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 Table of Contents  
Year : 2022  |  Volume : 14  |  Issue : 3  |  Page : 132-139  

In Silico and In Vitro Investigation of Anti Helicobacter Activity of Selected Phytochemicals

1 Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Yarmouk University, Irbid, Jordan
2 Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Jordan, Amman, Jordan
3 Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, JUST University, Irbid, Jordan; Department Pharmacological, Diagnostic Research Centre, Faculty of Pharmacy, Al Ahliyya Amman University, Jordan
4 Department Pharmacological, Diagnostic Research Centre, Faculty of Pharmacy, Al Ahliyya Amman University, Jordan

Date of Submission17-Dec-2021
Date of Decision14-Mar-2022
Date of Acceptance19-Jul-2022
Date of Web Publication19-Sep-2022

Correspondence Address:
Dr. Talal Aburjai
Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Jordan, Amman
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpbs.jpbs_850_21

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Introduction: Helicobacter pylori is Gram-negative helical bacteria that inhibit stomach mucosal lining and establish infection. Urease enzyme was confirmed to be pivotal target in which its suppression will prompt bacteria treatment and eradication. Methods: Series of naturally bioactive compounds were selected based on ethnobotanical and molecular modeling techniques with potential urease inhibitory effect. The selected phytochemical compounds were in-silico and in-vitro assayed against urease enzyme, minimal inhibitory concentrations (MIC) and a synergistic effect was studied and cultured specifically for H. pylori. Results: Terpineol was considered as the most active compound with an IC50 of 1.443 μg/ml (R2 = 0.9374). The synergistic effect of terpineol and metronidazole indicated a possible additive effect (fractional inhibitory concentration result is 0.78) with improvement of MIC results for both terpineol and metronidazole. Conclusion: This study suggests that terpineol is best to be considered as a lead compound for H. pylori infection treatment and could be a potent inhibitor when combined with metronidazole targeting urease enzyme.

Keywords: Antibiotics, bioactive ligands, H. pylori, terpineol, urease inhibition

How to cite this article:
Al Tawalbeh D, Aburjai T, Al Balas Q, Al Samydai A. In Silico and In Vitro Investigation of Anti Helicobacter Activity of Selected Phytochemicals. J Pharm Bioall Sci 2022;14:132-9

How to cite this URL:
Al Tawalbeh D, Aburjai T, Al Balas Q, Al Samydai A. In Silico and In Vitro Investigation of Anti Helicobacter Activity of Selected Phytochemicals. J Pharm Bioall Sci [serial online] 2022 [cited 2023 Feb 2];14:132-9. Available from:

   Introduction Top

Since its discovery in 1982 by Marshall and Warren, Helicobacter pylori has captured the attention of drug discovery researchers.[1] The impact of H. pylori on human health upgraded from causing stomach and peptic ulcer and chronic gastritis to be classified as the first pathogen that may cause carcinomas according to the WHO criteria.[2]

H. pylori is a Gram-negative microaerophilic bacterium that infects more than 50% of the world's population.[3] This infection follows geographic and socio-demographic distribution and it occurs markedly higher in developing countries.[4] Although the mode of transmission of H. pylori is not fully understood yet, but it seems that oral–oral, gastro–oral, and fecal–oral routes are possible routes for transmission while they are directly influenced by the level of hygiene and the socio-economic status of the country.[5]

The well-established treatment guidelines for H. pylori infection include the use of at least two antibiotics accompanied by a proton pump inhibitor. This combination was effective for the pathogen eradication for decades till the emergence of resistant strains of H. pylori attributed to treatment collapse.[6] This led to an instant demand to search for novel alternative treatment regimens that improve the eradication of the pathogen with alleviated toxicity and side effects on host cells.[7]

Nowadays, complementary and alternative modes of treatment originated from plant sources are considered attractive for being relatively safe and affordable.[8] Many in vitro and in vivo studies support the effectiveness of some plant extracts against H. pylori infection attributed to their wide spectrum anti-bacterial and anti-fungal properties.[9] Consequently, our research group has emanated from this fact in screening the literature for plants in which its extract has showed activity against H. pylori. This effort has resulted in selecting (–)-alpha-thujone, alpha-terpineol, (+)-alpha-pinene, D-limonene, and β-sitosterol that then being evaluated for their possible antimicrobial effectiveness in treating and eradicating H. pylori infection.

To investigate the efficiency of the selected compounds against H. pylori, our research group has found that H. pylori urease enzyme is a validated and pivotal target (PDB code: 1E9Y). Urease enzyme inhibition will lead to incapacitating the bacteria from being harmful [Figure 1]a. It was discovered that the active site of this enzyme is composed of a shallow binding pocket that is occupied with two nickel atoms as structural cofactors [Figure 1]b.
Figure 1: (a) A 3D representation of the docked pose compound acetohydroxamic acid (AHA) within the active site of the urease enzyme (PDB code 1E9Y). The binding site is represented according to the solvent accessible surface (SAS) and as shown, it is away from the solvent accessible area. Docked compound is shown with carbon atoms in gray and the Ni + 2 ion with gray spheres (b) A 2D interaction map showing the interaction between AHA and the active site of the enzyme. Residues are colored according to their type of reaction with the ligand. The interactions are shown as dashed lines

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Acetohydroxamic acid (AHA) is a hydroxamic acid derivative with appreciable inhibitory effect. It is considered as a reversible slow-binding inhibitor of H. pylori urease.[10] The mode of AHA–urease inhibition could be explained by the formation of bidentate complex between the carbonyl oxygen of the hydroximate moiety and the nickel ion. The structure of AHA slides to the active site perfectly without causing any unfavorable steric interaction with the surrounding amino acids. This co-ordinate bonding will lead to unsuitable conformational deformities in the active site, thus, inactivating the enzyme.[11] Unfortunately, compounds possessing hydroxamate scaffold have showed undesirable teratogenic side effects hindering its use in H. pylori disposal.

Because of the importance of urease in H. pylori colonization, we focused on investigating the possible inhibitory effect of the selected bioactive ligands on urease enzyme that can lead the future studies to improve and develop a bioavailable, selective, and effective treatment for H. pylori.

The essentiality of urease enzyme in H. pylori viability and colonization has prompted us to screen literature for compounds that have ethnobotanical propensity for its inhibition with the assistance of molecular modeling studies.

   Methods Top

Materials and chemicals

All chemicals, reagents, and solvents were of analytical grade and used directly without further purification as follows: thujone (TCI, Tokyo), terpineol (Santa Cruz Biotechnology, the Netherlands), pinene (TCI, Tokyo), limonene (Santa Cruz Biotechnology, the Netherlands), β-sitosterol (Sigma, USA), and dimethyl sulfoxide (DMSO) (Xilong Chemicals, China).

In silico design

Ligand preparation

All bioactive ligands were sketched using ChemDraw – Ultra 12.0 (United States Fremont, California), then exported into discovery studio (DS) 2019 from BioVia® (United States, San Diego, California), and then converted into their corresponding 3D structures using “prepare ligand” protocol. Within this protocol, assignment of proper bond orders, generation of ionization states, and assignment of chemical isomers and tautomer could be achieved by using default parameters. The ligands were prepared then minimized using “in situ ligand minimization” protocol.

Protein preparation

The X-ray structure of urease enzyme of H. pylori (1E9Y) in complex with AHA was retrieved from the Protein Data Bank ( (PDB code 1E9Y: resolution 3.00 Å) to serve as docking template.[12] The enzyme was checked for missing loops, alternate conformations, or incomplete residues using “prepare protein protocol” within DS. It was applied to clean the crystal structure, correct its connectivity and bond order, and to standardize atom names. The enzyme is a dimer that encompasses chains A and B, the later contains the active site that performs the hydrolysis step of urea with the aid of two nickel atoms. The B chain's two nickel atoms hybridization was modified into octahedral with +2 charge assignment. The protein was prepared using “prepare protein” protocol, then solvated by subjecting “solvation protocol” to be ready for the minimization step in the presence of the co-crystalized ligand using “protein minimization protocol.” The minimized protein was ready for docking after removing water molecules and other excessions.

Molecular docking

Molecular docking of the bioactive ligands against H. pylori urease was carried out using DS. The solvated and energy-minimized crystal structure was used after removing the co-crystallized ligand (AHA) that was used to define the binding site using the Define and Edit Binding Site tool. The radius of the generated sphere was chosen to be 12 Å radius centered around AHA. Subsequently, the dock ligands protocol (Libdock) was employed using default parameters.

Drug-likeness analysis

Drug-likeness analysis for our bioactive ligands was performed using DS by employing “ADMET (absorption, distribution, metabolism, and excretion) protocol” that predicts absorption, distribution, metabolism, excretion, and toxicity parameters of the study ligands. The parameters that have been studied are blood brain barrier (BBB) permeability, CYP2D6 inhibitor, AMES (Biological testing method) toxicity, TD50 (Median toxic dose) for rats, LD50 for rats, maximum tolerated feed dose, maximum tolerated gavage dose, Alogp98, plasma protein binding, hepatotoxicity, absorption level, BBB level, and PSA (Polar surface area).

In vitro design

Bacterial strain and culture conditions

Colombia blood agar base (Oxoid Ltd., Basingstoke, Hampshire, England) was sterilized by autoclaving (121°C for 15 min), then enriched with 7% (v/v) laked horse blood (Oxoid Ltd., Basingstoke, Hampshire, England), and supplied with H. pylori selective supplement (Dent) (Oxoid Ltd., Basingstoke, Hampshire, England). Our standard strain (NCTCC 11916) of H. pylori was used for testing.

After spreading the bacteria, plates were incubated in Candle jar 2.5L (Oxoid ltd., Basingstoke, Hampshire, England) with 98% humidity at 37°C, this environment achieved by using CampyGen Gas Pack (Oxoid Ltd., Basingstoke, Hampshire, England), and the incubation period was 7 days.[13]

The presence of H. pylori growth on plates was confirmed by typical spiral morphology and flagella presence of the colonies, Gram staining, and conventional biochemical tests, including urease, catalase, and oxidase reactions (positive for urease, catalase, and oxidase reactions).[14]

Anti-microbial sensitivity testing and minimum inhibitory concentration (MIC) determination

Disc diffusion method on solid Colombia agar media was used to determine the susceptibility of H. pylori to the selected bioactive compounds. Sterile blank discs (6 mm) (Oxoid Ltd., Basingstoke, England) were impregnated with 30 μl of tested bioactive compounds, amoxicillin and metronidazole discs were used as positive control. Discs placed on each plate and incubated for 7 days under suitable cultivation conditions.[15]

MIC were determined according to the Clinical and Laboratory Standards Institute (2009),[16] by agar dilution methods, the bacterial suspensions were prepared to the two McFarland's standard and inoculated into Colombia blood agar media supplemented with 7% (v/v) laked horse blood and the tested bioactive compound. The MIC of each compound was determined after 5 days of incubation under microaerophilic conditions.

The MIC was recorded for the lowest concentration that inhibited visible growth of H. pylori bacterium. The plates with metronidazole served as positive control and plates with DMSO served as negative control. Triplicate tests were performed for each bioactive compound.

Synergistic effect between α-terpineol and metronidazole

Checkerboard test was carried out to check the synergistic activity between terpineol and metronidazole,[17] with two-fold serial agar dilution of the prepared bioactive compound (terpineol) and metronidazole against reference strains of H. pylori.

Terpineol was dissolved in DMSO/PBS in ratio of 2:1 and serially diluted in PBS to obtain concentrations 25, 12.5, 6.25, 3.125, 1.563, and 0.7813 mg/ml, after that metronidazole (51.2 mg) was dissolved in 10 ml DMSO/distilled and serially diluted in PBS to obtain 5.12, 2.56, 1.28, and 0.64 mg/ml concentrations. A volume of 0.5 ml of diluted terpineol and metronidazole was mixed with 9.5 ml of molten Columbia agar media (supplemented with 7% laked horse blood) triplicity.

The fractional inhibitory concentrations (FICs) were calculated as follows:

FIC = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone)

The FIC index interpreted as: ≤0.5 synergy, 0.5 – 1 additive, 1 – 4 indifference, ≥4 antagonism.[17]

Urease inhibition assay

About 10 μl of 6 × 108 CFU/ml H. pylori suspension was mixed with an equal amount of serially diluted solution of terpineol in 96-well plate and incubated for 30 min at 37°C. After that, 200 μl of detecting reagent was added (50 mM PB pH 6.8, urea 500 mM0, and phenol red 0.02%) (Oxoid Ltd., CM 0053, England) for each well, the color change was monitored by measuring the OD at 555 nm in 5-min intervals for 2 h.

Bacterium alone was the negative control and terpineol without bacteria was the positive control. The percentage of inhibition was calculated as follows:

Percentage of inhibition = [(activity without terpineol – activity with terpineol)/(activity without terpineol)] × 100

The reference used was urease inhibitor AHA (Sigma, USA).

   Results and discussion Top

Within this study, both molecular docking and in vitro assay were executed to identify the activity of the candidate ligands against urease enzyme. The candidate ligands were selected according to the literature and ethnopharmacological studies in Jordan.[18],[19] The identified ligands were D-limonene, (+)-α-pinene, β-sitosterol, α-terpineol, β-terpineol, γ-terpineol, and (-)-α-thujone [Table 1].
Table 1: The 2D structure of the selected bioactive compounds and our reference ligand AHA available from PubChem

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Before starting our docking studies and to avoid any wastage of time and efforts, the selected ligands were subjected to strict selection criteria based on its docked parameters of candidate ligands which has been calculated and presented on [Table 2] and their obeyable characters to Lipinski's rule of 5 where showed in [Table 3]. However, the results revealed that the ADME parameters for the candidate ligands are suitable for being selected for the docking studies [Table 4].
Table 2: Calculated parameters of docked ligands using Libdock protocol

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Table 3: Physicochemical properties of ligands

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Table 4: ADMET prediction of ligands

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To understand the mode of interaction between the selected ligands and the urease active site, molecular repositioning of AHA was performed on the H. pylori urease enzyme (PDB code: 1E9Y). First, analysis of the binding site of the AHA ligand with urease enzyme was performed. The binding site comprises four His residues that are His248, His274, His221, and His138, KCX 219 (carbamylated lysine), Asp362, and Ala365 with the two nickel atoms Ni+23001 and Ni+23002 as showed in [Figure 2].
Figure 2: Binding mode of terpineol with H. pylori urease. (a) A 3D representation of the docked pose of terpineol within urease active site, the binding site is represented as hydrophilic area (blue is positively charged and brown is hydrophobic areas). Terpineol is in balls and sticks, carbons colored green, and Ni+2 colored in dark gray.(b) 2D interaction map showing the different interactions between terpineol and the active site as dashed line, residues are colored according to their type of interaction with the ligand

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The active pocket contains two positively charged nickel atoms and amino acids Ala365, Ala169, and Met366 forming partially positive-charged surface. AHA binds strongly to the active site after the oxime moiety being negatively ionized forming salt bridge. This ionic bond is expected to be crucial in reflecting the reported activity of AHA.[10]

To validate the docking parameters set by DS for the suitability of our work, the AHA was extracted from the crystal structure then re-docked into the active site using Libdock. The re-docked AHA ligand was then superimposed to its active urease enzyme structure. The root means square deviation achieved was 0.67 Å, and showed the same binding pattern and orientation. Thus, validation is essential to trust our docking results.[20]

[Figure 3] shows the best docked pose of terpineol to the active site of the enzyme, it appears that the hydroxyl moiety coordinates with the nickel ion in a bidentate fashion through the exocyclic hydroxy donor atom. The cyclic part of the structure provides an interaction area with hydrophobic region of Met366, Ala365, and Cys321 stabilizing the compound in the active site.
Figure 3: Docked pose of terpineol

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After docking the candidate ligands into the active site, the interactions between the enzyme and the docked ligands were investigated using docking scores and by reporting Libdock score and Libdock energy as shown in [Table 2].

The docking scores showed that the best fitting bioactive compounds are β-sitosterol, α-terpineol, and (-)-α-thujone, this is approved by more negative binding energy values and the high Libdock scores compared to our reference AHA. Also, it can be seen how electrostatic energy declined largely when α-terpineol fits with the receptor reflecting the high affinity of the candidate ligands toward the active site.

After studying the physicochemical properties of our ligands and comparing them to Lipinski's rule, we observed that all the ligands are not violating Lipinski's rule. Furthermore, ADMET results are in concordance with results presented in the in silico docking, and the α-terpineol and (-)-α-thujone can penetrate the CNS and cross BBB with possible mutagenic properties as seen in TOPKAT Ames as showed in [Table 5].
Table 5: TOPKAT dose prediction for ligand

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Among the selected candidate, terpineol and thujone showed strong inhibition activity against H. pylori in in vitro studies with MIC results of 0.125 and 0.25 mg/ml, respectively, whereas β-sitosterol showed minimal inhibitory effect with MIC of 1 mg/ml [Figure 4] and [Table 2].
Figure 4: in vitro studies of β-sitosterol MIC against H. pylori

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Although previous studies showed that plant extracts that contain β-sitosterol may inhibit H. pylori growth and act synergistically on the bacterium colonization,[21] there is no direct correlation between the presence of β-sitosterol and the antibacterial effect of those extracts and this supports our result of the weak inhibitory effect of β-sitosterol. The structure of β-sitosterol is similar to sterols that are present in the cell and this may explain how they can enter the cell and replace those sterols to exert their antimicrobial activity. However, their large structure and possessing only one hydroxide group reduce their antimicrobial effect to the minimum, making them not superior as antimicrobials to the current used medications.[22]

Akrout et al.[23] in their study observed that as the number of oxygenated monoterpenes increases in the essential oil, the antimicrobial activity becomes more pronounced. The position of hydroxyl group and the inclusion of unsaturated cyclohexyl group influence the effectiveness of the volatile oil activity. In another study, Dorman et al.,[24] confirmed that the hydroxyl group enhances the antimicrobial activity of the volatile oils, this can explain the pronounced effect of thujone as antibacterial agent when compared to D-limonene and α-pinene, although the presence of ketone ring improved the activity of thujone, its low water solubility and hydrogen bonding capacity Allow its entry into Gram-negative bacteria.

According to the MIC results, terpineol (IC50 1.443μg/ml) has higher potency than thujone (IC50 2.688 μg/ml) [Figure 4] and metronidazole (MIC is 0.512 mg/ml), and this can be explained by the presence of phenolic group in terpineol structure that improves the antimicrobial activity, this oxygen in the framework may play a significant role in improving the antimicrobial activity of terpineol when carbonylated, it interferes with the lipid bilayer of the outer membrane leading to a more permeable bacterial cell membrane with possible protein leakage.[25]

Although metronidazole remains the corner stone in H. pylori infection therapy, about 10–80% of worldwide patients complain of metronidazole resistance because of a point mutation in nitroreductase encoding genes rdxA and frxA in H. pylori, and this problem appears more frequent in females because of their excessive use of metronidazole in gynecological problems.[26]

After studying the standard checkerboard test for terpineol and metronidazole combination and as shown in [Figure 4], the MIC of metronidazole was improved from 0.512 to 0.069 mg/ml, and the MIC of terpineol improved from 0.125 to 0.078 mg/ml, thus, the calculated FIC value of this combination was 0.78 and indicates a possible additive effect as showed in [Table 6].
Table 6: MIC, FIC, and IC50 values of tested bioactive compounds

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The additive effect of terpineol and metronidazole could be because of their different pharmacological mechanisms. Terpineol is responsible for the morphological and fermentative changes of the bacteria,[27] in addition, in silico results in this work suggest that terpineol can bind the urease active site and fits it. Furthermore, inhibition of its activity and ability to colonize in the mucosa by decreasing the pH that would improve the penetration of metronidazole into the bacterium cell rupturing its DNA double chain, thus, improving metronidazole efficiency with lowered dose and decreased possible side effect.

   Conclusion Top

H. pylori is becoming an alarming health concern, and there is a necessity to find novel agent that can manage and eradicate the infection. This work has successfully introduced terpineol noticeable activity against H. pylori. It can bind to the active site of urease enzyme with its hydroxyl group, block the enzyme, and prevent the colonization of the bacterium.

Also terpineol could be considered as a good candidate to treat metronidazole-resistant H. pylori by decreasing the dose of metronidazole and improving its efficacy. This finding should be thoroughly investigated in future based on in vivo studies and to recommend adding it to the already established guidelines.

Contribution to the field statement

H. pylori is a known Gram-negative microaerophilic pathogen that causes peptic ulcer, chronic gastritis, and carcinomas to human. The antibiotic resistance is a struggle in treating this infection. Thus, several researchers are looking for finding alternative treatments that could be efficient with fewer side effects. One of the hot targets for inhibiting H. pylori colonization is urease enzyme. In our study, we found that terpineol had a promising result in silico and in vitro. Also, the combination of terpineol with metronidazole could achieve a new beneficial treatment method that can reduce the dose of metronidazole, thus, reduce its possible side effects.


Authors wish to thank Deanship of Academic Research, Deanship of Post Graduate Studies and Faculty of Pharmacy, University of Jordan, Amman, Jordan for providing the necessary facilities and funds for conducting this research. Also, authors wish to thank Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan and Faculty of Pharmacy, Petra University, Amman, Jordan for providing the necessary facilities for conducting this research.

Author contributions

Talal Aburjai designed the corresponding experiments, Qosai Al-Balas designed the docking designs, Deniz Al-Tawalbeh conceived and designed the microbiological activities, Deniz Al-Tawalbeh performed the designed experiments, Talal Aburjai contributed by providing reagents and materials, Ali Al-samydai wrote the paper.

Financial support and sponsorship

This research was funded by THE UNIVERSITY OF JORDAN.

Conflict of Interest

There are no conflicts of interest.

   References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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