In Vivo And In Vitro Approach To Study The Anti .

Published by :http://www.ijert.orgInternational Journal of Engineering Research & Technology (IJERT)ISSN: 2278-0181Vol. 8 Issue 11, November-2019chronic inflammation, both the steroidal and nonsteroidalanti-inflammatory drugs (NSAIDS) were used. Extensiveusages of synthetic drugs result in various side effects. Someof the side effects caused by the steroidal drugs includealteration in the metabolism of proteins, carbohydrates, saltand water imbalance, and various enzymatic reactions.Whereas the NSAIDS drugs excessive intake results incardiovascular, gastric and respiratory problems. So the studyfocuses on herbal anti-inflammatory drugs with moreeffective and fewer side effects which will regulate the proinflammatory conditions.Different phytochemicals isolated from the plants exertanti-inflammatory actions through the inhibition of variouspro-inflammatory mediators. Eugenia uniflora is a shrubwhich possesses various medicinal properties. Eugeniauniflora is known as the Brazilian cherry and it belongs to theMyrtaceae family. Eugenia uniflora leaf infusions are usedfor the treatment of stomach and digestive disorders, gout,yellow fever, and hypertension. Eugenia uniflora was foundeffective for reducing blood pressure, weight and it also actsas a diuretic [7]. Essential oils acquired from the leaves ofEugenia uniflora exhibited strong anti-fungal property [8].Preliminary screening studies of Eugenia uniflora showed thepresence of phytochemicals in it. The main aim of the studywas to evaluate the anti-inflammatory activity of EF18(Eugenia uniflora active fraction) on RAW 264.7macrophage cells and in vivo anti-inflammatory study in maleWistar rats by the carrageenan-induced method.chloroform, ethyl acetate, and ethanol. The stationary phaseused was silica gel of mesh size 60-120nm. The fractionsobtained (EF1 to EF18) were exposed to phytochemicalanalysis, antioxidant, and anti-inflammatory activity. Theresults showed that among various fractions of crudeethanolic extract of Eugenia uniflora the EF18 fraction wasfound to be the most active fraction and was subjected tofurther in vitro studies. This EF18 fraction was 100%ethanolic fraction.D. HR-LCMS analysis of EF18II. MATERIALS AND METHODSA. Plant collectionEugenia uniflora leaf samples were collected fromChenganoor (9.31830N, 76.61110E) Alappuzha district,Kerala, India. The plant material was authenticated by ataxonomist and a voucher specimen SBSBRL 25 was kept atSchool of Biosciences, M.G. University, Kottayam, Kerala,India.B. Preparation of crude ethanolic extract of EugeniaunifloraEugenia uniflora leaves collected were washed properlyand then shade dried and powdered and were stored in anairtight container for further analysis. Eugenia uniflorapowder (50g) was extracted (72 hours) in a soxhlet apparatuswith 500ml of various solvents with increasing polarity. Thesolvents used were petroleum ether, chloroform, ethylacetate, ethanol, methanol, and water. The extracts were thenfiltered through whatmann No 1 filter paper and wereconcentrated to dryness using a rotary evaporator and storedat 40C in sterile vials for further studies. The most activecrude ethanolic extract of Eugenia uniflora was obtained bythe phytochemical screening, evaluation of the antioxidantand anti-inflammatory activity. It was collected in largequantities and stored for further analysis.C. Preparation of Eugenia uniflora active fraction(EF18)For the preparation of Eugenia uniflora activefraction (EF18), the crude ethanolic extract of Eugeniauniflora was subjected to column chromatography partialpurification technique using solvents with increasing polarity.The mobile phases used comprises of petroleum ether,The LCMS analysis was performed using aquadrupole time –of –flight (Q-TOF) mass spectrometer (QTOF LC/MS 6550 I Funnel Q-TOFs series G6550A, AgilentTechnologies, USA) equipped with an ESI source. Massspectral data were collected from an Agilent 6200 series TOFand 6500 series Q-TOF LC/MS system B.05.01 (B5125).E. Cell line culture and treatmentsThe RAW macrophage cells (264.7 cells) wereprimarily obtained from the NCCS (National Centre for CellSciences, Pune, India). These cells were then maintained in aspecial medium named Dulbecco’s modified Eagles medium(DMEM) purchased from Sigma Aldrich, USA [9].IJERTV8IS110267HR-LCMS analysis of EF18 was performed using anAligent 1290 infinity series HPLC instrument (Agilenttechnologies USA) which was equipped along with a binarypump (G4220B, USA) and an autosampler (G4226A, USA)connected to a column compartment (G1316C, USA). Theeffective chromatographic separation was done with theAligent Zorbax C-18 column (2.1x50 mm, 18µm). The mobilephase consists of 0.1% formic acid in water which was used asthe solvent A and 90% acetonitrile {CH3CN 10% formicacid(0.01%)} in water as solvent B in a gradient elution mode.The mobile phase gradient was started with solvent A (95%):solvent B (5%) with a flow rate of 0.3ml per minute for about18 minutes. After that the solvent was changed to solvent Awith 0%: solvent B (100%) for 7 minutes. And finally, it wasreturned to solvent A 95%: solvent B (5%). The spectra of theanalysis were obtained by using UHPLC PDA detector in amass range of 120 to 1000(m/z) at the rate of 1spectra/second.The cells were cultured using a 25 cm tissue cultureflask with DMEM medium supplemented with L-glutamine,10%FBS, sodium bicarbonate from Merck, Germany. Anantibiotic solution containing Streptomycin (100µg/ml),Amphotericin B (2.5µg/ml) and Penicillin (100 µg/ml) werealso used for this cell culture. All the cultured cell lines werekept at 37 0C in a humidified 5% carbon dioxide incubator(NBS Eppendorf, Germany). The entire grown-up cells up to60% confluency were shadowed by the enrichment oflipopolysaccharide (LPS: 1µg/ml). These LPS stimulatedRAW 264.7 cells were exposed to various concentrations ofdiclofenac sodium (standard drug) and EF18 atconcentrations 25, 50, 100µg/ml. Diclofenac sodium wasconsidered as the standard anti-inflammatory drug in thisstudy. The LPS stimulated cells were incubated for 24 hours.After the incubation time, all the anti-inflammatory assayswere carried out using the cell lysate.www.ijert.org(This work is licensed under a Creative Commons Attribution 4.0 International License.)600

Published by :http://www.ijert.orgInternational Journal of Engineering Research & Technology (IJERT)ISSN: 2278-0181Vol. 8 Issue 11, November-2019F.MTT assayThe cells cultured in the DMEM medium wereseeded in 96 wells and were then treated with various sampleconcentrations (6.25µg/ml -100µg/ml). Later these cells werethen kept for incubation at 370C for 24 hours. After thetreatments, the medium was replaced and 30µl of MTTsolution was added subsequently to both the control andsample wells. This was then kept for incubation at 37 0C for 4hours.MTT solution was replaced after incubation.Formazan crystals were then dissolved in 100µl Dimethylsulfoxide which acts as a solubilization solution. Using anELISA microplate reader, the extent of reduction of MTT toformazan crystals was quantified by measurement of theabsorbance at 570nm.G. Estimation of Cyclooxygenase (COX) activityThe COX activity was estimated using the methodof walker [10]. Here 100µl cell lysate was incubated withTris-HCl buffer (pH 8), along with glutathione 5mM/L, andhemoglobin 5mM/L for about 1 minute at 25 C. Then thereaction was commenced by the addition of arachidonic acid200mM/L and incubation was carried out at 37 C for 20minutes. To this 200µl of 10% Trichloroacetic acid in 1Nhydrochloric acid was added. After that, the centrifugationwas done and 200µl of 1% thiobarbiturate was added, thetubes were then boiled for 20 minutes. Finally, after cooling,the tubes were again centrifuged for three minutes. COXactivity was evaluated by reading absorbance at 632 nm.H. Estimation of Lipoxygenase enzyme (LOX) activityLOX activity was determined according to theprocedure of Axelrod [11]. The reaction mixture was madeup to 2 ml final volume that contained Tris-HCl buffer (pH7.4), and sodium linoleate (200 µl) and 50µl of cell lysate.The absorbance was read at 234nm and this reflects the 5hydroxyeicosatetraenoic acid formation.I. Estimation of MyeloperoxidaseMyeloperoxidase activity was determined by themethod of Bradley [12]. Myeloperoxidase estimationinvolves a cell lysate that was homogenized in a solution methyl ammonium bromide (HTAB). Thesesamples were then centrifuged at 2000rpm for 30 minutes at4 C. After the centrifugation supernatant was taken and thenanalyzed for its Myeloperoxidase activity. In the sample,Myeloperoxidase was activated by the addition of 50 mMphosphate buffer of pH 6 (1.67mg/ml guaiacol and 0.0005%H2O2). Absorbance was monitored at 460 nm.Myeloperoxidase activity can be represented as units per mlof cell lysate. One unit of myeloperoxidase activity wasequivalent to 1µM of peroxide degradation in one minute at25 C.J. Estimation of nitric oxideNitric oxide synthase was evaluated by the methodof Salter [13]. Here the cell lysate was homogenized in 2mlof HEPES buffer. The assay system contained substrate 0.1mlL-Arginine, 0.1ml manganese chloride, 0.1ml 30µgdithiothreitol (DTT), 0.1ml NADPH, 0.1ml tetrahydropterin,0.1 ml oxygenated haemoglobin and 0.1ml enzyme (sample).An increase in absorbance was recorded at 401nm.IJERTV8IS110267K. Estimation of cellular nitrateThe cellular nitrite level was assessed by the methodof Lepoivre [14]. Sodium nitrite solution was used as thestandard for this reaction. In this procedure sulphosalicylicacid (0.1 ml) was added to cell lysate (0.5 ml) and wasvortexed for 30 minutes. After that, the samples werecentrifuged for 15 minutes at 5000 rpm. The supernatant(protein-free) was used for the evaluation of nitrite levels.30μl of 10% NaOH along with 300μl of Tris-HCl buffer wasadded to supernatant (200μl). All these were mixed well and530μl of Griess reagent was added to this mixture. This wasthen kept for incubation in dark for 10-15 minutes.Absorbance was measured at 540nm against the blank (Griessreagent). From the standard curve obtained the nitrite levelwas estimated.L. Gene expression studiesGene expression studies were carried out using rawmacrophage cells (264.7 cells). The expression of NF-kB andCOX-2 gene was evaluated. The positive control used wasGAPDH. The primers used for the study are depicted inTable 1. RAW 264.7 cells were placed in a 6-well plate. Afterattaining 70% confluency of cells in 6 well plates(approximately 4 x 105 cells), the cells were then treated withEF18 of concentration 100µg/ml and was incubated for 24hours. A set of untreated control cells was also incubated at370C for 24 hours in a CO2 incubator. After the incubationperiod, DMEM media was removed aseptically and 200µl ofTRIZOL reagent was added to culture well plate andincubated for 5 minutes [15]. These were then moved to afresh and sterile eppendorf. To this 200µl of chloroform wasadded and shaken for 15 seconds. After that it was incubatedat room temperature for 2-3 minutes. Then centrifugation wascarried out for 15 minutes at 4 0C at 14000rpm.The topaqueous layer was collected and 500µl of 100% isopropanolwas added to that. This was then incubated at roomtemperature for 10 minutes and after that centrifuged at 4 0 Cfor 15 minutes at 14000rpm.After centrifugation thesupernatant was discarded and pellet was taken. Pellet takenwas washed using 200µl of 75% ethanol and centrifugedagain for 5 minutes at 14000rpm at 40C in a coolingcentrifuge. The RNA pellet was then dried and suspended ina TE buffer. The cDNA synthesis was carried out by usingThermo scientific verso cDNA synthesis kit with the methodfollowed by Joshi [16]. To the RNAse free tube 4µl of 5 XcDNA synthesis buffers, dNTP mix (2µl), anchored oligo dT(1µl), RT enhancer (1µl), Verso enzyme mix(1µl) and 5μl ofRNA template (1nano gram of total RNA) were added. Bythe addition of distilled water which was sterile, total reactionvolume was made up to 20μl. The solution was then mixedproperly by using the technique of aspiration. The thermalcycler was programmed to undergo cDNA synthesis. ForcDNA synthesis at one cycle, the temperature was maintainedat 420C, for 30 minutes. The temperature of inactivation forone cycle was maintained for 2 minutes at 95 0C. Theamplification was carried out using the Thermo scientificamplification kit. Primary denaturation for 3 minutes at 95 0Cwas followed by the denaturation for the 30s at 950C,annealing at temperature for about 30s and extension at 72 0Cfor 1 minute which was continued for 35 cycles and the finalextension was at 720C for 5 minutes. After the amplification,www.ijert.org(This work is licensed under a Creative Commons Attribution 4.0 International License.)601

Published by :http://www.ijert.orgInternational Journal of Engineering Research & Technology (IJERT)ISSN: 2278-0181Vol. 8 Issue 11, November-2019the PCR product was detached by Agarose gelelectrophoresis. The stained gel was visualized using a geldocumentation system of E gel imager, Invitrogen.TABLE I. Primers used in Agarose Gel ElectrophoresisOligoPrimers used in Agarose Gel ElectrophoresisnameFORWARDREVERSESEQUENCE (5’ 3’)TmSEQUENCE (3’ TTGGCAGGTT(20)55.3COX F- TCTC54.8III. RESULTSA.HR-LCMS analysis of EF18HR-LCMS analysis revealed the presence of variousbioactive compounds in the EF18. Out of the differentbioactive compounds obtained Gallic acid andDihydromyricetin exhibited anti-inflammatory activity.Preliminary phytochemical screening studies of EF18fraction revealed the presence of various secondarymetabolites like alkaloids, flavonoids, tannins, saponins,and terpenoids in it. Phytochemicals exhibited the abilityto imp

In Vivo and in Vitro Approach to Study the Anti-Inflammatory Efficacy of Eugenia Uniflora L S. Syama 1, Meenu Thampi 2, Dr. M S Latha 3, * 1, 2, 3 School of Biosciences, Mahatma Gandhi University, Priyadarshini Hills, Kottayam, Kerala, India. *Corresponding author: School of Biosciences, Mahatma Gandhi University, Priyadarshini Hills, Kottayam, Kerala,India.