Anti inflammatory activity of Thymeolerosin – Raw 264 Macrophage cell linesType of manuscript:Original ResearchRunning Title:Anti Inflammatory Activity Of ThymeolerosinC.Shunmugam Kumar Mangal Undergraduate Student Saveetha Dental College, Saveetha University,ChennaiAnitha Roy Department HODDepartment of Pharmacology Saveetha Dental College, Saveetha University,Chennai Lakshmi ThangaveluAssistant Professor Department of PharmacologySaveetha Dental College, Saveetha University, Chennai Abstract:Aim:To estimate nitric oxide and nitric oxide synthase gene expression in macrophage cell lines in thyme oleoresinsObjective : To determine the nitric oxide and nitric oxide synthase gene expression in macrophage cell lines in thyme oleoresins Background: Thyme is an aromatic perennial evergreen herb with culinary, medicinal, and ornamental uses. The most common variety is Thymus vulgaris. Thyme is of the genus Thymus of the mint family . Oil of thyme, the essential oil of common thyme Thymol, an antiseptic, is an active ingredient in various commercially produced mouthwashes such as Listerine. Before the advent of modern antibiotics, oil of thyme was used to medicate bandages. It has also been shown to be effective against various fungi that commonly infect toenails. Thymol can also be found as the active ingredient in some all-natural, alcohol-free hand sanitizers.Materials and MethodsChemicalsLipopolysaccharide (LPS), Phenol free Dulbecco’s modified Eagle medium (DMEM), MTT, Dimethyl sulphoxide(DMSO), phosphate buffer saline (PBS), and antibiotic-antimycotic solutionCell cultureMacrophage RAW 264.7 cells were obtained fromthe NCCS, Pune with Passage no 16. Cells werecultured in phenol red-free Dulbecco’s modified Eaglemedium (DMEM) supplemented with 100units/ml penicillin, 100µg/ml streptomycin, and 10%heat-inactivated fetal bovine serum at 37 °C with5% CO2.Results:The effects of different concentrations of Thymeolerosin was assessed and the nitrous oxide levels was estimated. It was observed that with increased concentration of the ethanolic extract of Thymeolerosin the nitrous oxide Level was lowered and thus it is proven that Thymeolerosin possess anti inflammatory properties. Conclusion:Thymeolerosin extract possess anti-inflammatory activity showed by significantly decrease in production of pro-inflammatory mediator NO and also by the suppression by the iNOS gene expression even when enhanced with LPS.Key words: Anti inflammatory, Anti inflammation, Thymeolerosin , Anti Arthritic, Nitrous oxide Introduction: Nature has provided a complete store-house of remedies to cure all aliments of mankind 1.This is where, nature provides us drugs in the form of herbs, plants and algae’s to cure the incurable diseases without any toxic effect2. Research on medicinal plants is an important fact of biochemical research in India because of several reasons. Inflammation is a disorder involving localized increases in the number of leukocytes and a variety of complex mediator molecules 3. Prostaglandins are ubiquitous substances that indicate and modulate cell and tissue responses involved in inflammation.5 Their biosynthesis has also been implicated in the pathophysiology of cardiovascular diseases, cancer, colonic adenomas and Alzheimer’s disease 4. Medicinal plants are believed to be an important source of new chemical substances with potential therapeutic effects.6-8 Macrophages after LPS stimulation produce NO by up-regulating iNOS expression through mitogen-activated protein kinases (MAPK) and NF-?B signaling pathways. In response to macrophage activation, LPS stimulates a Toll-like receptor 4 (TLR4)-mediated myeloid differentiation factor (MyD88)-dependent pathway, which in turn activates the transforming growth factor-?-activated protein kinase 1 (TAK1), which subsequently results in activation of nuclear factor-?B (NF-?B) and activating protein-1 (AP-1), and produces inflammatory cytokines including TNF-?, IL-6, and IL-1? .9-10 Therefore, inhibition of these inflammatory mediators has been considered as an effective strategy for the development of anti-inflammatory drugs. The aim of the study is to evaluate the anti inflammatory activity of Thymeolerosin on Raw 264 Macrophage cell linesMaterials and MethodsChemicalsLipopolysaccharide (LPS), Phenol free Dulbecco’s modified Eagle medium (DMEM), MTT, Dimethyl sulphoxide(DMSO), phosphate buffer saline (PBS), and antibiotic-antimycotic solution (100U penicillin, 100µg streptomycin, and 0.25µg amphotericin B per ml) were purchasedfrom Sigma-Aldrich. Fetal bovineserum was purchased from GIBCO/BRL Invitrogen.Cell cultureMacrophage RAW 264.7 cells were obtained fromthe NCCS, Pune with Passage no 16. Cells werecultured in phenol red-free Dulbecco’s modified Eaglemedium (DMEM) supplemented with 100units/ml penicillin, 100µg/ml streptomycin, and 10%heat-inactivated fetal bovine serum at 37 °C with5% CO2. Cells were washed with DMEM mediumand detached with 0.25% trypsin-EDTA. The cells were seeded at a density of 5 x 105cells/well in 24 well plate and incubated for 18h at 37oCand 5% CO2. Then media of each well were aspirated andfresh FBS-free DMEM media were replaced. Different concentrationsofThymeextract (3.175–150µg/mL) were prepared in FBS-free DMEM to give a total volume of 500µlin each well of a microtiter plate. The cells were co – incubated with 1µg/ml of LPS for 24h. Estimation Nitric oxide (NO)The presence of nitrite, a stable oxidized product ofnitric oxide (NO), was determined in cell culturemedia using Griess reagent. Briefly, after treatment protocol, 50µl ofsupernatant from the test culture was mixed with50 µl of 1% (w/v) sulphanilic acid in 5% (v/v)phosphoric acid in a 96-well plate, followed by incubationfor 10 min at room temperature. After that50 µl 0.1% (w/v) N-1-naphthylethylenediamineHCl in distilled water was added and incubatedfor 10 min at room temperature. The optical densityat 540 nm was measured with a microplatereader. The NO concentration was calculated bycomparison with a NaNO2 (0–100 µM) standardcurve. The final concentration of DMSO was adjustedto less than 0.1% for all treatments. The results were expressed asinhibition of NO production compared to the control(LPS) using: (nitritec – nitritet)/nitritec,where nitritec and nitritet are the nitrite concentrationin the control and test sample, respectively.RNA Isolation and q – PCR AnalysisRAW macrophages were treated with 12.5µg/ml, 25µg/ml and 50µg/ml of Thyme extract with 1µg/ml of LPS and incubated for 24h.Total RNA was isolated using TRIzol reagent (Invitrogen) according to themanufacturer’s protocol, and 2?g of RNA was used for complementary DNA synthesis using M-MLV reverse transcriptase (Promega, Madison, WI, USA). Quantitative real-time polymerase chain reaction (q-PCR) wasperformed in an ABI 7500 Real-Time System with SYBR Green PCR Master Mix (Takara). Reactionswere initiated with an initial incubation at 50?C for two minutes and 94?C for 10 min, followed by40 cycles of 94?C for 5s, 60?C for 15s, and 72?C for 10 s. The relative gene expression levels werecalculated using the 2???Ct method. The specific primer sequences used were given below:INOS:Forward:5?-ATGTCCGAAGCAAACATCAC-3? Reverse:5?-TAATGTCCAGGAAGTAGGTG-3??-actin wasused as an internal reference gene between different samples.Statistical analysisData obtained from the experiments were expressed as Mean ± SEM. The Statistical analysis of the difference between the groups was evaluated by Dunnett’s following one way ANOVA Posthoc comparisons in Graph pad Prism 5.0 software version. p<0.001, p<0.01 and p<0.05 were considered to be statistically significant.ResultsEffect of Thyme extract on NO productionThe efficacy of Thyme extract on nitric oxide production in RAW macrophages was determined. Nitrite production wasdependent on the activating state of the cells. LPS unstimulated macrophages (Control) for 24h produced lowest levels of NO, whereas LPS stimulated group showed 89.61% of NO. Thyme extract at its tested concentrations exhibited dose – dependent decrease in the production of NO. Effect of Thyme extract on NO production in LPS stimulated RAW 264.7 macrophages S.No Concentration(µg/ml) % of NO production in LPS stimulatedRAW 264.7macrophages1 LPS (1µg/ml) 89.61±0.472 3.175 86.13±0.473 6.25 70.98±0.504 12.5 63.31±0.455 25 53.52±0.836 50 16.35±0.597 75 12.63±0.498 150 3.84±0.82Values are expressed Mean±SEM (n = 3)Graphical representation of effect of Thyme extract on production of NO production in LPS stimulated RAW 264.7 macrophages Gene Expression of iNOSTo determine whether Thyme extract inhibits NO productionat the level of transcription, we used RT - PCR to examinethe expression of the iNOS gene in activated macrophages. LPS stimulated RAW macrophages strongly upregulated the iNOS gene expression levels. In the presence of Thyme extract at three different doses of 12.5µg/ml, 25µg/ml and 50µg/ml, the iNOS levels was significantly suppressed, compared to that of LPS treatment only. Effect of Thyme extracton LPS – stimulated iNOS expression in RAW 264.7 macrophages Discussion: Several studies have demonstrated the properties of various compounds from plants tend to possess rich pharmacological properties that play beneficial roles in many different conditions, including inflammation-related diseases (11-13). Inflammation is a dynamic process involving proinflammatory cytokines such as nitrous oxide and it acts as important biological response toward injury (14-15). In the present study we have examined the anti inflammatory activity of Thymeolerosin on Raw 264 macrophage cell lines enhanced with LPS. NO is a free radical produced from l-arginine by nitric oxide synthases (NOSs), and an important cellular second messenger (16). The modulation of iNOS-mediated NO release is one of the major contributing factors during the inflammatory process (17). Thus suppression of the response of NO would In turn indicate the anti inflammatory activity of the particular extract.NO has the property to modify or generate intercellular signals and thus it has an effect on Immune cells, tumour cells and the cells of different tissues or organs. From the above results of the present study it is evident that Thymeolerosin has an anti inflammatory activity from table 1. On increasing the concentration of the extract the nitrous oxide level continues to decrease and thereby inhibiting the effect of iNOS mediated inflammation.Thus the anti inflammatory effect of Thymeolerosin has been confirmed. The limitations of the study would include that only the NO pro-inflammatory messenger system has been analysed in the current study. Other secondary messengers (18-19) in case of inflammatory reactions can be analysed with the same extract and a conclusive final analysis of the particular source can be obtained. Conclusion: Caralluma Fimbrata extract possess anti-inflammatory activity showed by significantly decrease in production of pro-inflammatory mediator NO and also by the suppression by the iNOS gene expression even when enhanced with LPS.