Phenolic compounds of Zanthoxylum armatum DC as potential inhibitors of urease and SARS-CoV2 using molecular docking approach and with simulation study

Abstract The anti-urease effects of active extract and three isolated phenolic compounds viz., chlorogenic acid, trans-ferulic acid, and gallic acid of leaves of Zanthoxylum armatum DC were evaluated. The compounds were identified based on HPLC-PDA, HR-MS, and NMR analysis. Molecular docking analysis revealed that these compounds significantly interacted with Helicobacter pylori urease and SARS-CoV2 vital proteins. Chlorogenic acid was found to show the strongest interaction with the H. pylori urease and coronavirus main protease (Mpro, also called 3CLpro), while gallic acid with five spike proteins (Cathepsin L) of SARS-CoV2. The compounds were checked for their drug-likeliness character and were found to pass the Lipinski filter and abide by Veber’s rule and passed through ADMET. Chlorogenic acid was simulated for 50 ns using GROMACS. The study shows that chlorogenic acid isolated from Z. armatum could be a significant antagonist of the H. pylori urease. Graphical Abstract


Introduction
Helicobacter pylori are one of the antibiotic-resistant ureolytic bacteria that possess a considerable danger to human life, as these infect the intestinal tract and cause gastro duodenal diseases resulting in peptic and gastric cancer (Hooi et al. 2017). In the stomach, the catalytic hydrolysis of urea by urease produces carbon dioxide and ammonia. The inhibition of urease is one of the effective strategies to combat the growth of these bacteria (H ribov a et al. 2014). Previous literature suggests that there are no effective mono-therapeutic substances that could treat H. pylori infections. Currently, available treatment depends on a combination of antibiotics, such as clarithromycin, amoxicillin, metronidazole, and acid-suppressive drugs. However, the use of these antibiotics is declining due to the increase in antibiotic resistance of H. pylori (Sharaf et al. 2022).
The fight against SARS-CoV2 and its different mutations is not yet finished. The search for a potent drug for treating SARS-CoV2 infections is still going on (Hassab et al. 2022). So, there are enormous possibilities of a plethora of medicinal plants possessing specialized metabolites that could play a vital role against SARS-CoV2. Therefore, we are approaching and focusing on drugs of plant sources that could have significant potential against SARS-CoV2 and H. pylori infections. There may be no exact evidence of a direct or indirect relationship between Coronavirus and H. pylori. But sample which contains active compounds that are effective against both Coronavirus and H. pylori may act as a promising candidate in the treatment of SARS-CoV-2 and H. pylori-borne diseases. Plenty of plant extracts were evaluated for their anti-urease activities (H ribov a et al., 2014).
Concerning all the aspects, one traditionally used highly medicinal plant, Zanthoxylum armatum DC (Family: Rutaceae) was considered for our study. The plant is used for the treatment of viral diseases and this is an essential ingredient in folk remedies for treating viral fever, diarrhoea, vomiting, gastrointestinal disorders, toothache, and so on (Brijwal et al. 2013;Phuyal et al. 2019). Earlier studies on Z. armatum DC revealed that leaves are a good source of phenolic compounds like hesperidin, vitexin, catechin, sesamin, and so on, that show antipyretic, antioxidant, antimicrobial, antispasmodic, cytotoxic, phytotoxic, insecticidal, anticancer, and antileishmanial effects (Alam and Najam us Saqib 2017;Phuyal et al. 2019).
This study aims to evaluate in vitro anti-urease activity and isolate some active compounds from Z. armatum DC leaves. This study also aims to establish a correlation between in vitro results and in silico molecular docking analysis of isolated compounds of Z. armatum DC with H. pylori urease and SARS-CoV2.

Results and discussion
Three compounds, that is, chlorogenic acid, trans-ferulic acid, and gallic acid were isolated from leaves of Z. armatum DC. In this study, in vitro urease inhibition effect was investigated followed by in silico molecular docking study of the identified compounds of Z armatum DC to correlate with the in vitro result. The isolated compounds were further checked for their drug-likeliness character and ADMET. In addition, the molecular docking efficacy of compounds was also evaluated with SARS-CoV2.
Urease inhibitory activity was found to be of moderate efficacy than the standard, that is, thiourea. Among all the extracts of Z armatum DC, butanol extract proved to be most effective against the urease enzyme with an IC 50 value of 152.31 mg/mL and the IC 50 value of thiourea was found to be 39.09 mg/mL. Butanol sub-fraction 3 (BSF3) was found to be quite effective against urease with an IC 50 value of 57.67 mg/mL (Table 1S, Supplementary Materials (SM)).
BSF3 was run through an HPLC instrument equipped with an analytical and semipreparative column to identify and isolate some of the responsible bioactive compounds. Accordingly, three compounds, viz., chlorogenic, trans-ferulic, and gallic acid were identified and isolated from BSF3 ( Figure 1S, SM). The concentrations of chlorogenic, trans-ferulic, and gallic acid were 0.028, 0.200, and 0.007 mg/mL (Table 2S, SM). The calibration curves of these compounds are shown in Figure 2S (SM). The isolation of the above compounds was carried out using column chromatography followed by semi-preparative HPLC analysis.
The isolated compounds were confirmed by analyzing HRMS data. The mass spectra of BSF3 showed the precursor peaks at m/z ¼ 163.1430 of chlorogenic acid (Molecular weight ¼ 354), m/z ¼ 177.1683 of trans-ferulic acid (M.W.¼194), and m/z ¼ 171.0571 of gallic acid (M.W.¼170) (Figure 3Sa, 3Sb, SM). The UV spectra of these compounds are shown in Figure 4S (SM). The structures of the isolated compounds in Figure 1 were further confirmed by NMR spectra (Figure 5S, SM).
The active sites of the urease enzyme were analyzed for potential interactions with chlorogenic, trans-ferulic, and gallic acid. The cDocker interaction energy of chlorogenic acid was À38.7069 kcal/mol, which formed five conventional H-bonds, two C-H bonds, one Pi-Alkyl bond and five van der waal interactions. trans-Ferulic acid was found to form two conventional H-bonds, while gallic acid formed three conventional H-bonds. The cDocker interaction energies of trans-ferulic acid and gallic acid with urease enzyme were found to be À19.7402 and À18.955 kcal/mol, respectively. Among these three molecules, chlorogenic acid was the most significant one (Table 3S, Figure  6S, SM).
The SARS-CoV2 main protease is responsible for the proteolysis of the viral proteins to form important structural proteins of the virus (Krishnamoorthy and Fakhro 2021). The crystal structure of coronavirus main protease (M pro , also called 3CLpro) complexed with Boceprevir was taken from RCSB-PDB. The cDocker interaction energy of chlorogenic acid was À43.3416 kcal/mol and formed four conventional H-bonds with residues of the M pro . The cDocker interaction energies of trans-ferulic acid and gallic acid were found to be À26.0357, and À26.5003 kcal/mol and both of them formed three and four conventional H-bonds, respectively. Chlorogenic acid had shown the lowest binding energy among the three compounds (Table 4S, Figure 7S, SM).
The molecular docking (MD) simulation runs for 50 ns on chlorogenic acid showed positive interaction with the active sites of the urease enzyme ( Figure 9S, SM). The analysis of the simulated complex validates the inference of the docking analysis, confirming the interaction between the pocket residues of the receptor and the ligand. The hydrogen bond analysis of the simulated protein-ligand complex showed the formation of about four H-bonds, out of which two of them remained pretty strong throughout the simulation ( Figure 10S, SM). The RMSD for protein in complex with chlorogenic acid showed stability after 15 ns of run and ranging below 2 nm ( Figure  11S, SM). The radius of gyration was within the range of 2.25-2.75 nm, indicating the backbone's stability ( Figure 12S, SM).

Conclusion
Three phenolic compounds viz., chlorogenic acid, trans-ferulic acid, and gallic acid were isolated from Z armatum DC. In silico molecular docking analysis established a good correlation with the in vitro urease inhibitory results. Chlorogenic acid interacted significantly with H. pylori urease and the stability of these interactions was verified using simulation studies. Gallic acid showed considerable interactions with four spike proteins of SARS-CoV2, while interaction with M pro by chlorogenic acid was the strongest one. Therefore, it can be concluded that Z armatum DC may act as a potential inhibitor of H. pylori and SARS-CoV2. Z armatum DC in vitro and in silico anti-urease activity could be seen as a fact of the traditional claim of treating stomach issues. This study suggests further exhaustive research on Z armatum DC that can reveal its immense possibilities.