A NETWORK PHARMACOLOGY OF LEMONGRASS ( Cymbopogon citratus ) ON COVID-19 CASES

Various ways and treatment efforts are carried out to avoid the severe impact of COVID-19 cases, one of which is using plants as natural immunomodulatory agents. One of the plants that is proven to act as a natural immunomodulator is lemongrass ( Cymbopogon citratus ). This study aimed to determine the protein tissue associated with the body's immune system activated by C. citratus . The secondary metabolites of C. citratus were identified using the KNApSAck and Dr. Duke databases. Target proteins associated with plant-secondary metabolite compounds from the SwissTargetPrediction database and immunomodulatory-associated target proteins were obtained from the GeneCards database. The intersected proteins were put into StringDB and analyzed using KEGG to obtain network pharmacology. 98 secondary metabolite compounds of C. citratus were obtained from the database. Proteins associated with C. citratus contain 1096 compounds, and those related to immunomodulators contain 1380 proteins. The intersection results obtained 244 proteins predicted to interact with C. citratus and are related to immunomodulators. From the results of KEGG analysis, five pathways related to C. citratus were obtained, namely PD-L1 expression and PD-1 checkpoint pathway in cancer, Fc epsilon RI signaling pathway, Th17 cell differentiation, T cell receptor signaling pathway, and IL-17 signaling pathway. MAPK 1, MAPK 3, and MAPK 14 proteins are predicted to be in all five related pathways, and Mol 13 compounds are predicted to be able to interact with these three proteins. Thus, it can be concluded that the compound Mol 13 is the compound that plays the most role in acting as an immunomodulator in C. citratus .


Backgroud
Corona viruses are known to cause disease in humans and animals, usually infecting only the upper respiratory tract and causing relatively minor symptoms (Syauqi, 2020).COVID-19 (Corona Virus Disease 2019), the disease that first appeared in Wuhan at the end of 2019 (Lena et al., 2023), can destroy lung cells and trigger local immune responses, recruiting macrophages and monocytes that respond to infection, release cytokines, and prime adaptive T and B cell immune responses.In most cases, this process can resolve the infection.However, in some cases, a dysfunctional immune response can lead to severe and even systemic lung pathologies (Tay et al., 2020).Various ways and treatment efforts are carried out to avoid the severe impact of COVID-19 cases, one of which is using plants as natural immunomodulatory agents (Morley, 2020).Immunomodulators are essential to the treatment process amid the current pandemic and disasters.Immunomodulators help the body optimize the immune system's function, the central system that plays a role in the body's defence against viruses (Erniati and Ezraneti, 2020).Immunomodulators are compounds that have a role in the human immune system, specifically and non-specifically, that can improve the immune system and increase cellular and humoral defences in the human body (Erniati & Ezraneti, 2020).In addition, immunomodulators also function in improving the immune system in the body by restoring immune system function, which is generally called immune restoration, stimulating a disturbed immune system (immunostimulant), then immunodepression, which suppresses and normalizes immune reactions that experience abnormal or abnormal conditions (Fujiati et al., 2022).One plant that is empirically used by the community to improve the body's immune system is lemongrass (Cymbopogon citratus).The ease of lemongrass in tropical regions such as Indonesia makes lemongrass a plant that is easily found in the yards of Indonesian people's homes and is often used as a spice for cooking.The phytochemical composition of lemongrass includes tannins, saponins, anthraquinones, phenols, flavonoids and alkaloids (Ernis et al., 2020).Exploration of the activity of a compound of a plant can be done through three approaches: in vitro, in vivo, and in silico tests.Research is carried out with the in silico method for developing new drugs.It is more effective, efficient and economical because it is tested by computer (Zhang et al., 2019).The use of in silico methods is more like a computational predictive model.The purpose of in silico prediction is as a preliminary test before proceeding to in vitro and in vivo testing, screening several test compounds before proceeding to the in vitro and in vivo stages to predict the toxicity of these compounds (Ekins et al., 2007).Network Pharmacology have been explicitly used to explore protein/gene-disease connectivity pathways.It can illustrate the complexity between biological systems, drugs and disease to network and provide a comprehensive approach.Recent years have seen an overall increase in traditional medicinal plant research due to their natural resources and the ability to use these plants to complement existing pharmacological approaches (Veda et al., 2023).Based on the background description that has been described, this study aims to determine the protein network associated with the body's immune system that is activated due to the administration of lemongrass (Sharma and Kaur, 2022)

Research Methods
The secondary metabolite compounds of plants were identified using the KNApSAck and Dr. Duke's Phytochemical and Ethnobotanical Databases, then looking for the SMILES code of each compound using PubChem (Lena et al., 2023).Proteins with a probability of > 0 that were predicted using SwissTargetPrediction were selected for further analysis (Daina et al., 2019).The immunomodulators-linked proteins have been searched using Genecards (Stelzer et al., 2016).Then, look for the intersection between the predicted proteins from SwissTargetPrediction and immunomodulator-linked proteins using Venny (Daina et al., 2019).The list of proteins that appear is then entered into the StringDB (Szklarczyk et al., 2021),after which predictions of proteins related to the immune system are searched using KEGG (Kanehisa et al., 2023) by looking at which proteins interact with the most with pathways related to the immune system and then looking at secondary metabolite compounds of lemongrass that interact with these proteins.

Identification and Bioavailability Prediction of Secondary Metabolites of Lemongrass
Secondary metabolites of lemongrass were obtained using the KNApSAcK Family and Dr. Duke's Phytochemicals and Ethnobotanical Databases.There are 76 (KNApSAcK Family) and 34 (Dr.Duke's Phytochemicals and Ethnobotanical Databases), with a total of 98 secondary metabolite compounds obtained from those databases (Table 1).KNApSAcK is the database with the highest number of compounds.KNApSAcK ranks first with more than 10,500 entries for therapeutic efficacy and biological activity records (Nguyen-Vo et al., 2020).Dr. Duke's Phytochemicals and Ethnobotanical Databases was widely used to characterize bioactive compounds in plants (Anand and Gokulakrishnan, 2012).

Immunomodulator-linked Protein That Predicted Interact With Lemongrass
A search for target protein predictions of each prediction compound was carried out using SwissTargetPrediction.The search results found that there are 909 proteins that are predicted to interact with secondary metabolites of the lemongrass.Immunomodulator-linked protein was searched using GeneCard.There were 1340 proteins obtained related to immunomodulators.Furthermore, intersections were carried out using Venny between proteins that were predicted to interact with secondary metabolites of lemongrass and immunomodulator-linked proteins, and there are 244 proteins that are predicted to interact with secondary metabolites of lemongrass related to immunomodulators (Figure 1).
Figure 1.The protein is predicted to interact with secondary metabolites of lemongrass with proteins related to immunomodulators

Network Pharmacology Analysis
The protein obtained from the results of the Venn diagram intersection is then further analyzed using StringDB, which aims to create a network of interactions between selected target proteins and analyze immunostimulating biological pathways influenced by these proteins (Figure 3) (Lena et al., 2023;Veda et al., 2023).StringDB is a database of known and predicted proteinprotein interactions integrating functional relationship data from multiple sources, including more than nine million proteins (Grabowski and Rappsilber, 2019) After that, KEGG enrichment analysis was carried out.KEGG (Kyoto Encyclopedia of Genes and Genomes) is a collection of manually drawn pathway maps representing our knowledge of molecular interactions and reaction networks (Kanehisa et al., 2023).The five highest strength values of immunomodulators-linked pathways were selected for further analysis (Table 3).Three proteins are connected to five immunomodulator-linked pathways: MAPK1, MAPK3, and MAPK14.In further analysis, there is one compound (Mol 13) that has the most potential to be developed into immunomodulators (Table 4).This compound (alpha-pinen) connect to three immunomodulator-linked protein.Therefore, lemongrass has the potential to be further explored and developed as a promising immunomodulating agent.