ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. A SYSTEMATIC ANALYSIS OF COMPOUNDS PRESENT IN OCIMUM TENUIFLORUM (TULSI) REGARDING ITS ANTI-INFLAMMATORY PROPERTIES USING IN-SILICO TECHNIQUES JINAY PATEL, SONIA ARORA (FACULTY ADVISOR) ✵ ABSTRACT The objective of this study was to gather data, create a database of the compounds present in Ocimum tenuiflorum (O. tenuiflorum), and gather related literature on the compounds found. A thor- ough literature search was performed to gather in- formation on compounds present in O. tenuiflorum, including chemical structures, relative abundance, presence in different plant parts, and availability from chemical supply vendors. The compounds’ chemical structures were refined using Discovery Studio Visualizer and Chimera software for future in- silico docking studies. The structures with cleaned structural geometry were obtained through D.S. Vis- ualizer for docking in the future. From the literature search of previously presented articles, it was found that methyl eugenol had the greatest percent com- position in O. tenuiflorum. After searching the Pro- tein Data Bank, COX-1, COX-2, and NF Kappa B were found to be the main protein targets of O. ten- uiflorum compounds in the arachidonic acid inflame matory pathway. Thus, the anti-inflammatory proper- ties of O. tenuiflorum have been analyzed in this ar- ticle for future in silico docking. 1 INTRODUCTION Tulsi is a plant of the species Ocimum, scien- tifically known as Ocimum tenuiflorum L. and more commonly known as “the holy basil.”[5] It is native to India and parts of North and Eastern Africa, China, Hainan Island, and Taiwan, where it is also titled as "the elixir of life" or "the queen of herbs."[5] Numer- ous parts of O. tenuiflorum, such as the leaves, stems, and flower spikes, are traditionally used in Ayurveda and Siddha medicine for treating condi- tions such as coughs, bronchitis, fever, and bile dis- turbances.[5] Ayurveda and Siddha medicines are two of the most ancient medicinal branches of India based on herbal and mineral compounds. O. tenui- florum has also been vividly known for its anti-inflam- matory, antiseptic, and other numerous organ pro- tective properties.[5] Based on its prominent medici- nal uses, the Ocimum tenuiflorum was chosen for fur- ther investigation. In this study, the focus was on in- flammation because it is a well-known symptom of numerous infectious and non-infectious diseases. Various in vitro and in vivo studies have documented the anti-inflammatory effects of O. tenuiflorum, and it has been suggested that O. tenuiflorum has many bioactive secondary metabolites that help inhibit certain inflammatory pathways synergistically or alone.[6] Arachidonic acid is the major polyunsatu- rated fatty acid present in mammalian systems, which is oxygenated by three important pathways— the cyclooxygenase (COX), the lipoxygenase (LOX), and the epoxygenase pathways.[10] The inhibition of COX and LOX pathways in arachidonic acid metab- olism in conjunction with major O. tenuiflorum com- pounds like eugenol and linoleic acid contributes to the anti-inflammatory action of O. tenuiflorum, ob- served in both acute and chronic inflammatory mod- els in animals.[4] This observation suggests that cer- tain compounds present within O. tenuiflorum are responsible for anti-inflammatory responses in ani- ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III mal models. This implication can be further explored to determine which compounds in O. tenuiflorum may induce anti-inflammatory activity in humans. As a result, O. tenuiflorum has demonstrated anti-in- flammatory effects normally observed in nonsteroi- dal anti-inflammatory drugs (NSAID) such as phenyl- butazone, ibuprofen, naproxen, aspirin, and indo- methacin.[4] The inhibition of the cyclooxygenase en- zymes COX-1 and COX-2 is what drives the NSAID mechanism.[1] Although NSAIDS are effective, they have a few common side effects, such as nausea, dyspepsia, vomiting, and skin reactions. On the con- trary, herbal medications are devoid of such side ef- fects, which illustrates the importance of conducting a study of the compounds found in O. tenuiflorum: it may open the path for herbal NSAIDs without these side effects.[1] O. tenuiflorum has various transcription fac- tors, one of which is the nuclear factor-Kappa B (NF Kappa B). NF Kappa B is one of the major regulators of inflammation, cellular transformation, tumor cell survival, proliferation, invasion, angiogenesis, and metastasis as compared to the other transcription factors.[11] Plants of the Ocimum species are known to have an abundance of terpenes.[7] Additionally, plant isolates containing terpenoids (a modified class of terpenes) have been found to suppress NF kappa B signaling, a protein complex linked to the pathogenesis of inflammatory diseases, cancer, viral infection, and autoimmune diseases.[7] Thus, the study of O. tenuiflorum for anti-inflammatory proper- ties is vital. There are numerous compounds present in the Ocimum species amongst which methyl euge- nol, β-selinene, γ-murolene, rosmarinic acid (phe- nolic), ursolic acid, and camphene are some major ones based on their percentage of makeup. These and other compounds from O. tenuiflorum have been characterized and analyzed in this study to form an extensive database. This database is used to determine which of the compounds identified would interact with desirable molecular targets and de- crease inflammation in future studies. Therefore, in this study, an in-silico approach was used to refine the compounds present in O. ten- uiflorum and to develop a highly detailed database to be used for future purposes, such as investigating how an individual compound from O. tenuiflorum extract will interact with these molecular targets. This interaction would be detailed using the in-silico docking mechanism, which is a molecular model- ing technique that is used to predict how a protein (enzyme) interacts with small molecules (ligands) to deduce their mechanism of action on targets found in future dry labs. 2 METHODOLOGY First, a literature search was conducted through Pubchem to create a list of well-known com- pounds present in O. tenuiflorum and their percent composition in the part of the plant they are found in.[8] The vendors that distribute the compound in the USA were identified from PubChem as well (TABLE 1).[8] Next, the collection of the virtual structures of all the compounds was created and the files were saved in the SDS (2D) formats (FIGURE 1). Then, within the structural data, the pharmacology and biochem- istry of the compounds were reviewed for each com- pound from PubChem in order to get an idea of the mechanism of action and the aspects of human in- teraction associated with the individual com- pounds.[8] This was an adaptation process, so new compounds were added to the list when and if they were discovered throughout the study. Later, software such as Discovery Studio (D.S.) Visualizer (BIOVIA Dasasult Systemes, San Di- ego, CA, USA) and Chimera were downloaded.[9] Chimera was used to convert the mol format files into sybyl mol 2 format files for compatibility within D.S. Visualizer. The sybyl mol 2 format files were then opened in D.S. visualizer, wherein hydrogen atoms were added to the structure. The geometry of the hy- drogen atoms was cleaned and 3D coordinates were assigned to them. The three-dimensional structure was then saved as sybyl mol 2 format files (FIGURE 2). The formats sybyl mol 2 and SDS were employed to have a better approximation of the structure of the compounds, and to notice the 2D and 3D differ- ences in the molecules, which would visually assist in the molecular docking. And finally, another literary search was con- ducted on the Protein Data Bank for the inflamma- tory molecular pathways and O. tenuiflorum within ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III COMPOUNDS PERCENTAGE FOUND VENDORS PART OF PLANT 1. 1,8-CINEOLE TRACE(T) ALL LEAVES 2. 1,10 DI-EPI-CUBENOL 1.8 N/A LEAVES 3. 3,4-DIMETHOXYCINNAMIC ACID (PHENOLIC) N/A ALL N/A 4. 3.4.5-TRIMETHOXYCINNAMIC ACID N/A 1,2,4 N/A 5. 4.4′-METHYLENE-BIS (2-METHYL ANILINE) N/A N/A N/A 6. Α-ELEMENE 0.5 2 FLOWER SPIKES 7. Α-HUMULENE 0.2 4 LEAVES 8. Α-PINENE 0.2 2 LEAVES 9. Α-TERPINEOL TRACE(T) 1,2,3 LEAVES 10. ALLOAROMADENDRENE 1.16 N/A N/A 11. APIGENIN N/A 2 LEAVES 12. Β-BOURBONENE 0.2 N/A LEAVES 13. Β-CUBEBENE 0.1 2,4 FLOWER SPIKES 14. Β-PINENE 0.1 2,3 LEAVES 15. Β-SELINENE 3.3 2,4 LEAVES 16. Β- SESQUIPHELLANDRENE 0.2 N/A LEAVES, FLOWER SPIKES 17. BAICALIN (LAVONOIDS) N/A 2,4 N/A 18. BENZALDEHYDE 0.44 2,4 LEAVES 19. BICYCLOGERMACRENE TRACE(T) N/A LEAVES 20. CAFFEIC ACID (PHENOLIC) N/A ALL LEAVES 21. CAMPHENE 0.1 1,2,3 LEAVES 22. CAMPHOR 0.1 2,3 LEAVES 23. CARNOSIC ACID N/A 2,4 N/A 24. CHRYSOERIOL (FLAVON) N/A 1,2,4 N/A 25. CI Α-COPAENE 1.9 N/A LEAVES, FLOWER SPIKES 26. CI Α-CUBEBENE TRACE(T) 4 FLOWER SPIKES 27. CI Β-CARYOPHYLLENE 4.1 2,3,4 LEAVES 28. CI Β-GURJUNENE TRACE(T) N/A LEAVES 29. CI BORNEOL 2.4 2 LEAVES 30. CI TERPIN-4-OL 0.1 2,3 LEAVES, STEM 31. CUBEBOL 0.3 N/A LEAVES 32. DI (ETHYLHEXYL) PHTHALATE N/A 1,2 N/A 33. DI-N-BUTYL PHTHALATE, DIBUTYL PHTHALATE N/A 1,2,4 LEAVES TABLE 1: This table shows various compounds found in specific parts of O. tenuiflorum such as the leaves, stem, or flower spikes. It also includes their percentage composition, the vendors who supply them in the USA, and their PubChem SID and Purchas- able Chemical ID. Some of the compounds have trace(t) in their “Percentage Found” column, which implies that the compound is present in a quantity too small to be measured. PubChem[8] ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III COMPOUNDS PERCENTAGE FOUND VENDORS PART OF PLANT 34. DIOSMETIN (FLAVONE GLYCOSIDE) N/A 1,2,4 N/A 35. (E)-Α-BERGAMOTENE 0.72 2,4 LEAVES, FLOWER SPIKES 36. (E)-Β-OCIMENE 1.9 2,4 LEAVES 37. E-METHYL CINNAMATE 1.5 ALL LEAVES, STEM 38. EPI-Α-CADINOL 1.03 N/A LEAVES 39. ESTRAGOL N/A 1,2,4 LEAVES, FLOWER SPIKES 40. EUGENOL 0.9 ALL LEAVES 41. Γ-MUROLENE 5.82 2 LEAVES 42. GERMACRENE A 0.7 N/A LEAVES 43. GERMACRENE D 2.3 2,4 LEAVES 44. GERANIOL TRACE(T) ALL LEAVES 45. GLOBULOL 1.05 2,4 N/A 46. ISOSAKURANETIN (LAVANONE) N/A 4 N/A 47. KAEMPFEROL N/A ALL N/A 48. LINALOOL 0.5 3 LEAVES 49. LIMONENE 0.2 1,2 LEAVES 50. LUTEOLIN (FLAVONOID) 0.5 N/A LEAVES 51. METHYL CHAVICOL TRACE(T) 1,2,3 LEAVES 52. METHYL EUGENOL 82.9 2,3,4 LEAVES 53. MYRTENAL N/A 2 LEAVES 54. NEVADENSIN (FLAVONES, GLYCOSIDES) N/A 2,4 N/A 55. P-CYMENE TRACE(T) 1,2,3 FLOWER SPIKES 56. PEDUNCULIN N/A N/A N/A 57. PERMETHRIN N/A 1,2 N/A 58. PEINIDIN (ANTHOCYANIDINS) N/A N/A N/A 59. ROSMARINIC ACID (PHENOLIC) 0.27 2,4 LEAVES, STEMS 60. SABINENE TRACE(T) 2 LEAVES 61. Δ-CADINENE 1.1 2,4 LEAVES 62. (TRANS)-Β-FARNESENE 0.12 2,4 LEAVES 63. (TRANS)-Β-GUAIENE 0.29 N/A LEAVES 64. URSOLIC ACID 2.5 1,2,4 LEAVES 65. XANTHOMICROL N/A 2 STEM 66. (Z)-3-HEXANOL 1.8 1,2 LEAVES VENDORS 1) TIM TEC 2) MUSECHEM 3) ACROS ORGANICS 4) ZINC TABLE 1 CONTINUED ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III the context of inflammation and its molecular targets concerning humans.[2] This search was conducted to determine which molecular target should be further used to analyze the docking compatibilities of the compiled O. tenuiflorum compounds. 3 RESULTS A detailed database was created in TABLE 1 to compile an extensive list of 66 compounds in O. ten- uiflorum. TABLE 1 also displays information about the percentage of a particular compound present in cer- tain parts of O. tenuiflorum, such as the leaves, stem, flower spikes, etc. In TABLE 1, it is evident that most of the compounds were available from the list of US- based vendors like Tim Tec, Musechem, Acros or- ganics, and ZINC. The PubChem SID and Purchasa- ble Chemical ID were also available for all of the compounds. The highest percentage of composi- tion for a compound in O. tenuiflorum was for methyl eugenol, about 82.9% within the leaves, which was available from Musechem, Acros organics, and ZINC (TABLE 1). Overall, a total of 66 compounds were iden- tified to be present in O. tenuiflorum. Out of those compounds, many were in trace amounts and some did not have any data on the percentage of their presence (TABLE 1). Next, 2D structures in SDS format files were obtained from PubChem in the form of 2D ChemDraw files (FIGURE 1). FIGURE 1 shows the chemical structure of a few major compounds found in O. ten- uiflorum. The compounds seen were not ready for docking since they had not been refined yet to allow for further in-silico processes due to their structural ambiguity. It would be difficult to dock them; there- fore, further work on each compound was needed, such as adding hydrogens and removing unwanted ions (FIGURE 1). Next, the refined structures were ob- tained from D.S. Visualizer in order to transfer the co- ordinates to proceed to the in-silico docking step (FIGURE 2). The refined structure of several O. tenuiflo- rum compounds are portrayed in FIGURE 2. Finally, the literature search on the Protein Data Bank yielded that COX-1, COX-2, and NF Kappa B were the anti-inflammatory target proteins that interact with certain compounds found in O. ten- uiflorum. However, within the Protein Data Bank the COX-2 protein was the most prevalent target found in humans and COX-1 had the least human entries. These targets will be explored in future studies with the collected O. tenuiflorum database compounds. FIGURE 1: The 2D structures of compounds present in O. tenuiflorum are shown here, such as ursolic acid, methyl eugenol, γ-murolene, rosmarinic acid, camphene, and β-selinene. Some of the figures that were downloaded from PubChem portray the oxygen and hydroxide atoms in red fonts. FIGURE 2: 3D structures of a few of the major compounds present in O. tenuiflorum, such as camphene, methyl eu- genol, rosmarinic acid, β-selinene and γ-murolene with hy- drogen atoms added and their geometry cleaned. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 4 DISCUSSION & CONCLUSION For this study, a thorough database was cre- ated for various compounds present in different parts of O. tenuiflorum. This database is being stored on the computers in the Arora lab to be used for future studies. During the literary search of these structures, it was found that methyl eugenol was the most abundant compound and γ-Murolene was the second most abundant compound. Both com- pounds are present in the leaves of O. tenuiflorum. These compounds are significant because they are present in such high abundance, which suggests that they might have an important role in the anti-in- flammatory effect of O. tenuiflorum. To test this state- ment, these structures will have to be run through in- silico docking modules to determine whether the abundance has any correlation with the anti-inflam- matory effect. Moreover, the purpose of attaining the 2D files was to make the structures available before re- finement so that they could be reviewed when re- quired. The refined structures with cleaned geome- try were obtained so that their docking sites were clear of any hindrance and to ensure that docking could be done properly in the future. Also, the target proteins were identified from the Protein Data Bank so that they could be used along with their ligands to dock. Thus, the future goal is to use in-silico ap- proaches to discover how compounds will interact with various molecular targets and inflammatory pathways, specifically COX-2, as it is the most preva- lent target yielded in most searches related to hu- man query within the Protein Data Bank. The data- base gathered in this study will be crucial for future studies to identify which compounds present in O. tenuiflorum are attributed to its anti-inflammatory properties. This information could in turn allow for the development of herbal anti-inflammatory medi- cation with minimal side effects∎ 5 ACKNOWLEDGEMENTS I would like to thank my mentor and supervi- sor Dr. Sonia Arora for her invaluable support and knowledge behind this paper and research. Her pas- sion for the subject, along with attention to detail has been greatly inspiring, allowing me to diligently work on this research project. Her ingenious remarks and constructive criticism for various drafts of this pa- per has allowed for this research paper to be suc- cessfully completed. I will always be extremely grate- ful to Dr. Arora for imparting me with the knowledge and wisdom about in-silico approaches, which can be used to create comprehensive databases in any research work. 6 REFERENCES [1] Ahmad A., Abuzinadah M.F., Alkreathy H.M., Banaganapalli B., & Mujeeb M. (2018). Ursolic acid rich Ocimum sanctum L leaf extract loaded nanostructured lipid carriers ameliorate adjuvant induced arthritis in rats by inhibition of COX-1, COX-2, TNF-α and IL-1: Pharmacological and docking stud- ies. PLoS ONE, 13(3), E0193451. [2] Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., & Bourne, P. E. (2020). The Protein Data Bank. Nucleic acids research, 28(1), 235–242. [3] Biovia, Dassault Systemes, Discovery Studio Visualizer, Ver- sion 20.1, San Diego: Dassault Systèmes, (2020). [4] Cohen M. M. (2014). Tulsi—Ocimum sanctum: A herb for all reasons. Journal of Ayurveda and integrative medicine, 5(4), 251–259. [5] Flegkas A., Milosević Ifantis T., Barda C., Samara P., Tsitsilonis O., & Skaltsa H. (2019). Antiproliferative Activity of (-)- Rabdosiin Isolated from Ocimum sanctum L. Medicines, 6(1), 37. [6] Jamshidi, N., & Cohen, M. M. (2017). The Clinical Efficacy and Safety of Tulsi in Humans: A Systematic Review of the Literature. Evidence-based complementary and alternative medicine. Evidence-based Complimentary and Alternative Medicine: eCAM, 2017, 9217567. [7] Kapewangolo, P., Omolo, J. J., Bruwer, R., Fonteh, P., & Meyer, D. (2015). Antioxidant and anti-inflammatory activity of Ocimum labiatum extract and isolated labdane diterpe- noid. Journal of inflammation (London, England), 12, 4. [8] Kim, S., Chen, J., Cheng, T., Gindulyte, A., He, J., He, S., Li, Q., Shoemaker, B. A., Thiessen, P. A., Yu, B., Zaslavsky, L., Zhang, J., & Bolton, E. E. (2019). PubChem 2019 update: Im- proved access to chemical data. Nucleic acids research, 47(D1), D1102–D1109. [9] Pettersen EF, Goddard TD, Huang CC, Couch GS, Green- blatt DM, Meng EC, Ferrin TE. (2004). UCSF Chimera—A vis- ualization system for exploratory research and analysis. J Comput Chem. 25(13):1605-1612. [10] Reddy, K. K., Vidya Rajan, V. K., Gupta, A., Aparoy, P., & Red- danna, P. (2015). Exploration of binding site pattern in ara- chidonic acid metabolizing enzymes, Cyclooxygenases and Lipoxygenases. BMC research notes, 8, 152. [11] Yadav, V. R., Prasad, S., Sung, B., Kannappan, R., & Aggarwal, B. B. (2010). Targeting inflammatory pathways by triterpe- noids for prevention and treatment of cancer. Toxins, 2(10), 2428–2466. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III Jinay Patel is Rutgers University graduate, having B.S degree in Biotechnology and Plant Science from School of Environmental and Biological Science. His research was on O. tenuiflorum to gather the data on the compounds present within it and study its anti-inflammatory properties using In- Silico techniques. He is currently working as a QC analyst at Roche Molecular Systems within enzy- matic department and in the future, he would like to attend graduate school and study drug devel- opment. Education in drug development for him would entail discovering new medicines and treating diseases to support better patient care. The following research on O. tenuiflorum is one of the crucial steps for him in attaining this goal.