Immunomodulatory and Antiallergic Potentials of the Bioactive Compounds of Ginger

Immunomodulatory and Antiallergic Potentials of the Bioactive Compounds of Ginger


INTRODUCTION
Allergy and related immune hypersensitivity diseases have become one of the most common health issues, affecting about 20% of the global population, 1 and are coincidentally increasing with more civilized lifestyles. 2 As any external or internal particle can be a potential allergen, allergy can take many different forms and sometimes can be fatal, with an estimate of 19 death per 10,000 people every year. 3Depending on the type of allergens, allergic reactions follow diverse molecular pathways and involve inflammatory immune cells such as mast cells, neutrophils, basophils, and eosinophils. 4major molecular pathway of allergic reaction is the binding of histamines, released by the inflammatory cells, to histamine H1 receptors (H1R).The ligand-H1R binding is exploited by allergic medications such as cetirizine, chlorpheniramine, diphenhydramine, levocetirizine, and pheniramine that bind to H1R as antagonists or inverse agonists, thereby preventing the binding of histamine to H1R and abrogating the cascade of molecular pathway to initiate allergic reaction. 5,6ysteinyl leukotriene receptor 1 (CysLTR1) is another receptor that is activated in many allergic responses.The most important and potent agonist of CysLTR1 is cysteinyl leukotriene D4 (LTD4), an anti-inflammatory lipid mediator that is released during degranulation of mast cell and leads to histamine production. 7Leukotriene C4 (LTC4) and leukotriene E4 (LTE4) are also CysLTR1 agonists.Common drugs like montelukast, zafirlukast and pranlukast are CysLTR1 antagonists.For enhanced allergic suppression, combination drugs are often used to target both H1R and CysLTR1. 8yond the general use as food condiment, ginger, Zingiber officinale Roscoe (family Zingiberaceae), is known to possess several therapeutic properties such as analgesic, anticancer, antidiabetic, antiinflammatory, antiemetic, anthelminthic, antihyperglycaemic, and antimicrobial activities. 9,10xperimental studies are reported for some of the major medicinal applications.Its major chemical constituents, 6-gingerol and 6-shogaol are shown to be promising lead compounds as anticancer drugs.Its specific use as antiallergic agent is notable.It is variously recorded as a remedy for arthritis, food poisoning, 12 cough, antiallergic, anti-irritant, and anti-inflammatory activities. 13Other bioactive compounds have also been determined including polyphenols such as 6-dehydrogingerdione, gingerenone-A, paradols, quercetin, and zingerone; terpenes such as β-bisabolene, α-curcumene, α-farnesene, β-sesquiphellandrene and zingiberene.Although the major pharmacological properties, anti-inflammatory and anti-cancer activities, are quite empirically established, 15,16 there is no information on the biological activity of any of the ginger compounds at the molecular level.This study is therefore an attempt to show the molecular picture of the interaction between ginger compounds with the key cell receptors involved in allergic reactions, and the possible targets in other cellular activities.

ABSTRACT
Background: Allergy is an ever-increasing immune disorder and is often fatal under certain circumstances.Lack of total curative medication prompts the search for various compounds as the lead molecules.Ginger, Zingiber officinale Roscoe, is a well-established medicinal plant in different traditional practices.Its use as antiallergic or anti-inflammatory agent has been vindicated but the underlying mechanism of action is yet unknown.Method: In this study, we analyzed the phytocompounds characterized from ginger for their binding affinities on cysteinyl leukotriene receptor 1 (CysLTR1) and histamine H1 receptor (H1R) by molecular docking.The molecular interactions were compared against known agonists and antagonists of the two receptors.Results: The data indicate that ginger compounds have high binding affinity for both LTR1 and H1R comparable to those of antiallergic medications.Protein retrieval and processing X-ray crystal structure of cysteinyl leukotriene receptor 1 (CysLTR1, PDB code: 6RZ5) and histamine H1 receptor (H1R, PDB code: 3RZE) were retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB)-PDB database (www.rcsb.org).To obtain clear molecular interactions, molecules attached to the proteins such as co-factors, water and unique ligands were removed with Molegro Molecular Viewer software. 17

Molecular docking
Molecular docking of the ginger compounds to each of the recovered proteins was carried out on the AutoDock Vina platform, which is appreciated as the most powerful molecular modelling tool for ligandreceptor interactions. 18Polar hydrogens and Kollman charges were added to all the proteins in the AutoDockTool-1.5.6 before saving the data in protein data bank, partial charge (Q) and atom type (T) (PDBQT) format.On the docking platform, the compounds of ginger were docked to CysLTR1 and H1R.In addition, leukotriene D4 and montelukast were docked to CysLTR1; histamine and cetirizine to H1R (Trott and Olson 2010). 19Flexible docking was performed on all the proteins.Grid boxes were prepared for CysLTR1 (size_x=40, _y=46, _z=66, center_x=11.082,_y=15.676,_z=-8.069)and H1R (size_x=40, _y=40, _z=50, center_x=16.801,_y=35.487,_z=24.168) to cover all the possible protein binding sites.With an exhaustiveness of 8, the ligands were docked to the proteins and the outcomes were saved for visual analysis.

Visualization and analysis of interaction
Visualization of molecular interactions and docking analyses were performed on BIOVIA Discovery Studio Visualizer 2016 v16.1.0.15350.The software is a comprehensive tool suitable for micro-to macromolecules for all types of molecular interactions in pharmacological studies. 20The ligand output and protein PDBQT formats were accessed and defined.Non-bond interactions and ligand interactions were selected and labels were added to each of the residues.The files were then saved and converted to image files.The molecular docking showing lowest binding energy and the root-mean-square deviation (RMSD) were selected for each ligand-protein interaction.

Prediction of activity spectra for substances
To predict to biological activities of the ginger compounds based on their structural resemblance to already known molecules, an online tool, prediction of activity spectra for substances (PASS) (http://www.way2drug.com/passonline/predict.php) was employed. 21The simplified molecular-input line-entry system (SMILES) data of the compounds retrieved from PubChem as previously mentioned were uploaded for the prediction.Predicted probable biological activities were given with their probability to be active (Pa) and their probability to be inactive (Pi).Pa of more or equal to 0.7 were considered in this study.

RESULTS AND DISCUSSION
Molecular docking results showing the docking score expressed in kcal/mol, amino acid residues of receptors and types of interactions involved in the interaction of the compounds of ginger with CysLTR1 and H1R are shown in Table 1 and Table 2 respectively.Leukotriene D4 and montelukast bind to similar region on CysLTR1 although the amino acid residues they interacted differ (Figure 1, Table 1).Ginger compounds, 6-dehydrogingerdione, gingerenone A, gingerol, paradol, quercetin, zingerone, zingiberol and α-farnesene shared the binding sites with leukotriene D4 and montelukast (Figure 2).Zingiberene, α-curcumene, β-bisabolene, and β-sesquiphellandrene bind to CysLTR1 on different sites and do not interfere with the binding of leukotriene D4 or montelukast, both of which bind to the same site (Figure 3).For H1R, cetirizine and histamine bind to different regions (Figure 3A).6-Dehydrogingerdione, gingerol, paradol, zingiberene, α-farnesene, β-bisabolene and β-sesquiphellandrene bind in the same binding pocket as cetirizine on the H1R while gingerenone-A, quercetin, zingerone and α-curcumene share similar binding pocket with histamine.However, zingiberol neither binds to similar region on H1R as histamine nor with cetirizine (Figure 3H).
Our result shows that montelukast and leukotriene D4 can bind to similar region of CysLTR1, however, montelukast exhibited lower binding affinity (-9.7 kcal/mol) than leukotriene D4 (-6.5 kcal/mol) (Table 1).Even though leukotriene D4 is released during mast cell degranulation, this may be the reason montelukast acts effectively as an antiallergic medication.In a comparable manner, our result showed that the molecular docking score of histamine to H1R is -4.5 kcal/mol while the docking score of cetirizine, an H1R antagonist, is -7.2 kcal/ mol (Table 2).
A clinical trial had shown that ginger extract treatment alleviates the symptoms of allergic rhinitis and the result was found to be comparable to loratadine which is known to directly target H1R. 22A study in mice indicated that gingerol is the main molecule that suppresses the production of cytokines and subsequent reactions in allergic rhinitis symptoms. 23The compound is also reported to suppress eosinophilia and interleukin-1 beta-induced MUC5AC gene expression in human airway epithelial cells and also reduced intestinal allergic reactions in irritable bowel syndrome. 24,256-Gingerol, 10-gingerol and 6-shagaol have been reported to relax the smooth muscles of the airway by acting as β-agonists and inhibit phosphodiesterase 4D and phosphatidylinositolspecific phospholipase C. 26 Our result indicates that gingerol has lower affinity for CysLTR1 than leukotriene D4, but a higher affinity for H1R than histamine which may imply that the antiallergic property of gingerol may be through competitive inhibition of H1R.
Another compound of ginger, quercetin, exerts antiallergic property by inhibiting the production of histamine and other pro-inflammatory mediators, 27 which is at par with our results wherein quercetin has high binding affinity for both CysLTR1 and H1R (-8.mol respectively).Quercetin is also known to decrease reactive oxygen species (ROS) and TNF-α-induced oxidative stress, apoptosis and inflammation and also suppresses the expression of matrix metalloprotease-9 (MMP9) and intercellular adhesion molecule-1. 28,29ur result indicates that quercetin may act similarly as HIF1A expression inhibitor, JAK2 expression inhibitor, MMP9 expression inhibitor and histamine release inhibitor (Table 4).
Although the role of gingerenone-A in allergy and inflammation is not well established, our result shows that it has high affinity for both CysLTR1 and H1R and may act as JAK2, HIF1A and MMP9 expressions inhibitor (Table 3).Similarly, zingiberol exhibited relatively high affinity for both LTR1 and H1R (Table 1 & 2).The biological activities predicted for zingiberol include anti-inflammatory and immunosuppressant properties.Zingerone has been demonstrated to have protective effects against oxidative stress, inflammation, asthma, thrombosis and histopathological alterations. 30It exhibits lower binding energy than histamine for H1R, but has higher binding energy for CysLTR1 than leukotriene D4 suggesting the anti-inflammatory property of zingerone may be through H1R and not CysLTR1.Zingerone has also been predicted to have probable biological activities as JAK2 expression inhibitor, MMP9 expression inhibitor and TNF expression inhibitor (Table 3).
6-Dehydrogingerdione has been reported to have anti-inflammatory, antiallergic, antitumor and anti-atherosclerotic properties. 31Paradol is a potent anticancer, chemopreventive, and anti-inflammatory compound. 32,33Zingiberene is an established anticancer and antiinflammatory compound. 34α-Farnesene is a molecule of wide applications from medicine to mechanical appliances. 35An essential oil, β-bisabolene is used in food flavouring and has anticancer activity. 36β-sesquiphellandrene has strong anti-neoplastic property. 37ur data indicates that 6-dehydrogingerdione, paradol, zingiberene, α-curcumene, α-farnesene, β-bisabolene and β-sesquiphellandrene also bind to the same receptor pocket of CysLTR1 as leukotriene D4, they all exhibited higher binding energy and thus may not be able to compete with leukotriene D4 (Table 1).However, all these phytocompounds has lower binding energy for H1R than histamine (Table 2).6-Dehydrogingerdione and paradol are predicted to act as JAK2 expression inhibitors.Additionally, 6-dehydrogingerdione may also act as HIF1A, TNF and MMP9 expressions inhibitor (Table 3).β-Sesquiphellandrene may have the potential to be prostaglandin-E2 9-reductase inhibitor; while α-farnesene may act as both G-protein coupled receptor kinase and β-adrenergic receptor kinase inhibitor (Table 4).

CONCLUSION
Our molecular models show that the bioactive compounds of ginger interact well with CysLTR1 and H1R indicating that they can play direct role in the antiallergic and anti-inflammatory pathways.Some ginger phytocompounds such as 6-dehydrogingerdione, gingerenone-A, paradol, quercetin, and zingerone are predicted to be potential inhibitors of JAK2, a protein involved in allergic reactions via JAK-STAT pathway.HIF1A is also known to be activated during allergic reactions by IL-4 and IL-13, however, 6-dehydrogingerdione, gingerenone-A and quercetin are predicted to inhibit HIF1A expression.TNF and MMP9 are also involved in allergic reactions and both these may be inhibited by 6-dehydrogingerdione, gingerenone-A, paradol, quercetin and zingerone.
3 and -9.0 kcal/ B. Lalruatfela, et al.Immunomodulatory and Antiallergic Potentials of the Bioactive Compounds of Ginger

Figure 1 :Figure 2 :Figure 3 :
Figure 1: Zingiber officinale growing in Aizawl, Mizoram, India, and the chemical structures of the bioactive compounds used for molecular modelling of the pharmacological properties.

Table 1 : Molecular interactions of leukotriene D4, montelukast and compounds of ginger with cysteinyl leukotriene receptor 1 (CysLTR1).
aa-R: Amino acid residues on receptor aa-P: Position of amino acid residues MDS: Molecular docking score Pharmacognosy Journal, Vol 15, Issue 6, Nov-Dec, 2023 aa-R: Amino acid residues on receptor aa-P: Position of amino acid residues MDS: Molecular docking score

Table 2 : Molecular interactions of histamine, cetirizine and compounds of ginger with histamine H1 receptor (H1R).
. Lalruatfela, et al.Immunomodulatory and Antiallergic Potentials of the Bioactive Compounds of Ginger

Table 3 : Probable biological activities of polyphenols from ginger.
Lalruatfela, et al.Immunomodulatory and Antiallergic Potentials of the Bioactive Compounds of Ginger