DISCOVERY OF INTERLEUKIN-6 INHIBITORY PROPERTIES OF THE COMPOUNDS DETECTED FROM METHANOLIC LEAF EXTRACTS OF PLECTRANTHUS AMBOINICUS (LOUR) SPRENG. THROUGH MOLECULAR DOCKING

DISCOVERY OF INTERLEUKIN-6 INHIBITORY PROPERTIES OF THE COMPOUNDS DETECTED FROM METHANOLIC LEAF EXTRACTS OF PLECTRANTHUS AMBOINICUS (LOUR) SPRENG. THROUGH MOLECULAR DOCKING

Suchita Muthukrishnan and Sathyabhama Muthuswamy *

Department of Biotechnology, PSG College of Arts & Science, Coimbatore, Tamil Nadu, India.

ABSTRACT: Plectranthus amboinicus, locally called Karpooravalli in Tamil Nadu, India, is known for its various traditional therapeutic properties. Rheumatoid arthritis is a chronic autoimmune disease where uncontrolled inflammatory markers are released to induce bone damage and pain. In this study, the methanolic extract of P. amboinicus was characterized phytochemically and subjected to in-silico analysis to evaluate its anti-inflammatory properties. The methanolic extract of P. amboinicus was subjected to gas chromatography-mass spectrometry (GCMS) analysis. The detected compounds were subjected to in-silico analysis with biomarkers specific for rheumatoid arthritis (RA) that is interleukin 6. Of the 86 profiled compounds, three belonging to flavonoid, porphyrin, and heterocyclic triazoles classification had the highest affinities for interleukin-6 and was compared with common DMARs given during RA treatment which had significantly low affinities to above 3 highest affinities bioactive compounds. The ADMET profile, drug-likeness, and bioactivity score were also calculated for all the GCMS profiled compounds, revealing adequate drug-like properties. In-silico analysis of the leaf extract of P. amboinicus compounds showed a promising result for its anti-inflammatory activities and indicates the possible use of P. ambionicus extract in treatment of Rheumatoid arthritis.

Keywords: Plectranthus amboinicus, Gas chromatography-mass spectrometry, In-silico analysis, Rheumatoid arthritis (RA), Anti-inflammatory, Interleukin-6

INTRODUCTION: Plectranthus amboinicus (Lour.) Spreng is a perennial, shrubby herb also called Coleus amboinicus that belongs to the Lamiaceae family originates from tropical and warm regions of Asia, Africa, and Australia ^{1, 2}. Along with beneficial essential oils, this edible herb has an aromatic nature used in folk medicine to treat conditions such as cough, cold, sore throat, nasal congestion, breast milk stimulant ³, asthma, fever, and headache ⁴.

The extract is also effective against other diseases, such as flu, bronchitis, epilepsy, and skin diseases. A total of 76 volatiles and 30 non-volatile’s of different classes were detected and these plants are also known to contain highly bioactive compounds such as monoterpenoids ¹.

In recent years, plants have gained scientific importance in pharmaceutical research for evaluating phytochemicals and discovering potential compounds that can treat various diseases. One study revealed that carvacrol in the ethyl acetate extract of P. amboinicus modulates the expression of inducible nitric oxide synthase, cyclooxygenase 2, interleukin 1β, the histamine 1 receptor gene, and the nuclear factor kappa B protein thus exhibiting anti-inflammatory and antinociception properties ⁵. Another study demonstrated that high doses of P. amboinicus decrease the production of proinflammatory cytokines ⁶. Hsu and colleagues also postulated that rosmarinic acid from P. amboinicus inhibits osteoclastogenesis by inhibiting the activity of RANKL, an apoptotic regulatory gene, thus preventing bone destruction ⁷.

Inflammation is an essential biological response and part of the body’s healing process. One of the first responses of the immune system is inflammatory cells travel to the site of injury or infection ⁸. When this response occurs for an extended period it leads to chronic inflammation, which is the primary response in most autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disease in which inflammation and bone destruction is the primary prognoses ⁹. It has been presumed that genetically inclined individuals exposed to environmental stimuli acts as triggering factors for the progression of RA. RA is associated with systemic pathology, including (i) triggering stages, including the production of inflammatory cells. The environment also triggers the production of inflammatory cells. (ii) In the maturation stage the inflammatory class activates MHC class II-dependent T-cells in lymphoid tissue, which produce B cells and many additional inflammatory cells that, in turn, induce pain, bone loss, and inflammation. The next step is (iii) targeting, during which leukocytes and proinflammatory cells infiltrate the synovial compartment and interact with synovial fibroblasts to produce an inflammatory cascade. The final stage is called the (iv) fulminant stage and is characterized by a hyperplastic synovium caused by the dysfunction of synovial fibroblasts ¹⁰.

The hallmark immune components responsible for the pathogenesis of RA are macrophages, neutrophils, CD4+ and CD8+ T cells and their associated cytokines such as tumor necrosis factor-α (TNF-α), interleukin- IL-1, IL-6, IL-17, and IL-23 ¹¹. The administration of non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and disease-modifying anti-rheumatic medications (DMARDs) are frequently used treatment methods. Physical therapy is also recommended to sustain joint mobility ¹⁰. In-silico analysis of phytochemicals tremendously helps rapid screening of the vast amounts of compounds in medicinal plants which could have potential in the pharmaceutical industry. With the ability to treat many diseases, diverse plant kingdoms have gained significant attention from researchers seeking new drugs with the help of computational technologies. In-silico analysis has also provided insight in to the immune response and its interaction with ligands and possible outcomes as well as how to narrow down the target protein, thus helping to modify ligands to improve interactions with targeted proteins and increase disease inhibiting activity.

In this study, we targeted P. amboinicus methanolic leaf extract as an herbal medicine for treating RA. Phytochemical analysis was performed, and an evaluation of the antiarthritic properties via in-silico analysis was performed.

MATERIALS AND METHODS:

Sample Collection, Preparation and Extraction: Plectranthus amboinicus leaves were collected from Coimbatore, Tamil Nadu (Lat. 11.022053⁰, Long. 77.017328⁰). The plants were extracted using a modified protocol from Samad et al., 2019. P. amboinicus leaves were cleaned, shade-dried for 48 hours, and ground using an electric grinder. Then, the dried leaf powder was soaked for 5 hours in 30% methanol, and the solvent to solid ratio was 30mL/g. Then, the samples were irradiated for 2 minutes via microwave-assisted extraction (IFB Solo microwave, 2450MHz) ¹².

Gas Chromatography-mass Spectroscopy Analysis: The methanolic plant extract was subjected to GC-MS analysis on an Agilent 7000 D GC/TQ instrument by a DB 35-MS capillary standard nonpolar column using helium gas at a flow rate of 1.0 mL/min and an initial temperature of 70°C after which the temperature increased to 260°C at a rate of 6°C to identify the compounds present.

In-silico Analysis (Molecular Docking):

Docking: Compounds identified by GC-MS analysis were downloaded from the PubChem database in SDF format. Similarly, protein-interleukin 6 (Protein ID: IL6) was downloaded from the RCSB Protein Data Bank in PDB format. The compounds were converted to PDBQT formatted using Open Babel GUI software 2006 v3.1.1. In contrast, after docking parameters such as deleting the water molecule, adding Kollman charges, computing Gasteiger, and assigning the AD4 type were applied, the proteins were converted to the PDBQT format in the AutoDock tool.

Docking was performed using AutoDock vina 1.5.7 via the command prompt method. A dock file was created in which ligand and protein files in PDBQT format were saved with the configuration file. The configuration file consists of the protein and ligand file names with the grid box configuration, the log output file name, and the exhaustiveness of the number of docking runs. A grid box was generated and set at 80 × 80 × 80 with the coordinates X-0.166 Y-0.191 Z-0.438 for the protein IL6. Ligand efficiency ¹³ was calculated using the following formula:

Ligand efficiency =∆G / (Heavy atoms)

Moreover, the inhibition constant (Ki) 14 was calculated using the following formula:

Inhibiton constant Ki = Exp × (∆G / RT)

Where ∆G is the binding energy, R is the universal gas constant (1.985× 10^-3 kcal/mol/K), and T is the temperature (298.15 K).

FIG. 1: BIOMARKER OF RA RETRIEVED FROM THE RCSB PDB; INTERLEUKIN 6 (IL6)

Receptor-ligand Interaction: Docking results and receptor-ligand interaction, such as the number of H-bonds formed with amino acid residue, were visualized using the BIOVIA Discovery Studio visualizer 2021V21.1.0.20298.

ADMET Profile: The parameters for ADMET (absorption, distribution, metabolism, excretion, and toxicity) were Human intestinal absorption (HIA) for absorption, blood-brain barrier (BBB), Plasma protein binding (PPB) for distribution, cytochrome P substrate/inhibition for metabolism, Time ½ (half-life) for excretion, and AMES, Carcinogenity, LD50 in rats for toxicity were profiled for the ligand derived from Pubchem using ADMETSAR 2.0 website(http://lmmd.ecust.edu.cn/admetsar2). The half-life was predicted using ADMETLab 2.0 (https://admet.scbdd.com/calcpre/index_sys/) and the LD₅₀ toxicity in rats were determined using the Gusar-Way2Drug (http://way2drug.com/Gusar/acutoxpredict/). 

Drug-likeness: SwissADME (http://www.swissadme.ch/index.php) was used to predict the drug-likeness of the ligands. The topological polar surface area (TPSA), solubility log S, C log P, molecular weight (MW), and Lipinski’s rule for the drug-likeness of the given ligands was predicted.

Bioactivity Score: The molinspiration online predictor tool (https://www.molinspiration.com/cgi-bin/properties) was used to calculate the predicted values of various bioactivities such as G-protein-coupled receptor (GPCR), ion channel modulator, kinase inhibitor, nuclear receptor inhibitor, protease inhibitor, and enzyme inhibitor for the given ligands.

RESULTS:

GC-MS Analysis: The identified compounds were further functionally characterized to determine their pharmacological activity and were subjected to in-silico analysis. GC-MS profiling of the extracts revealed a total of 86chemical compounds. Table 1 shows the compound list, retention time classification, and pharmacological characteristics.

Fig. 2 shows the chromatogram of methanolic P. amboinicus extract. The compounds are classified as terpenoids, alkaloids, phenols, and flavonoids. Pteridinone, thymol, diphenhydramine, α-farnesene, hydropapaveroline, and β-Bergamotene are some of the major compounds. A study revealed the presence of α-bergamotene, carvacrol, caryophyllene, ρ-cymene, and γ-terpinene as major constituents ¹⁵.

FIG. 2: MASS SPECTRA OF P. AMBOINICUS EXTRACTS                               

TABLE 1: P. AMBOINICUS COMPOUNDS IDENTIFIED BY GC-MS ANALYSIS

S. no.	Compound Name	RT	Structure	Formula	Classification	Pharmacological Property
1.	(S)-Laudanine	35.8524		C20H25NO4	Phenols	Antioxidant anti-protozoal antimicrobial16
2.	(Z)-3-Hexenyl Angelate	14.5748		C11H18O2	undetermined	unspecified
. 3.	1,1,1,5,5,5-Hexafluoropentan-3-one	21.9468		C5H4F6O	Organofluorine	Unspecified
4.	1,2,3-Triphenyl-3-methyl-cyclopropene	35.3912		C22H18	undetermined	unspecified
5.	1,3-Benzenediol, o-(2-furoyl)	35.6205		C11H8O4	esters	Unspecified
6.	diphenhydramine	19.4390		C­17H21NO	Ethylamines	anti-allergic, antiparkinson , antipruritic, antidyskinesia 17, antitussive, oneirogen 18
7.	1,3-Diphenyl-2-propyl imidazolidine	27.1891		C18H22N2	Heterocyclic compounds	Antibacterial Anti-leishmanial19
8.	1-Hexene, 4,4-diethyl	13.4897		C10H20	Volatile hydrocarbans	unspecified
9.	1-Phenyl-2,4-pentadiyn-1-one(demethylcapillin)	19.1240		C11H6O	Flavonoids	Anti-proliferative 20
10.	1-phenyl-2-methyl-6,7-dimethoxy-1,2,3,4-tetrahydroxyquinoline	33.0116		C­18H21NO2	Alkaloids	Anti-angiogenic21
11.	2,3-Dihydroxynaphthalene	26.9708		C10H8O2	Phenols	Anti-proliferative22
12.	2,4-Dimethyldecane	15.8288		C12H26	Alkanes	Antimicrobial Antioxidant23
13.	2,7-Bis-allyloxyfluoren-9-one	31.7128		C19H16O3	undetermined	Antimicrobial24
14.	2-Anilino-3-chloro-1,4-naphthoquinone	34.0346		C16H10CINO2	Phenols	Antitrypanosomal 25 Antitumor 26
15.	2-butyl-10H-acridin-9-one	32.3700		C17H17NO	Aminoacridines	Antibacterial Antiprotozoal Anticancer Antiviral 27
16.	2-Butynoic acid	24.9594		C4H4O2	Unsaturated fatty acid	unspecified
17.	2-Ethylhexanol	8.5879		C8H18O	Hexanols	Food additives28
18.	2-Ethylpyrazine	16.6790		C6H8N­2	Peptide alkaloids	Antioxidant29
19.	2-Morpholin-4-yl-1,5-diphenyl-7-p-tolylimino-5-trifluoromethyl-1,5,6,7-tetrahydro-[1,2,4]triazolo[1,5-c]pyrimidine-8-carbonitrile	16.7173		C30H26F3N7O	Heterocyclic triazoles	unspecified
20.	2-Oxo-pentanedioic acid 1-ethyl ester	8.7695		C7H10O5	Linear dicarboxylic acid	Antibacterial Antitumor Antioxidant30
21.	2-Pentanoylfuran	24.8601		C9H12O2	Aromatic ketone	Antioxidant29
22.	2-Phenylethyl docosanoate	12.3695		C30H52O2	Esters	Antioxidant31
23.	2-Phenylquinoline	27.3080		C15H11N	Organic heterocyclic compound	Antimicrobial32 Antioxidant33
24.	2-Phenylquinoxaline	33.8430		C14H10N2	N-heterocyclic compound	Antifungal, antibacterial34,antiviral,35anticancer36
25.	3(2H)-Furanone, 5-methyl-2-octyl- (cepanone)	23.2901		C13H22O2	oxolanes	Antioxidant37
26.	3-(3,5-di-tert-butylphenyl)pentanedioic acid	34.1681		C19H28O4	linear dicarboxylic acid	Unspecified
27.	3,4-Bis(methoxycarbonyl)benzoic acid	32.2998		C11H10O6	aromatic carboxylic acid	Unspecified
28.	3',4'-Desoxy-3,4-dihydropapaveroline	36.4835		C16H15NO2	Alkaloids	Anticancer38
29.	3-(4-nitrophyenyl_-4,5-dihyro-1.2.4-oxadiazol-5-one	33.0011		C8H5N3O4	oxopyridine	Antimicrobial, anti-inflammatory, anticancer39
30.	3,4-Epoxy-3-ethyl-2-butanone	20.1784		C6H10O2	Un-determined	unspecified
31.	3-Acetylpyrrole	27.2731		C6H7NO	Azoles alkaloids	Unspecified
32.	3-Ethyl-3-methylheptane	15.8421		C10H22	branched alkane	Antioxidant Antimicrobial23
33.	3-Hydroxybenzylhydrazine	4.2565		C7H10N2O	Phenols	Central Nervous System Agents40
34.	3-Methyl-4-methylenebicyclo(3.2.1)oct-2-ene	19.7241		C10H14	Sesquiterpenoids	Antifungal41 Anticancer42
35.	2-(2,4-dihydroxyphenyl)-5-(e)-propenylbenzofuran	35.0606		C17H14O3	Heterocyclic compound	Anti-inflammatory Antioxidant43
36.	4-Methoxyformanilide	19.4691		C8H9NO2	Undetermined	Unspecified
37.	5-Aminotetrazole	20.6271		CH3N5	Azoles	Anticancer44
38.	5-Hydroxy-7-methoxy-2-methyl-3-phenyl-4-chromenone	35.3794		C17H14O4	flavonoids	Unspecified
39.	6-chloro-2H-pyridazin-3-one	16.5656		C4H3CLN2O	Pyridazines derivative	Unspecified
40.	6-Demethoxytangeretin	35.7121		C19H18O6	Flavonoids	Anti-inflammatory Anti-allergic45
41.	7(8H)-Pteridinone	27.2053		C6H4N4O	Heterocyclic compound	Anti-proliferative46
42.	9,10-Dihydro-9,10-dimethyl-9,10-ethanoanthracene-11,12-dicarboxylic acid	36.3748		C22H22O4	Polycyclic aromatic hydrocarbons	Unspecified
43.	9-Ethyl-3,6-dimethoxy-10-methylphenanthrene	34.9583		C19H­20O2	polycyclic aromatic hydrocarbon	Unspecified
44.	9-Oxo-10(9H)-acridineacetic acid	32.1181		C15H11NO3	Heterocyclic Compounds	Anticancer Antimicrobial47
45.	Amphetamine, N-pentafluoropropionyl	32.3174		C12H12F5NO	Organofluorines	Central nervous system stimulant48 Antioxidant49
46.	beta-Bergamotene	19.4418		C15H24	sesquiterpene	Antioxidant Anti-inflammatory50
47.	Benzphetamine	19.7269		C17H21N	Ethylamines	Adrenergic Agents, Central Nervous System Stimulants51
48.	Bis[(2.3)paracyclophano][1,2-b:1’,2’-i]anthraquinone	5.3099		C44H32O2	Flavonoids	anticancer, anti-inflammatory, diuretic, anti-arthritic52
49.	Carbamic acid, N-(3-oxo-4-isoxazolidinyl)-, benzyl ester	5.1489		C11H12N2O4	undetermined	Unspecified
50.	Carbonic acid, octadecyl vinyl ester	21.5138		C21H40O3	Fatty acid vinyl ester	Anti-diabetic53,anti-inflammatory54
51.	Chromeno(4,3-c)chromene-5,11-dione	35.7456		C16H8O4	Phenylpropanoids	anti-nociceptiveand anti-inflammatory55
52.	Cinnamolaurine	33.0264		C18H19NO3	Alkaloids	Antioxidant, anti-inflammatory, anti-proliferative56
53.	Crotonyl isothiocyanate	13.1665		C5H5NOS	organosulfur compound	Anticancer57 Anti-inflammatory, antioxidant,58 Antimicrobial59
54.	Cyclohexyl benzoate	20.1184		C17H16O2	aromatic carboxylic acid	Unspecified
55.	Cyclopentane, 1-butyl-2-propyl-	18.7852		C12H24	Fatty acid	Antioxidant60, antimicrobial, Anti-inflammatory38
56.	Cyclotetracosene	6.8518		C24H46	Branched unsaturated hydrocarbon	Anti-inflammatory Antioxidant61
57.	dibutyl phthalate	27.2009		C16H22O4	Phthalic acid	Antiparasitic62
58.	Diisobutyl phthalate	25.9845		C16H22O4	Phthalic acid	Anti- inflammatory63, antimicrobial64,
59.	DL-Laudanosine	35.6852		C21­H27NO4	Isoquinolines	Central Nervous System Agents neuromuscular relaxant65
60.	Egomaketone	37.7898		C10H12O2	Monoterpenoid	Antioxidant Anti-inflammatory Antibacterial Antiallergic66
61.	Eicosane, 10-methyl	20.7849		C21H44	Volatile hydrocarbon	Antimicrobial Antioxidant67
62.	Flufenamic acid	35.1844		C14H10F3NO2	Organofluorine	Analgesic Anti-inflammatory Antipyretic 68
63.	Furfural	4.1476		C5H4O2	heteroaromatic aldehyde	Anti- tyrosinase Antimicrobial69
64.	gamma atlantone	22.3299		C15H22O	Sesquiterpenoids	Antioxidant70, anti-inflammatory71
65.	I-Leucine, N-(2-chloroethoxycarbonyl)-N-methyl-, tetradecyl ester	32.1576		C24H46ClNO4	Ester	Anti-inflammatory72
66.	Imazapyr, N,O-dimethyl	32.0524		C15H19N3O3	Amine ester	Herbicide73
67.	Isoxazolidine	16.6046		C3H7NO	Saturated organic compound	Anti-inflammatory Anticancer Antiviral Antibacterial74
68.	N-(4-methoxyphenyl)cyclobutanecarboxamide	27.1719		C12H15NO2	Phenols	Unspecified
69.	N-Acetyl-2,5-dimethoxy-4-ethylamphetamine	34.4682		C15H23NO3	Alkaloids	Hallucinogenic75
70.	N-Cinnamylideneaniline	33.6032		C15H13N	Cyclopentanes	Antibacterial76
71.	Nonane, 2,2,4,4,6,8,8-heptamethyl	23.9368		C16H34	Acyclic hydrocarbons	Anticancer77
72.	Norflurane	18.2201		C2H2F4	Fluorinated Organic Compound	Unspecified
73.	Pentandioic acid, (p-t-butylphenyl)	18.4623		C15H20O4	linear dicarboxylic acid	Antimicrobial78
74.	Phthalic acid, 2-ethylcyclohexyl heptyl ester	32.9246		C23H34O4	Aromatic carboxylic acid	anti-trypanosomal, anti-inflammatoryand antimicrobial agents79
75.	Pimelic acid, di(2-nitro-5-fluorophenyl) ester	32.1270		C19H16F2N2O8	Aromatic Organofluorine	Unspecified
76.	Semioxamazide	8.6178		C2H5N3O2	Glyoxylates	Antimicrobial80
77.	s-Triazole, 3-propyl	22.2861		C5H9N3­	heterocyclic organic compound	Anti-inflammatory Anti-proliferative81
78.	Tetraphenylporphyrin	25.6537		C44H30N4	porphyrins	Anti-inflammatory antinociceptive82
79.	Thiophene, 3-methyl-5-octadecyl-2-pentadecyl	19.0156		C38H72S	organosulfur heterocyclic compound	Unspecified
80.	Thymol	18.1194		C10H14O	Monoterpenes	antiseptic, antibacterial antifungal Anti-inflammatory 83
81.	trans-4'-Ethyl-4-(methylthio)chalcone	35.4057		C18H18OS	unsaturated ketone	Anti- inflammatory,analgesic, antioxidant84
82.	Undecane, 3,5-dimethyl	8.9449		C13H28	Fatty Acyls	Anti-inflammatory Anti-allergic59
83.	Yttrium, tris(1,3-diphenyl-1,3-propanedionato	6.8518		C45H36O6Y	Yttrium oxide	Antioxidant Anti-inflammatory85
84.	α-Farnesene	19.7427		C15H24	Sesquiterpene	antioxidant, antimicrobial, and antifungal86
85.	1,2,4,5-Tetramethyl-6-methylenespiro[2.4]heptan	20.1142		C12H20	Undetermined	Unspecified
86.	Hexacosane, 1,26-bis(4'-benzoylphenyl)	26.7183		C52H70O2	Aliphatic saturated hydrocarbons	Unspecified
RT= Retention time

In-silico Analysis:

Docking: The compounds were subjected to molecular docking with the biomarker IL6 in AutoDock Vina via the command prompt method. The results show that the binding energies of the compounds ranged from -1.8 to -9.3. The compounds with the highest binding energy were Bis [(2.3) paracyclophano] [1,2-b:1’,2’-i] anthraquinone, Tetraphenyl porphyrin, 2-Morpholin – 4 – yl - 1, 5 – diphenyl -7-p-tolylimino-5-trifluoromethyl-1, 5, 6, 7-tetrahydro-[1,2,4] triazolo[1,5-c]pyrimidine-8-carbonitrile which had affinities of -9.3, -7.9, and -7.0 respectively. The DMAR, hydroxychloroquine sulfate (HCQs) had a -5.3 affinity for IL6. The further binding energies of the other compounds and their ligand efficiencies and inhibition constants are summarized in Table 2. A study in 2014 targeted IL-6, as it has a profound role in treating RA, and performed docking with 5 compounds, out of which margaric acid exhibited good affinity ⁸⁷. Another study conducted in silico studies on the ability of the database-derived phytochemicals of Murraya koenigii to treat TNF-α of RA, revealed two compounds with high binding scores ⁸⁸. Maste and colleagues screened P. ambionicus against COVID-19 and showed two compounds in combination, i.e., plectranthol A and B had high viral anti-inflammatory activity ⁸⁸. (See supplementary data for the docking profiles of the remaining 76 compounds).

TABLE 2: LIGAND BINDING EFFICIENCY OF GC-MS PROFILED COMPOUNDS WITH INTERLEUKIN-6 DERIVED FROM AUTODOCK

Receptor-ligand Interaction: BIOVIA Discovery Studio helps to visualize the position of compounds interacting with the targeted protein. It also provides a 2D diagram of interactions at the amino acid level. Van der Waal formation was the primary bond found in the docking results. The amino acids found in most of the interactions were ARG, TRP, VAL, PHE, TYR, MET, GLN, ASN, and LYS. The significant bonds that formed were Pi-alkyl and conventional hydrogen bonds. (See supplementary data for the rest of the figures). A study was conducted on flavonoid compounds from Glycyrrhiza glabra as anti-inflammatory drugs against the inflammatory marker interleukin 1 in periodontitis using Autodock tools and the BIOVIA Discovery Studio Visualizer ⁹⁰.

FIG. 3: A: BIS[(2.3)PARACYCLOPHANO][1,2-B:1’,2’-I]ANTHRAQUINONE B: TETRAPHENYLPORPHYRIN, C: 2-MORPHOLIN-4-YL-1, 5-DIPHENYL-7-P-TOLYLIMINO-5-TRIFLUOROMETHYL - 1, 5, 6, 7 - TETRAHYDRO-[1,2,4]TRIAZOLO[1,5-C]PYRIMIDINE-8-CARBONITRILE, D: 1,2,3-TRIPHENYL-3-METHYL-CYCLOPROPENE, E: FLUFENAMIC ACID, F: DIPHENHYDRAMINE, G: CYCLOTETRACOSENE, H: 3',4'-DESOXY-3,4-DIHYDROPAPAVEROLINE, I: CHROMENO(4,3-C)CHROMENE-5,11-DIONE, J: 2-(2,4-DIHYDROXYPHENYL)-5-(E)-PROPENYLBENZOFURAN

ADMET Profile: ADMET was predicted using the ADMETSAR online tool, which is freely available. An HIA below 0.3 indicates low or no absorption, but the values of all the compounds are high; hence, the compounds have high intestinal adsorption. Compounds with a value greater than 0.1 are positive for the BBB. All the other compounds had a positive blood-brain barrier except for6-demethoxytangeretin, chromeno (4,3-c) chromene-5,11-dione, and flufenamic acid. Cytochrome P plays a role in drug metabolism. Table 3 (the 10 compounds with the highest binding efficiency remaining available in supplementary data) shows whether the compounds are substrates or inhibitors. A positive value indicates a substrate and inhibitor, while a negative value indicates a non-substrate and non-inhibitor. Positive/negative indicates substrate/non-inhibitor, whereas negative/positive indicates non-substrate/ inhibitor. The time ½ (ADMETLab) indicates the rate of elimination or excretion; all of the compounds have values less than 3hours and hence have a low elimination rate. PPB signifies that compounds with high protein-bound efficiency have a low therapeutic index; only 10 among the other compounds have low plasma-protein binding values. AMES predicts the mutagen city of the compound; 20 compounds are predicted to be mutagenic, and 33 are predicted to be carcinogenics. Acute toxicity prediction for the compounds was done using LD₅₀ on Gusar- online way 2 drugs tool and subcutaneous administration of the compounds to rats was chosen because the hydrogel will be applied onto the skin. A recent study of the anti-inflammatory activity of lavender (Lavandula officinalis) essential oil bioactive compounds against COX-1 and COX-2, inflammatory markers, used the ADMETSAR tool to predict the drug-likeness and risk assessment of the compounds with high affinities for these compounds.⁹¹Another study used ADMETLab 2.0 for further analysis to evaluate potential candidates against SARS-CoV-2. This study designed 1389 protease inhibitors among which one compound was selected to be most favourable ⁹². A study used GUSAR software to predict the toxicity of xenobiotic metabolites using quantitative structure–activity relationship (QSAR) models created for acute rat toxicity after the intravenous administration ⁹³.

TABLE 3: ADMET PROFILE OF THE COMPOUNDS FROM PLECTRANTHUS AMBOINICUS EXTRACT

Druglikness: Drug-likeness refers to qualitative concepts such as the structure and physio-chemical properties of a compound and is used to assess its bioavailability as a drug. The TPSA represents the quality of the transport of compounds into the cell membrane; molecules with high TPSA are transported more easily than are those with low TPSA. Pimelic acid, di (2-nitro-5-fluorophenyl) ester, 3,4-Bis (methoxycarbonyl) benzoic acid, Semioxamazide, 3-(4-nitrophyenyl-4,5-dihyro-1.2.4-oxadiazol-5-onehad the highest TPSA value compared to the others. C Log P represents the hydrophilicity of the compound; high logP values indicate low hydrophillicity and poor absorption and permeation. 5-Aminotetrazole and semioxamazide are the compounds that were predicted to have low logP values. Solubility is an important parameter for the absorption of compounds and their subsequent distribution in the body, and is measured using solubility logS. The lower the value of logS is, the greater the solubility. Among the 86 compounds, hexacosane, diphenhydramine highly soluble compounds and rest are moderately soluble. The molecular weight is directly related to the absorption rate; the lower the molecular weight is, the greater the absorption rate. The average molecular weight of the drug is between 160 and 480 g/mol and only seven compounds have molecular weights above the average. Lipinski’s rule states that a compounds is drug-like if it has no more than one violation ⁹⁴, and in Table 4 (the 10 highest binding efficiency compounds remaining available in supplementary data), yes indicates that it passes the rule, whereas no means it does not, and the number indicates how many rules are not passed out of five; hence all the other six compounds pass this rule. A study conducted using the SwissADME tool for pharmacological and pharmacognostical profiling of Butea momosperma Lam. Taub ⁹⁵.

TABLE 4: DRUGLIKENESS PREDICTION OF THE COMPOUNDS FROM PLECTRANTHUS AMBOINICUS EXTRACT

Bioactivity Score: The bioactivity score was calculated using the free tool from Molinspiration. Table 5 (other 76 compounds; supplementary data) lists of the compounds with bioactivity scores for GPCRs, ion channel modulators, kinase inhibitors, nuclear receptor inhibitors, protease inhibitors, and enzyme inhibitors; yellow indicates a moderate score, whereas green indicates a high score. The compounds that had good bioactivity scores were Carbamic acid, N-(3-oxo-4-isoxazolidinyl)-, benzyl ester, β-Bergamotene, gamma atlantone, Carbonic acid, octadecyl vinyl ester, Amphetamine, N-pentafluoropropionyl, 9-Oxo-10(9H)-acridineacetic acid, 3-(3,5-di-tert-butylphenyl) pentanedioic acid. A study conducted in Cassia auriculata reported bioactivity scores for phytochemicals predicted via Molinspiration software, which resulted in high scores for to copherol compounds across all parameters ⁹⁶. In another study in which Molinspiration software was used to predict bioactivity scores of terpenoids and fatty acid esters extracted from the Tiliaceae shrub Triumfetta pentandra ⁹⁷.

TABLE 5: BIOACTIVITY SCORE OF THE COMPOUNDS FROM PLECTRANTHUS AMBOINICUS EXTRACT

DISCUSSION: P. amboinicus has potential therapeutic phytocompounds which is supported by published research. In a recent study on the anti-inflammatory potential of a Coleus amboinicus-based gel formulation, a good inhibitory effect was detected via an albumin denaturation assay ⁹⁸. A study conducted in 2019 investigates the anti-inflammation effects of Plectranthus amboinicus extract (PA-F4) on THP-1 monocytic leukemia cells. PA-F4 inhibited ATP-induced caspase-1, IL-1β, and IL-18 release from LPS-primed cells, blocking p65 NF-κB activation and ATP-induced signaling pathways. The constituents rosmarinic acid, cirsimaritin, salvinorin, and carvacrol may explain PA-F4's inhibitory activity ⁹⁹. Another study evaluates the antioxidant in a murine macrophage model, showing that PaE reduces nitric oxide production and inhibits DPPH free radicals and anti-inflammatory using the in-silico method of Indian borage (Plectranthus amboinicus) ethanol extract ¹⁰⁰.

The hallmark inflammatory markers involved in RA are interleukins (IL-1, IL-6, IL-17, and IL-23) ¹¹. These inflammatory markers are also responsible for inflammation and detrimental activities in fibroblast-like synoviocytes (FLSs) among which IL-6 is involved, and other cells regulate fibroblast activated protein-α (FAP-α) in FLSs during RA ¹⁰¹. IL-6 also plays a critical role in B cell differentiation into plasma cells and is a potent growth factor for plasmacytoma and myeloma ¹⁰², and it induces excess production of VEGF, leading to enhanced angiogenesis and increased vascular permeability ¹⁰³. Another critical role of IL-6is to cause bone resorption by inducing osteoclast formation via the induction of RANKL in synovial cells, and cartilage degeneration ¹⁰⁴. Hence IL-6 was targeted for this study to evaluate anti-inflammatory effects of phytocompounds from P. amboinicus extract on IL-6 via an in-silico method. In this investigation, the constituents of P. amboinicus methanolic extract were profiled using GC-MS analysis and the anti-inflammatory effects of the compounds were analyzed via molecular docking with the IL-6 of RA. The analysis revealed that the extract had significant anti-inflammatory effects on IL-6 and had a greater binding energy than the prescription medication for RA. Thus these compounds are potential agents for treating RA.

CONCLUSION: We subjected methanolic leaf extracts of P. amboinicus to microwave-assisted extraction and used analyzed phytochemical compounds via GC-MS, revealing many bioactive compounds belonging to terpenoids, alkaloids, and phenols classification. Furthermore, the 87 chemical compounds identified were analyzed via the in-silico method.

Molecular docking revealed that three compounds namely Bis[(2.3) paracyclophano] [1,2-b:1’,2’-i] anthraquinone, Tetraphenylporphyrin, 2 – Morpholin – 4 -yl-1,5-diphenyl-7-p-tolylimino-5-trifluoromethyl-1, 5, 6,7-tetrahydro-[1,2,4] triazolo [1,5-c] pyrimidine-8-carbonitrile, had high binding affinities, with affinities of -9.3, -7.9, and -7.0, respectively. Hydroxychloroquine sulphate, a common DMAR, was also docked for comparative purposes with IL6, resulting in a lower binding score (-5.3) than that of the above mentioned compounds. The ADMET profile and drug-likeness scores showed  that in addition to Bis[(2.3) paracyclophano] [1,2-b:1’,2’-i] anthraquinone, Hexacosane, 1,26-bis(4'-benzoylphenyl), Tetraphenylporphyrin, Thiophene, 3-methyl-5-octadecyl-2-pentadecyl, α-Farnesene, all the other compounds had adequate drug-like properties. Finally, it can be concluded that phytochemicals from P. amboinicus have potent anti-inflammatory effects on rheumatoid arthritis.

ACKNOWLEGGEMENTS: The authors are thankful to the management of the PSG College of Arts & Science, Coimbatore, India for providing the required infrastructure support.

Author Contribution: The experiments were designed out by SMK and MS. The in-silico analysis was performed by SMK. The article was framed by MS and SMK.

Funding: The author(s) received no financial support for the research.

CONFLICTS OF INTEREST: The authors express no conflicts of interest.

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     How to cite this article:

     Muthukrishnan S and Muthuswamy S: Discovery of interleukin-6 inhibitory properties of the compounds detected from methanolic leaf extracts of Plectranthus amboinicus (lour) spreng. Through molecular docking. Int J Pharm Sci & Res 2024; 15(8): 2463-83. doi: 10.13040/IJPSR.0975-8232.15(8).2463-83.

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