Helicobacter pylori infection is a WHO class 1 carcinogenic factor of gastric adenocarcinoma. In the past decades, many studies have demonstrated the increasing trend of antibiotic resistance and pointed out the necessity of new effective treatment. This study was aimed at identifying phytochemicals that can inhibit H. pylori and possibly serve as adjuvant treatments. Here, in silico molecular docking and drug-like properties analyses were performed to identify potential inhibitors of urease, shikimate kinase and aspartate-semialdehyde dehydrogenase. These three enzymes are targets of the treatment of H. pylori. Susceptibility and synergistic testing were performed on the selected phytochemicals and the positive control antibiotic, amoxicillin. The in-silico study revealed that oroxindin, rosmarinic acid and verbascoside are inhibitors of urease, shikimate kinase and aspartate-semialdehyde dehydrogenase, respectively, in which, oroxindin has the highest potency against H. pylori, indicated by a minimum inhibitory concentration (MIC) value of 50 μg/mL. A combination of oroxindin and amoxicillin demonstrated additive effects against H. pylori, as indicated by a fractional inhibitory concentration (FIC) value of 0.75. This study identified phytochemicals that deserve further investigation for the development of adjuvant therapeutic agents to current antibiotics against H. pylori. Helicobacter pylori infections may cause chronic gastritis, peptic ulcer, gastric cancer and other non-gastric related disorders, such as lymphoma [H. pylori as a class 1 carcinogen [H. pylori, and the prevalence rate in Africa, Latin America and Asia is substantially increasing [H. pylori is a global public health concern. The main treatment option for H. pylori is the standard triple therapy, combining two antibiotics with one proton pump inhibitor, such as clarithromycin and amoxicillin with omeprazole [lymphoma . The Worrcinogen . A recencreasing . Thus, Heprazole . Due to eprazole . This caeprazole ,7. Some H. pylori infection have been identified, and they are generally related to H. pylori’s morphological structure, survival mechanisms and toxic factors. One well-known example of these targets is urease. H. pylori requires urease and the H+-gated urea channel to survive in the low pH environment of human gastric fluid. Ureases help to generate a layer of ammonia, which neutralises the stomach acid and resists the damage caused by acidic environments [H. pylori as it catalyses the formation of shikimic acid in the shikimate pathway [H. pylori that produces some major amino acids and metabolites [Many pharmacological targets against an ronments . Another pathway . The thiabolites ,11. ThisH. pylori abilities through in vitro, in vivo and randomised control clinical studies [H. pylori infections and peptic ulcers. The results demonstrated that the use of Wenweishu together with the standard triple therapy can significantly increase the healing rate, but the eradication rate was not statistically different. Another example is the in vitro study of the leaf extract of Allium ascalonicum [H. pylori effects. One of the downfalls of using plant extracts as medicine is the imprecise type and amount of the active ingredients. This is because many factors could affect the number of active ingredients of a plant, including climate, soil type and harvesting time [H. pylori compounds in these plants may help to produce more predictable responses and accurate dosing regimens.Many phytomedicines had been investigated for their anti- studies . Some of studies ,13,14; h studies . This raing time . Also, tH. pylori.In silico molecular docking and drug-like properties analysis is an efficient method to screen bioactive compounds from a pool of phytochemicals . DockingH. pylori: urease, shikimate kinase and aspartate-semialdehyde dehydrogenase. Phytochemicals were selected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). GOLD v5.5 was used as the docking suite. The identified inhibitors were oroxindin, rosmarinic acid and verbascoside, respectively. In vitro susceptibility and synergistic testing against H. pylori were then performed on these three phytochemicals and the parallel positive control antibiotic (amoxicillin) to calculate their minimum inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) values.This study performed molecular docking and drug-like properties analysis to identify phytochemicals that may inhibit the three pharmacological target enzymes of The accuracy of the docking procedures varies substantially between different docking suites. Here, we validated our docking procedures on urease and shikimate kinase using receiver operating characteristic (ROC) analysis; their area under the curve (AUC) values were 0.90 and 0.77, respectively . An AUC Molecular docking and drug-like properties analysis were performed on the three target enzymes to select phytochemicals for in vitro studies. The drug-like properties were classified into three categories: ADME , physicochemical and drug safety see . We seleH. pylori effect [H. pylori effect have not been identified. Oroxindin could be one of Radix Bupleuri’s active phytochemicals against H. pylori. Oroxindin has similar chemical structures with similar functional groups to quercetin and baicalin, which have been experimentally demonstrated with H. pylori inhibitors [H. pylori in the human gastric region without too much systematic absorption into the bloodstream.Oroxindin obtained a high urease binding score of 84.9, which is comparable to most of the known urease inhibitors with binding scores ranging from 57.1 to 111.8. The botanical source of oroxindin is Radix Bupleuri, which has been shown to have an anti-i effect . Howeverhibitors ,25,26. RMelissa officinalis and Ocimum basilicum; Melissa officinalis exhibits a gastroprotective effect against gastric ulcers in animal studies [Ocimum basilicum has been shown to significantly inhibit H. pylori in an in vitro study [Enterobacter species [Escherichia coli [Aspergillus niger [Staphylococcus aureus [H. pylori than many other phytochemicals and was selected for further in vitro studies.The docking score of rosmarinic acid on shikimate kinase was 79.1, which is comparable to the scores of many known inhibitors which range from 57.8 to 89.2. Rosmarinic acid achieved an excellent drug-like profile, with appropriate ADME, physicochemical and drug safety properties . Similar studies , and Ociro study . Again,  species , Escherihia coli , Aspergius niger , and manus niger . Rosmaris aureus . Hence, Aloysia triphylla, which exerted antibacterial effects against Proteus mirabilis [Staphylococcus aureus [H. pylori [Staphylococcus aureus and Escherichia coli [+/K+-ATPase. This indicates that verbascoside has the potential to mimic the action of a proton pump inhibitor in H. pylori treatment [Verbascoside was the phytochemical selected to target aspartate-semialdehyde dehydrogenase. It obtained a docking score of 82.31 and ranked 11th out of 4450 herbal compounds. Similar to the other two phytochemicals, verbascoside has acceptable drug-like properties, such as good water solubility, non-CNS penetrant and non-inhibitor to all CYPs and hERG.  High conirabilis , Staphyls aureus  and H. p. pylori  in in vihia coli . Verbascreatment .H. pylori.Due to the above-mentioned high docking scores, favourable drug-like properties and antibacterial effects suggested by the literature, we selected oroxindin, verbascoside and rosmarinic acid for further in vitro studies. According to our knowledge and literature searches in various databases, including the Cochrane Library, Embase, Medline and the China National Knowledge Infrastructure database, there is no experimental proof on the inhibitory effects of these three phytochemicals against H. pylori effects  [H. pylori effect of oroxindin.The MIC value of oroxindin was 50 μg/mL, which is comparable with some well-known anti-0 μg/mL) , chelery0 μg/mL) , and pro0 μg/mL) . However0 μg/mL) , fuscaxa0 μg/mL) , palmati0 μg/mL)  and alli0 μg/mL) . CertainH. pylori effects, many of them have undesirable side effects or unfavourable drug-like properties [H. pylori and has antiviral, anticancer and antihyperlipidemic effects [H. pylori infection can irritate the stomach and cause gastritis, and oroxindin has demonstrated its gastroprotective effects by suppressing the inflammatory response and conserving the gastric barrier function [Although many phytochemicals have suggested anti-operties . For exaoperties , but it operties . Another effects . However effects . In contfunction .H. pylori. The current recommended treatment of H. pylori is triple therapy, which contains two antibiotics, such as amoxicillin, clarithromycin, levofloxacin and metronidazole. In a study by Lee et al. [H. pylori (ATCC 43504) as in this study, and the MIC values of amoxicillin, clarithromycin and metronidazole were 0.029, 0.06 and 21.6 μg/mL, respectively. These referenced MIC values suggested that oroxindin (MIC: 50 μg/mL) may have less potency than amoxicillin and clarithromycin, but higher potency than metronidazole. For some antibiotic resistant clinical strains, these MIC values were much higher. Recent studies from different countries have reported that MIC values of amoxicillin, clarithromycin and metronidazole on clinical H. pylori strains were 256 µg/mL [H. pylori effects. Also, as the anti-H. pylori pharmacological mechanisms are different between the antibiotics and phytochemicals, the co-administration of them may improve their efficiency and reduce side effects by decreasing the dosage typically administered.The MIC values of oroxindin, verbascoside and rosmarinic acid are higher than the positive control, amoxicillin . This ine et al. , in vitr56 µg/mL ,50. The 56 µg/mL . Hence, H. pylori cell walls. Whereas, this in silico study suggests that oroxindin binds to H. pylori urease, which is responsible for converting stomach urea to ammonia and neutralise the gastric acid and protect H. pylori from the surrounding strong acids [H. pylori before the phytochemicals produced a significant effect. However, in an antibiotic resistant strain, the difference between their potency will be reduced, and hence there will be a higher chance for producing synergistic or additive effects. Certainly, further studies on clinically obtained antibiotic resistance H. pylori strains are required to prove this theoretical idea.This study demonstrated an additive inhibitory effect between oroxindin and amoxicillin. This could be due to their different pharmacological mechanisms, in which amoxicillin is a beta-lactam antibiotic that binds to penicillin-binding proteins and inhibits the synthesis of peptidoglycan of ng acids . The sugH. pylori targets, urease, shikimate kinase and aspartate-semialdehyde dehydrogenase. In vitro anti-H. pylori assays were performed to test the inhibitory abilities of these selected phytochemicals. We also investigated the synergistic anti-H. pylori effects between the phytochemicals and an antibiotic.This study performed molecular docking and drug-like properties analysis to select phytochemicals with desired pharmacokinetic properties that may interact with three anti-H. pylori X-ray crystallographic structures of aspartate-semialdehyde dehydrogenase, the homology structure built by SWISS-MODEL [https://swissmodel.expasy.org/repository/uniprot/O25801#none. The number of herbs and phytochemicals involved in the aspartate-semialdehyde dehydrogenase docking simulations were 32 and 4541, respectively. The docking procedures of shikimate kinase and aspartate-semialdehyde dehydrogenase were the same as that of the urease.Docking has been used to identify inhibitors from phytochemicals by analysing the interactions between the target protein and ligands. Several molecular docking software programmes have been successfully applied in computational drug design (CADD). Here, the automated docking suite, GOLD v5.5 ,54 was uSS-MODEL  with seqhttps://www.ccdc.cam.ac.uk) and researchers [50 less than 100 µM were also docked with the urease and shikimate kinase, respectively. These inhibitors served as the ‘positive’ hits, whereas the 11,421 ZIM ligands were ‘negative’ hits. ROC analysis statistically revealed the ability of the docking methods to distinguish between the ‘positive’ and ‘negative’ ligands. All the experimentally approved inhibitors were identified from the BindingDB database [The accuracy of molecular docking varies substantially between different docking algorithms, scoring functions and the type of protein–ligand interactions . The docearchers . GOLD haearchers ,11,19,20earchers . A totaldatabase , and ourhttps://www.acdlabs.com/products/percepta/index.php.Drug-like properties analyses of the phytochemicals were performed using the ACD/Percepta 14.0 software . The anaH. pylori. MIC is the lowest concentration of the phytochemicals required to inhibit the visible growth of H. pylori. The strain of H. pylori used in this study was ATCC-43504, which were recovered according to the supplier product information sheet (https://www.atcc.org/~/ps/43504.ashx), wherein the frozen strain was thawed in a water bath at 37 ℃ and inoculated in solutions (100 μL) containing 6% sheep blood Colombian agar  and 5% foetal bovine serum  in a facultative anaerobic environment for 3 days. The H. pylori solution were added to cryopreservation media containing brain heart infusion broth and glycerine and stored at −80 ℃. To identify the H. pylori strain, a combination of the test was performed. The microscopy visualisation of colony morphology was used to assess the needle-like translucent appearance of H. pylori. Three biochemical assays, urease, oxidase and catalase tests , were performed to confirm which H. pylori strains were present.The minimum inhibitory concentration (MIC) was used to evaluate the inhibitory abilities of the selected phytochemicals against H. pylori were added to 0.85% normal saline to produce a solution with 0.5 (approximately 1.5 × 108 CFU/mL) turbidity under the microbial turbidimeter . The solution was then diluted 50 times to make an approximately 3.0 × 106 CFU/mL H. pylori turbidity solution.After identification, colonies of H. pylori McFarland Turbidity solution (approximately 3.0 × 106 CFU/mL) was then added to each of the phytochemical solutions and incubated at 37 ℃.The inhibitory effects of the selected phytochemicals were obtained using microdilution methods, in which sterile Brucella broth  was used to dilute each of the most concentrated phytochemical solutions by six half-dilutions to produce solutions with a range of different concentrations. Two microliters of the prepared H. pylori or phytochemicals, only H. pylori  and only phytochemicals (no H. pylori), respectively. These experiments were also performed with a commonly prescribed antibiotic (amoxicillin) for H. pylori eradication. Amoxicillin served as a parallel positive control, and the MIC value of amoxicillin was calculated for comparison with those of the phytochemicals. All experiments followed the guidelines from the Clinical and Laboratory Standards Institute [The MIC experiments were performed in sterile 96-well microliter plates  at 37 ℃. The optical density (OD) values were collected from a spectrophotometer  at an absorbance of 540 nm after 72 h. These experiments were performed with quality, negative and blank controls, which contained no nstitute .90 value is the minimum concentration of phytochemicals that inhibit 90% of H. pylori. The inhibition percentage was calculated using the following equation:The MICH. pylori. FIC values are measurements of synergistic, additive or antagonistic effects. An FIC value of less than 0.5 indicated synergistic action, between 0.5 and 1.0 indicated additive action, 1.1 to 4.0 meant indifferent and larger than 4.0 indicated antagonism [Synergistic testing was used to study whether the additive effect of two compounds or antibiotics was superior to that of the effect of the individual compounds. An additive effect may help to reduce the dose of each compound and may reduce adverse pharmacological effects. Here, the MIC values of the phytochemicals and antibiotics obtained from the susceptibility testing were used to calculate the concentrations required for the synergistic testing. Various combinations of two compounds or antibiotics at different concentrations were tested. Their concentrations were 1/8 MIC, 1/4 MIC, 1/2 MIC, 1 MIC and 2 MIC. The checkerboard methods  were usetagonism ,69. All p-values for comparing the OD, MIC and FIC values of the sample groups and control groups at different concentrations. A p-value of less than 0.05 was considered to be statistically significant. The SPSS software  was used.A paired t-test was used to calculate the H. pylori effects, in which oroxindin had the highest potency. Their MIC values were higher than the current anti-H. pylori treatment, amoxicillin. Hence, their potential use as monotherapy of H. pylori treatment cannot be justified here. In our synergistic testing, oroxindin demonstrated its additive effects with amoxicillin. We believe further investigations on these phytochemicals as an adjuvant therapy with current antibiotics treatment on both resistant and non-resistant strains are well-intentioned.This study performed validated in silico techniques to identify a urease, a shikimate kinase and an aspartate-semialdehyde dehydrogenase inhibitor from 5015, 1548 and 4541 phytochemicals, respectively. The three identified inhibitors with appropriate drug-like properties were oroxindin, verbascoside and rosmarinic acid. In our in vitro susceptibility testing, all three phytochemicals were shown to have anti-