, with each having four hydrogen bondings. Furthermore, xanthotoxin showed many hydrophobic interactions, while bergapten formed one Pi-anion interaction. Concerning docking in the urease enzyme, the compounds showed mild to moderate binding affinities as compared to the ligand. Thus, based on docking results and good binding scores observed with the HpIMPDH active site, an in-vitro HpIMPDH inhibition assay was done for the compounds. Citropten showed the most promising inhibitory activity with an IC₅₀ value of 2.4 μM. Conclusion: The present study demonstrates that C. sinensis L. leaves are a good source for supplying coumarins that can act as naturally effective anti-H. pylori agents.

Keywords: Bergapten; Citropten; Citrus sinensis; Helicobacter pylori; HpIMPDH; Molecular docking; Urease; Xanthotoxin.

Fig. 1

Ramachandran plot of the homology model of HpIMPDH enzyme.

Fig. 2

Isolated coumarins from C. sinensis L. leaves.

Fig. 3

MS/MS fragmentation patterns of bergapten, xanthotoxin, and citropten.

Fig. 4

Percentage of H. pylori growth inhibition at different concentrations of the extract, the isolated compounds (bergapten, xanthotoxin, and citropten) as compared to clarithromycin.

Fig. 5

Percentage of H. pylori biofilm eradication at different MIC-based concentrations of citropten.

Fig. 6

3D representation of predicted binding mode for C91, clarithromycin bergapten, xanthotoxin & citropten with 3D-structure of HpIMPDH.

Fig. 7

3D representation of predicted binding mode for compounds SHA, clarithromycin, bergapten, xanthotoxin & citropten with Urease (PDB ID: 6zja) active site.

Fig. 8

The HpIMPDH % inhibition of the isolated compounds and clarithromycin at different concentrations.