Anti-Inflammatory Activity of Phytoconstituents of Ginseng Plant-Insilico Approach

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Ginseng is an herbal traditional plant that is a short, slow-growing plant with fleshy roots having 11 different varieties. It is an herb that is having light-colored, cleft-shaped roots, long stems, and green oval-shaped leaves 1 . American ginseng (Panax quinquefolius) and Asian ginseng (Panax ginseng) are the most popular types of ginsengs. Other types are Korean ginseng and South China ginseng. Ginseng can be used as a component in health drinks, hair tonics, and cosmetic products. Ginseng can be also used as a possibly effective aid in lowering blood pressure and respiratory infections. In most cases, ginseng can be used as alternative medicine, other uses include breast cancer, fatigue, menopausal symptoms, memory loss, bleeding disorders, and digestive disorders.
The major bioactive compound produced by ginseng is a saponin with a structure of dimmerene terpenoid ie., ginsenosides 2 . Ginsenosides are used to maintain stable blood pressure, mental stress reduction, and boost immune function 3 . The ginseng saponins can exert various pharmacological effects like antiinflammatory, antiviral, cardiovascular activity, and immunomodulatory effects 4 . Polysaccharides present in ginseng have immunomodulation, anti-fatigue, antitumor, antiadhesive, antioxidant, antiulcer, hepatoprotective, hypoglycemic, and antihyperlipidemic activities 5 . The effects of remaining volatile and non-volatile components that are present in ginseng include anti-inflammatory, cardioprotective, neuroprotective, antiaging, antitumour, anti-coagulation, and treatment of diabetes mellites 6 .
According to Saleem et al., in Pharmacological analysis of Indian ginseng ( Wi t h a n i a s o m n i f e r a ) , w i t h a n o l i d e a pharmacologically active steroidal lactone, an alkaloid isolated from the root of the plant is present. The principal withanolide extracted from the plant is withanolides A and D, found in India, which have antitumor, and cytotoxic properties. Withaferin A, an alkaloid extracted from the root of Withania somnifera, extracted and purified, exhibits anti-inflammatory activity by inhibiting NFêâ activity and targeting CYS 179 8 . In addition to withanolide, Indian Ginseng contains other bioactive compounds such as glycosides, phytophenols, flavanoids, steroids, and phenols 9,10 . Also, it is used in traditional medicine formulations as an antiinflammatory, adaptogenic, and antipyretic agent 11,12 . W. somnifera has been shown to possess antiinflammatory properties in many animal models of inflammation like carrageenan-induced inflammation, and cotton pellet granuloma 13 . But no docking studies are still carried out for establishing the data.
Anti-inflammatory drugs can interact with the pathogenesis of inflammation seeking to provide patient comfort with a variety of actions such as non steroidal anti-inflammatory drugs, corticosteroids, colchimes, penicillamines, and immunosuppressive agents. The most difficult and essential step in drug discovery and development is to execute drug metabolism and pharmacokinetics (DMPK) studies, often referred to as ADMET 14 .
In pharmacology, ADMET stands for "absorption, distribution, metabolism, excretion, and toxicity". ADMET properties have a pertinent role in determining the effectiveness of clinical candidates that can act as good standard as a drug. Nonsteroidal anti-inflammatory drugs can be used worldwide, used to treat pain resulting from the inflammatory process 15 . The main mechanism of NSAIDs is the inhibition of COX action in a selectively in the production of thromboxane and prostaglandins which have side effects 16 . Specific modifications of anti-inflammatory effects and side effects are associated with the existence of COX 1 and COX 2 genes 17 . Thus, in this scenario, the present study focused on the interaction of phytoconstituents of ginseng plants other than ginsenosides with COX 1 and COX 2 genes using the insilico approach.
The main objective of the study is to examine the Inhibitory action of phytoconstituents against COX-1 and COX-2, an in silico approach, Drug-likeness prediction, and ADMET analysis of phytoconstituents of ginseng plant.

Ligand molecule preparation
The ginseng plant's phytoconstituent's three-dimensional structure was retrieved from the National Library of Medicine PubChem in SDF format. It was converted using Open Babel GUI software to PDB format.

Preparation of the receptor molecule structure
The FASTA format of cyclooxygenase 1 (P23219) and cyclooxygenase 2 (P35354)was obtained from UniProt (https://www.uniprot.org). The FASTA formats were copied to the Swiss model and were searched for templates. The PDB structure of the receptor was downloaded. The repository of COX 1 was 6Y3C (Human COX-1 Crystal Structure)and COX2 was 4RRW.

ADMET and drug-likeness evaluation
The simplified molecular-input line-entry systems (SMILE) of phytoconstituents of plant were submitted to the SwissADME tool to evaluate molecular properties and in silico pharmacokinetic parameters 18 . The ADME predictions were computed for Log Kp of skin permeation value, blood-brain barrier permeability, cytochrome-P inhibitors, gastro-intestine absorption, and P-GP substrate. The toxicological endpoints and organ toxicities of the ligands like hepatotoxicity, immunotoxicity, carcinogenicity, cytotoxicity, mutagenicity, LD50and irritant properties were predicted using Osiris software and Pro Tox II.

Receptor-ligand docking
In-silico docking studies were performed using the AutoDock server (https://vina.scripps. edu/) 19 . The autodock result was opened in the MyPresto program. The desired ligand in the structure was selected and was run for the delta G value. The scores of dockings were documented and the poses were visualized. The hydrogen bonds and other interactions involved in docking and their respective amino acid positions and distances were evaluated by using chimera 1.5.3.
The role of COX genes in inflammation has been studied for decades. Although antiinflammatory drugs are available for treatment, the standard drugs are reported to have adverse effects on long time usage. Studies are being done to find improved anti-inflammatory drug quality.    The binding energy of myricetin to COX 1 is -6.13kcal/mol. Epicatechin requires -5.22kcal/mol to bind to COX-1. The binding energy required by the chlorogenic acid to bind to COX-1 was -4.54kcal/mol while gallic acid requires -2.82kcal/mol to bind to COX-1. The interaction of Myricetin against COX-1 resulted in two hydrogen bonds with amino acid residues CYS47 with a bond length of 2.98A o and GLN461 with a bond length of 3.36A o . Two amino acid residues GLN465 with a bond length of 2.22A o and CYS41 with a bond length of 2.21A o were bound to epicatechin. Interaction of chlorogenic acid against COX-1 resulted in two hydrogen bonds with amino acid residues GLY45 with a bond length of 2.9A o and CYS47 with a bond length of 1.6A o while in the case of gallic acid, two amino acid residues SER530 with a bond length of 2.20A o and 3.10A o were bound to COX-1. (Figure.1).
The binding energy required by the gallic acid to bind to COX-2 was -5.2kcal/mol. The binding energy of quercetin to COX-2 was -6.19kcal/mol while myricetin required -4.94kcal/ mol. Apigenin required 4.64kcal/mol to bind to  (Figure 2).

DISCUSSIONS
In this study, investigated molecules possessed several favorable drug-likeness properties ( Table 1). The molecular weights of all the phytoconstituents were found to be less than 500 and thus these molecules can easily be transported, distributed, and immersed. The number of hydrogen bonds acceptors except for epicatechin and the number of hydrogen bond donors for myricetin, epicatechin, and chlorogenic acid were by Lipinski's rule of five, which describes it should be less than 10 and 5 respectively. Thus it can be predicted that according to Lipinski's rule of five these compounds are likely to be orally active. TPSA values were higher than the default range. Except for stigmasterol, log P values of all the compounds were found to be less than 5 and are in acceptance of Lipinski's rule of five, suggesting permeability across cell membrane justifying that they can be orally used (TABLE 1).
The toxicity of a chemical can be measured in terms of toxicity endpoints, and toxicity parameters such as mutagenicity, carcinogenicity, mutagenicity, and many other endpoints. It can be further measured both quantitatively and qualitatively. ProTox, a web server published in 2014 for rodent oral toxicity. The web server classifies the different levels of toxicities such as carcinotoxicity, cytotoxicity, toxicological endpoints (such as mutagenicity, and immunotoxicity), oral toxicity, organ toxicity (hepatotoxicity), toxicological pathways (AOPs), and toxicity targets, which provide a deep idea about the possible molecular mechanisms and its toxic responses 20 .
In the above study, the phytoconstituents gallic acid and myricetin showed high antiinflammatory action against cells among various phytoconstituents in the ginseng plant and it is evident that these phytoconstituents showed high binding affinity may be due to the electrostatic force of attraction on COX1 and COX2 genes. Even though the other phytoconstituents that show antiinflammatory action are quercetin and apigenin showed high binding energy due to the attraction on COX2 genes and epicatechin and chlorogenic acid on COX1 genes.
According to Cheo et al.,2006, radical scavenging of gallic acid -linolenic acid was compared to those of gallic acid and ascorbic acid and tyrosine inhibition effect. Gallic acid did not show tyrosinase activity and the result of the COX inhibition effect showed that gallic acid have higher selectivity in COX1 inhibition, thus it could be used as a functional reagent for anti-inflammatory effects. According to Ratna et al.,2020, based on the study of molecular docking of chlorogenic acid and its isomers in atherosclerosis, it is reported that the strongest bond is found in the docking result of chlorogenic acid with COX2 and the smallest binding energy value was also obtained from the result of COX2 docking with chlorogenic acid compared to its isomers, so that it has the potential as an antiinflammatory agent. According to Peng and Yun, 2017, based on the study on the anti-inflammatory effect of myricetin and other plant compounds in neonatal rats, it is reported that Myricetin, and fisetin formed strong bonds and interactions with the ligand-binding sites of TNF-á, COX-1 and COX-2 and can suppress the enzymes responsible for inflammation. Therefore, myricetin, and fisetin can be used as alternatives to existing NSAIDs and an anti-inflammatory agents. According to Jee et al.,2007, the study on the Inhibition of Cyclooxygenase-2 Expression, Adhesion of Monocytes to Human Umbilical Vein Endothelial Cells, and Expression of Cellular Adhesion Molecules on apigenin, it is showed that apigenin inhibited Nitric Oxide production and COX-2 expression, and collagenase activity involved in rheumatoid arthritis. These inhibitory activities of apigenin on the inflammatory responses suggest that it may be useful as an alternative medicine to help treat inflammatory symptoms. According to Subramaniya et al.,2017, based on the study of Differential cytotoxic activity of Quercetin on colonic cancer cells depending on ROS generation through COX-2 expression, it is reported that increased generation of reactive oxygen species (ROS) was observed only in Quercetin treated cells, which is due to overexpression of COX-2, as COX-2 silencing inhibited Quercetin induced apoptosis and ROS generation. Insilico analysis provided evidence that Quercetin could partially inhibit COX-2 enzyme by binding to subunit A which has peroxidase activity and serves as a source of ROS. Quercetin depends on COX-2dependent ROS generation that induces apoptosis and inhibits cell survival, thus quercetin and its derivatives can be used as an anti-inflammatory agent. According to Rajesh et al.,2019, in the case of epicatechin, they can effectively inhibit the LPS inhibited the release of TNF alpha, IL6, NO and PGE2 production mediated by the LPS-stimulated macrophages suggesting that the epicatechin has anti-inflammatory properties.
According to Lestari, it is studied that, according to molecular docking studies, aspirin showed higher binding affinity towards COX2 and the presence of a hydrogen bond of ARG120, which is important for COX2 interaction. Similarly, in this study, the myricetin and gallic acid showed higher binding energy and there is the presence of hydrogen bonds of ARG44 and ARG333 was observed, which indicates the interaction of COX2. According to Lestari, Aspirin had higher effectiveness as an inhibitor of COX1 and COX2. The interaction of COX2 with aspirin formed 1 hydrogen bond with GLN529 and the interaction with COX1 formed hydrogen with SER, GLU, ARG, and TRP. In this study, it is indicated that the quercetin interact with COX2 forming a hydrogen bond with GLN461 and the gallic acid and epicatechin interacts with COX1 and a formed hydrogen bond with SER 530 and GLU461 respectively. In the process of competitive binding, it is found that alginate is more easily bound to COX2 due to smaller binding energy, thus it is considered as an excellent potential as an inhibitor of COX2 27 . In this study it is thought that apigenin more easily interacts with COX2 because of the smaller binding energy compared to quercetin, myricetin and gallic acid; likewise, chlorogenic acid easily interact with COX1 because of the smaller binding energy compared to myricetin and epicatechin, thus these are considered as excellent potential for the interaction of COX1 and COX2. According to Lestari, alginate interacts with COX1 formed a hydrogen bond with GLN374 thus alginate is considered one of the inhibitors of COX1; Similarly, in this current study, it is considered that the myricetin bind to GLN461 by hydrogen bond, thus it is also considered as one of the inhibitors of COX1.

CONCLUSION
Ginseng has been widely used as a traditional medicine for many years in East Asian Regions generally as a stimulant, and adaptogenic medicine. Though all the parts such as fruit, stem, leaves, flowers, and roots of the ginseng plants have medicinal value, the roots are used most extensively for medicinal purposes, especially for their remedial properties. The phytoconstituents of the ginseng plant have several properties such as anti-inflammatory, antibacterial, antiviral, antidiabetic, etc. To evaluate the efficiency and safety of ginseng plant consumption, more and more ginseng clinical trials have been conducted recently. From the modern research studies, ginseng possesses a variety of bioactive compounds including ginsenosides, polysaccharides, and peptides that have been used effectively for neuroprotective, Immunomodulatory, antiinflammatory, antidiabetic, antiglycaemic, and anticancer effects. In the present study, the phytoconstituents present in the ginseng plant were studied for their anti-inflammatory action against COX genes. Precisely, the in-silico approach showed that the ginseng plant and its phytoconstituents show anti-inflammatory properties against COX genes. The phytoconstituents like gallic acid, myricetin, apigenin, epicatechin, chlorogenic acid, and quercetin can potentially be used as anti-inflammatory agents. Even though the phytoconstituent of ginseng plant myricetin and apigenin show irritation, further more studies are needed to conclude the anti-inflammatory property of these compounds. The interaction of these phytoconstituents may provide useful insight for efforts to design new NSAIDs with novel properties providing an important field for future research on drug development.