Investigations on Leucas cephalotes (Roth.) Spreng. for inhibition of LPS-induced pro-inflammatory mediators in murine macrophages and in rat model
Identifieur interne : 000047 ( Pmc/Corpus ); précédent : 000046; suivant : 000048Investigations on Leucas cephalotes (Roth.) Spreng. for inhibition of LPS-induced pro-inflammatory mediators in murine macrophages and in rat model
Auteurs : Neeraj K. Patel ; Mohd. Shahid Khan ; Kamlesh K. BhutaniSource :
- EXCLI Journal [ 1611-2156 ] ; 2015.
Abstract
Silica gel column chromatography fractionation of the dichloromethane extract (LCD) of
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DOI: 10.17179/excli2014-667
PubMed: 26535039
PubMed Central: 4616245
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Investigations on Leucas cephalotes (Roth.) Spreng. for inhibition of LPS-induced pro-inflammatory mediators in murine macrophages and in rat model</title><author><name sortKey="Patel, Neeraj K" sort="Patel, Neeraj K" uniqKey="Patel N" first="Neeraj K." last="Patel">Neeraj K. Patel</name><affiliation><nlm:aff id="A1">Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S Nagar, Mohali-160062, Punjab (INDIA)</nlm:aff></affiliation></author><author><name sortKey="Khan, Mohd Shahid" sort="Khan, Mohd Shahid" uniqKey="Khan M" first="Mohd. Shahid" last="Khan">Mohd. Shahid Khan</name><affiliation><nlm:aff id="A1">Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S Nagar, Mohali-160062, Punjab (INDIA)</nlm:aff></affiliation></author><author><name sortKey="Bhutani, Kamlesh K" sort="Bhutani, Kamlesh K" uniqKey="Bhutani K" first="Kamlesh K." last="Bhutani">Kamlesh K. Bhutani</name><affiliation><nlm:aff id="A1">Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S Nagar, Mohali-160062, Punjab (INDIA)</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26535039</idno><idno type="pmc">4616245</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4616245</idno><idno type="RBID">PMC:4616245</idno><idno type="doi">10.17179/excli2014-667</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000047</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Investigations on Leucas cephalotes (Roth.) Spreng. for inhibition of LPS-induced pro-inflammatory mediators in murine macrophages and in rat model</title><author><name sortKey="Patel, Neeraj K" sort="Patel, Neeraj K" uniqKey="Patel N" first="Neeraj K." last="Patel">Neeraj K. Patel</name><affiliation><nlm:aff id="A1">Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S Nagar, Mohali-160062, Punjab (INDIA)</nlm:aff></affiliation></author><author><name sortKey="Khan, Mohd Shahid" sort="Khan, Mohd Shahid" uniqKey="Khan M" first="Mohd. Shahid" last="Khan">Mohd. Shahid Khan</name><affiliation><nlm:aff id="A1">Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S Nagar, Mohali-160062, Punjab (INDIA)</nlm:aff></affiliation></author><author><name sortKey="Bhutani, Kamlesh K" sort="Bhutani, Kamlesh K" uniqKey="Bhutani K" first="Kamlesh K." last="Bhutani">Kamlesh K. Bhutani</name><affiliation><nlm:aff id="A1">Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S Nagar, Mohali-160062, Punjab (INDIA)</nlm:aff></affiliation></author></analytic><series><title level="j">EXCLI Journal</title><idno type="eISSN">1611-2156</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Silica gel column chromatography fractionation of the dichloromethane extract (LCD) of <italic>Leucas cephalotes </italic>(Roth.) Spreng. led to the isolation of five compounds namely β-sitosterol (<bold>1</bold>) + stigmasterol (<bold>2</bold>), lupeol (<bold>3</bold>), oleanolic acid (<bold>4</bold>) and laballenic acid (<bold>5</bold>). Also, gas chromatography-mass spectrometry (GC-MS) analysis of sub-fraction (LCD-F1) of this extract showed the presence of eleven (<bold>6</bold>-<bold>16</bold>) compounds. In addition to this, <bold>3</bold>-<bold>5</bold> and LCD-F1 were evaluated for lipopolysachharide (LPS)-induced nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-1β production in RAW 264.7 and J774A.1 cells. Results directed that <bold>4</bold> and <bold>5</bold> were found to inhibit these mediators at half maximal inhibitory concentration of 17.12 to 57.20 μM while IC<sub>50</sub> for LCD-F1 was found to be 15.56 to 31.71 μg/mL. Furthermore, LCD at a dose of 50, 100 and 400 mg/Kg was found to reduce significantly LPS induced tumor necrosis factor (TNF)-α and interleukin (IL)-1β production in female Sprague Dawley (SD) rats. All the results findings evoked that the anti-inflammatory effects of <italic>Leucas cephalotes</italic> is partially mediated through the suppression of pro-inflammatory mediators and hence can be utilized for the development of anti-inflammatory candidates.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Baburao, B" uniqKey="Baburao B">B Baburao</name></author><author><name sortKey="Reddy, Arn" uniqKey="Reddy A">ARN Reddy</name></author><author><name sortKey="Kiran, G" uniqKey="Kiran G">G Kiran</name></author><author><name sortKey="Reddy, Yn" uniqKey="Reddy Y">YN Reddy</name></author><author><name sortKey="Mohan, Gk" uniqKey="Mohan G">GK Mohan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bagby, Mo" uniqKey="Bagby M">MO Bagby</name></author><author><name sortKey="Smith, Cr" uniqKey="Smith C">CR Smith</name></author><author><name sortKey="Wolff, Ia" uniqKey="Wolff I">IA Wolff</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bavarva, Jh" uniqKey="Bavarva J">JH Bavarva</name></author><author><name sortKey="Narasimhacharya, Avrl" uniqKey="Narasimhacharya A">AVRL Narasimhacharya</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bhandari, P" uniqKey="Bhandari P">P Bhandari</name></author><author><name sortKey="Patel, Nk" uniqKey="Patel N">NK Patel</name></author><author><name sortKey="Bhutani, Kk" uniqKey="Bhutani K">KK Bhutani</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bhandari, P" uniqKey="Bhandari P">P Bhandari</name></author><author><name sortKey="Patel, Nk" uniqKey="Patel N">NK Patel</name></author><author><name sortKey="Gangwal, P" uniqKey="Gangwal P">P Gangwal</name></author><author><name sortKey="Sangamwar, At" uniqKey="Sangamwar A">AT Sangamwar</name></author><author><name sortKey="Bhutani, Kk" uniqKey="Bhutani K">KK 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sortKey="Prasanna, K" uniqKey="Prasanna K">K Prasanna</name></author><author><name sortKey="Bhutani, Kk" uniqKey="Bhutani K">KK Bhutani</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hamdan, Di" uniqKey="Hamdan D">DI Hamdan</name></author><author><name sortKey="Mohamed, Me" uniqKey="Mohamed M">ME Mohamed</name></author><author><name sortKey="Abdulla, Rh" uniqKey="Abdulla R">RH Abdulla</name></author><author><name sortKey="Mohamed, Sm" uniqKey="Mohamed S">SM Mohamed</name></author><author><name sortKey="El Shazly, Am" uniqKey="El Shazly A">AM El-Shazly</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Held, S" uniqKey="Held S">S Held</name></author><author><name sortKey="Schieberle, P" uniqKey="Schieberle P">P Schieberle</name></author><author><name sortKey="Somoza, V" uniqKey="Somoza V">V Somoza</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Keyzers, Ra" uniqKey="Keyzers R">RA Keyzers</name></author><author><name sortKey="Davies Coleman, Mt" uniqKey="Davies Coleman M">MT Davies-Coleman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kripa, Kg" uniqKey="Kripa K">KG Kripa</name></author><author><name sortKey="Chamundeeswari, D" uniqKey="Chamundeeswari D">D Chamundeeswari</name></author><author><name sortKey="Thanka, J" uniqKey="Thanka J">J Thanka</name></author><author><name sortKey="Uma Maheswara Reddy, C" uniqKey="Uma Maheswara Reddy C">C Uma Maheswara Reddy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Miyaichi, Y" uniqKey="Miyaichi Y">Y Miyaichi</name></author><author><name sortKey="Segawa, A" uniqKey="Segawa A">A Segawa</name></author><author><name sortKey="Tomimori, T" uniqKey="Tomimori T">T Tomimori</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nadkarni, Km" uniqKey="Nadkarni K">KM 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<front><journal-meta><journal-id journal-id-type="nlm-ta">EXCLI J</journal-id><journal-id journal-id-type="iso-abbrev">EXCLI J</journal-id><journal-id journal-id-type="publisher-id">EXCLI J</journal-id><journal-title-group><journal-title>EXCLI Journal</journal-title></journal-title-group><issn pub-type="epub">1611-2156</issn><publisher><publisher-name>Leibniz Research Centre for Working Environment and Human Factors</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26535039</article-id><article-id pub-id-type="pmc">4616245</article-id><article-id pub-id-type="publisher-id">2014-667</article-id><article-id pub-id-type="doi">10.17179/excli2014-667</article-id><article-id pub-id-type="publisher-id">Doc508</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Investigations on Leucas cephalotes (Roth.) Spreng. for inhibition of LPS-induced pro-inflammatory mediators in murine macrophages and in rat model</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Patel</surname><given-names>Neeraj K.</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Khan</surname><given-names>Mohd. Shahid</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Bhutani</surname><given-names>Kamlesh K.</given-names></name><xref ref-type="corresp" rid="COR1">*</xref><xref ref-type="aff" rid="A1">1</xref></contrib></contrib-group><aff id="A1"><label>1</label>Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S Nagar, Mohali-160062, Punjab (INDIA)</aff><author-notes><corresp id="COR1">*To whom correspondence should be addressed: Kamlesh K. Bhutani, Department of Natural Products, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S Nagar, Mohali-160062, Punjab (INDIA), E-mail: <email>kkbhutani@niper.ac.in, kkbhutani@gmail.com</email></corresp></author-notes><pub-date pub-type="epub"><day>10</day><month>4</month><year>2015</year></pub-date><pub-date pub-type="collection"><year>2015</year></pub-date><volume>14</volume><fpage>508</fpage><lpage>516</lpage><history><date date-type="received"><day>11</day><month>11</month><year>2014</year></date><date date-type="accepted"><day>03</day><month>3</month><year>2015</year></date></history><permissions><copyright-statement>Copyright © 2015 Patel et al.</copyright-statement><copyright-year>2015</copyright-year><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p><pmc-comment>CREATIVE COMMONS</pmc-comment>This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>) You are free to copy, distribute and transmit the work, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:type="simple" xlink:href="http://www.excli.de/vol14/Bhutani_10042015_proof.pdf">This article is available from http://www.excli.de/vol14/Bhutani_10042015_proof.pdf</self-uri><abstract><p>Silica gel column chromatography fractionation of the dichloromethane extract (LCD) of <italic>Leucas cephalotes </italic>(Roth.) Spreng. led to the isolation of five compounds namely β-sitosterol (<bold>1</bold>) + stigmasterol (<bold>2</bold>), lupeol (<bold>3</bold>), oleanolic acid (<bold>4</bold>) and laballenic acid (<bold>5</bold>). Also, gas chromatography-mass spectrometry (GC-MS) analysis of sub-fraction (LCD-F1) of this extract showed the presence of eleven (<bold>6</bold>-<bold>16</bold>) compounds. In addition to this, <bold>3</bold>-<bold>5</bold> and LCD-F1 were evaluated for lipopolysachharide (LPS)-induced nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-1β production in RAW 264.7 and J774A.1 cells. Results directed that <bold>4</bold> and <bold>5</bold> were found to inhibit these mediators at half maximal inhibitory concentration of 17.12 to 57.20 μM while IC<sub>50</sub> for LCD-F1 was found to be 15.56 to 31.71 μg/mL. Furthermore, LCD at a dose of 50, 100 and 400 mg/Kg was found to reduce significantly LPS induced tumor necrosis factor (TNF)-α and interleukin (IL)-1β production in female Sprague Dawley (SD) rats. All the results findings evoked that the anti-inflammatory effects of <italic>Leucas cephalotes</italic> is partially mediated through the suppression of pro-inflammatory mediators and hence can be utilized for the development of anti-inflammatory candidates.</p></abstract><kwd-group><kwd>Leucas cephalotes</kwd><kwd>GC-MS</kwd><kwd>anti-inflammatory</kwd><kwd>nitric oxide</kwd><kwd>tumor necrosis factor-alpha</kwd><kwd>interleukin-1beta</kwd></kwd-group></article-meta></front><body><sec sec-type="intro"><title>Introduction</title><p><italic>Leucas cephalotes</italic> (Roth.) Spreng. (Family: Lamiaceae) also known as 'Dronapushpi' in Sanskrit, is an edible rainy season weed. In Ayurveda, it has been recommended for inflammation, psoriasis, scabies, chronic skin eruptions, edema, diaphoresis, chronic malaria, asthma, eye diseases, jaundice, paralysis and obstinate urinary troubles (Dash, 1987[<xref rid="R8" ref-type="bibr">8</xref>]; Bhoria and Kainsa, 2013[<xref rid="R6" ref-type="bibr">6</xref>]; Nadkarni, 2007[<xref rid="R16" ref-type="bibr">16</xref>]; Chandel et al., 1996[<xref rid="R7" ref-type="bibr">7</xref>], Miyaichi et al., 2006[<xref rid="R15" ref-type="bibr">15</xref>]). The plant is reported to have anti-filarial activity, anti-bacterial, anti-diabetic activity and hepato-protective activity (Bhoria and Kainsa, 2013[<xref rid="R6" ref-type="bibr">6</xref>]; Rastogi and Mehrotra, 1991[<xref rid="R21" ref-type="bibr">21</xref>]; Bavarva and Narasimhacharya, 2010[<xref rid="R3" ref-type="bibr">3</xref>]). Leaves of this plant is applied in the form of paste for snake bite treatment and also recommended in case of chronic rheumatism (Samy et al., 2008[<xref rid="R22" ref-type="bibr">22</xref>]; Bhoria and Kainsa, 2013[<xref rid="R6" ref-type="bibr">6</xref>]). Yadav et al., (2012[<xref rid="R23" ref-type="bibr">23</xref>]) reported that non-polar fraction of methanolic extract of <italic>L. cephalotes </italic>significantly reduced the increased levels of TNF-α, IL-1β and IL-6 in mouse ear tissue homogenate. Baburao et al. (2010[<xref rid="R1" ref-type="bibr">1</xref>]) also found that at doses of 200 and 400 mg/Kg body weight <italic>p. o</italic> methanolic extract of this plant demonstrated anti-inflammatory activity in a carrageenan induced rat paw edema model. Considering the anti-inflammatory potential of <italic>Leucas cephalotes</italic> and its related species <italic>viz.</italic><italic>Leucas aspera</italic> (Kripa et al., 2011[<xref rid="R14" ref-type="bibr">14</xref>]; Baburao et al., 2010[<xref rid="R1" ref-type="bibr">1</xref>]), we evaluated previously the dichloromethane (LCD), ethyl acetate (LCE) and methanol extracts (LCM) of this plant for <italic>in vitro</italic> pro-inflammatory cytokines (TNF-α and IL-1β) and nitric oxide (NO) inhibition in RAW 264.7 cells. Results revealed that LCD exhibited significant inhibition of NO, TNF-α and IL-1β production with an IC<sub>50 </sub>of 49.3, 46.8 and 49.8 µg/mL respectively as compared to LCE and LCM (IC<sub>50 </sub>= 50 to > 100 µg/mL) (Patel et al., 2014[<xref rid="R19" ref-type="bibr">19</xref>]). In the light of this, the present study was designed with an aim to isolate and identify compounds from LCD and afterwards evaluate them towards inhibition of pro-inflammatory mediators.</p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><sec><title>Plant material</title><p><italic>Leucas cephalotes </italic>(Roth.) Spreng. (whole plant) was procured from Arya Vastu Bhandar, Dehradun, Uttarakhand, India. A voucher no; NIP-NPM-CD-017 has been deposited at the Department of Natural Products, NIPER, Punjab, India after proper authentication by a botanist. </p></sec><sec><title>Instruments</title><p>Ultraviolet (UV) spectra were measured on a DU<sup>®</sup> 7400 spectrophotometer (Beckman, München, Germany). <sup>1</sup>H and <sup>13</sup>C Nuclear magnetic resonance (NMR) spectra were recorded on Ultrashield 400 MHz and 100 MHz spectrometer respectively (Bruker DPX, Faellanden, Germany). Mass spectra were recorded on a mass spectrometer (Thermo Quest Finnigan, San Jose, CA, USA). For <italic>in vitro</italic> experiments, ultracentrifuge (Sigma, St. Louis, MO, USA), CO<sub>2</sub> incubator (WTC Binder, Tuttlingen, Germany), Biosafety cabinet (Clean air, Chennai, India), autopipettes, ELISA plate reader (Labsystems, Helsinki, Finland) and Neubauer chamber (HBG, Gießen, Germany) were used. Extracts were dried <italic>in vacuo</italic> using a vacuum rotary evaporator (Buchi R-210, Flawil, Switzerland). </p></sec><sec><title>Chemicals and reagents</title><p>Silica gel (230-400 mesh; Merck (Mumbai, India) was used for column chromatography. TLC plates, pre-coated with silica gel 60 F<sub>254</sub> thickness 0.2 mm and solvents (laboratory grade) were obtained from Merck (Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2<italic>H</italic>-tetrazolium bromide (MTT), antibiotic solution and other chemicals for the biological experiments were purchased from Hi-media Limited (Mumbai, India). Lipopolysaccharide (<italic>Escherichia coli</italic> 026:B6) (LPS), dimethyl sulfoxide (DMSO), dexamethasone and curcumin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse and rat TNF-α and IL-1β ELISA kits were supplied by Krishgen Biosystems (Mumbai, India). </p></sec><sec><title>Extraction and isolation </title><p>Dried and pulverized whole plants (2.8 Kg, 20-30 mesh) was subjected to sequential maceration with 6 L (3x) hexanes (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) for 72 h at 25 °C to yield hexanes (LCH, 23.6 g), dichloromethane (LCD, 32.3 g), ethyl acetate (LCE, 21.6 g) and methanol (LCM, 18.3 g) extracts respectively. Vacuum liquid chromatography (VLC) of LCD (48.7 g) over silica gel (230-400 mesh) column chromatography (CC) was performed using a stepwise gradient of hexanes containing increasing amounts of EtOAc (from 0 % up to 100 %). Three pooled fractions; LCD-F1 (22.67 g, 0-25 % EtOAc/Hex), LCD-F2 (1.24 g, 25-40 % EtOAc/Hex) and LCD-F3 (8.37 g, 40-60 % EtOAc/Hex) were obtained on the basis of similar thin layer chromatography (TLC) profiles. LCD-F1 was found to be oily in nature and hence subjected for GC-MS analysis after derivatization. LCD-F2 (1.24 g) was fractionated by CC on silica gel (230-400 mesh) using a gradient of hexanes and ethyl acetate (4:1-7:3) and on crystallized with aqueous methanol provided <bold>1 </bold>and<bold> 2 </bold>(76 mg, mixture). Similarly, LCD-F3 (8.37 g) was loaded on a repeated silica gel column (230-400 mesh) and eluted gradient wise, using hexanes and EtOAc (13:7-1:1) yielded <bold>3 </bold>(18 mg),<bold> 4 </bold>(38 mg) and<bold> 5</bold> (10 mg). </p></sec><sec><title>GC-MS analysis</title><p>GC-MS Clarus 600 Gas chromatograph with Clarus 600 C mass spectrometer, column: ELITE 5MS 0.25 id, 30 m (Perkin Elmer) was used for analysis. Inlet line temperature was 270 °C and source temperature was 200 °C. Injector temperature: 220 °C, Runtime: 36.50 min, gas chromatography started at 35 °C saturated it for 10 min and then gradual increase of temperature by 10 °C/min up to 200 °C and then here saturated for 10 min and the obtained chromatogram was scanned by mass spectrometry by mass range scanned 10-600 EI. 10 mg of LCD-F1 was dissolved in 20 mL dry methanol, then 5 to 6 drops of concentrated sulphuric acid (H<sub>2</sub>SO<sub>4)</sub> was added, subsequently refluxed at 50 °C to 80 °C for 12 h. Thereafter, methanol was evaporated and residue was neutralized by adding sodium bicarbonate solution, and then extracted with chloroform. Organic layer was taken, dried with sodium sulphate and then concentrated under reduced pressure using rotary evaporator.</p></sec><sec><title>Cell culture, cell viability and nitric oxide release assay</title><p>RAW 264.7 and J774A.1 cells were obtained from the National Centre for Cell Sciences (NCCS, Pune, India) and were cultured in 250 mL culture flasks containing DMEM supplemented with heat inactivated 10 % FBS, 10,000 units/mL pencillin and 10 ng/mL streptomycin in 0.9 % saline, in a CO<sub>2</sub> incubator (5 % CO<sub>2</sub> in air) at 37 °C. MTT assay was carried out to check the cell viability by measuring the purple formazan formation in the mitochondria of living cells. NO release in the culture medium was quantified using the Griess reaction as previously reported by us (Bhandari et al., 2014[<xref rid="R4" ref-type="bibr">4</xref>][<xref rid="R5" ref-type="bibr">5</xref>]; Patel and Bhutani, 2014[<xref rid="R18" ref-type="bibr">18</xref>]). Briefly, cells were pre-incubated with different concentrations of the test samples for 1 h at 37 °C, and then stimulated with 1 µg/mL of LPS. After 24 h incubation, supernatants were mixed with an equal amount of modified Griess's reagent. After further incubation for 30 min, optical density (OD) was measured at 540 nm. </p></sec><sec><title>TNF-α and IL-1β assays</title><p>Cytokine levels in supernatants were measured by sandwich immunoassays using the supplier's protocol (Krishgen Biosystems, Mumbai, India) (Patel and Bhutani, 2014[<xref rid="R17" ref-type="bibr">17</xref>]; Patel et al., 2014[<xref rid="R19" ref-type="bibr">19</xref>]; Bhandari et al., 2014[<xref rid="R4" ref-type="bibr">4</xref>][<xref rid="R5" ref-type="bibr">5</xref>]). Briefly, cells were pre-incubated with different concentrations of the test samples for 1 h at 37 °C, and then stimulated with 1 µg/mL of LPS (<italic>Escherichia coli </italic>026:B6) in a 96 well microtitre plate. In the case of TNF-α and IL-1β, the cells were further incubated for 6 h and 12 h, respectively and cytokine level in supernatants were measured at 450 nm.</p></sec><sec><title>Measurement of TNF-α and IL-1β production in plasma of SD rats</title><p>The experimental study was approved by the Institute Animal Ethics Committee (IAEC) under approval number IAEC/13/46 and performed according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) given on animal experimentation. Female Sprague Dawley (SD) rats (aged 6-8 weeks, 200-250 g, <italic>n = </italic>5 in each group) were issued from the Central Animal Facility (CAF), NIPER and separated into three different groups (control, test and a positive control group) and were permitted to have free water and normal diet <italic>ad libitum</italic>. Cytokines (TNF-α and IL-1β) concentrations were determined as per the method previously described by us (Patel and Bhutani, 2014[<xref rid="R17" ref-type="bibr">17</xref>][<xref rid="R18" ref-type="bibr">18</xref>]). Briefly, a control group received <italic>p. o.</italic> vehicle (10 % Tween 80), test group received LCD (50, 100 and 400 mg/kg) and a positive control group received dexamethasone (5 mg/kg) respectively at an interval of 12 h. LPS (5 mg/Kg) was administered <italic>i. p.</italic> after 2 h of the final dose. After LPS administration, blood samples were collected later 2 h and 6 h in case of TNF-α and IL-1β respectively. Animals were monitored for any unusual behavior and signs of toxicity at treated doses of test samples. Sandwich ELISA kits specific for rats were used to determine the cytokines (TNF-α and IL-1β) levels in the plasma.</p></sec><sec><title>Statistical analysis</title><p>The results are expressed as the mean ± SD for all triplicates experiments. Half maximal concentration was calculated from concentration <italic>vs</italic> percent inhibition curves. Statistical analysis was performed by One-Way ANOVA followed by a Dunnett's post test (commercially available software SigmaStat 3.5). A level of <italic>p</italic> < 0.05 was used as the criterion of statistical significance.</p></sec></sec><sec><title>Results and Discussion</title><sec><title>Characterization of isolated compounds</title><p>The present study deals with the identification of the anti-inflammatory constituents present in <italic>L. cephalotes</italic> using inhibition of LPS induced NO, TNF-α and IL-1β levels in RAW 264.7 and J774A.1 cells. LCD exhibited significant inhibition of NO, TNF-α and IL-1β production with an IC<sub>50 </sub>of 49.3, 46.8 and 49.8 µg/mL respectively as compared to LCE and LCM (IC<sub>50 </sub>= 50 to > 100 µg/mL) (Patel et al., 2014[<xref rid="R19" ref-type="bibr">19</xref>]). Five compounds namely β-sitosterol (<bold>1</bold>) + stigmasterol (<bold>2</bold>) (mixture) (Patel et al., 2014[<xref rid="R19" ref-type="bibr">19</xref>]), lupeol (<bold>3</bold>), oleanolic acid (<bold>4</bold>) (Bhandari et al., 2014[<xref rid="R4" ref-type="bibr">4</xref>][<xref rid="R5" ref-type="bibr">5</xref>]; Galipalli et al., 2014[<xref rid="R10" ref-type="bibr">10</xref>]) and laballenic acid (<bold>5</bold>) (Bagby et al., 1965[<xref rid="R2" ref-type="bibr">2</xref>]) were isolated and identified from LCD (Figure 1<xref ref-type="fig" rid="F1">(Fig. 1)</xref>). Also, GC-MS analysis of sub-fraction (LCD-F1) revealed the presence of eleven (<bold>6</bold>-<bold>16</bold>) compounds (Table 1<xref ref-type="fig" rid="T1">(Tab. 1)</xref>, Figures 1<xref ref-type="fig" rid="F1">(Fig. 1)</xref> and 2<xref ref-type="fig" rid="F2">(Fig. 2)</xref>).</p></sec><sec><title>Effects of fraction/isolated compounds on NO production and cell viability in macrophages</title><p>LCD-F1 and isolated compounds from <italic>L. cephalotes</italic> were evaluated for the inhibition of LPS induced NO production using RAW 264.7 and J774A.1 cells. As visualized from the Table 2<xref ref-type="fig" rid="T2">(Tab. 2)</xref>, among the tested compounds, oleanolic acid (<bold>4</bold>) (IC<sub>50 </sub>= 20.74 to 25.22 µM) and laballenic acid (<bold>5</bold>) (IC<sub>50 </sub>= 22.03 to 23.21 µM) were found to be slightly more potent than lupeol (<bold>3</bold>) (IC<sub>50</sub>=34.51 to 37.36 µM) in inhibiting NO production in macrophages. Similarly, LCD-F1 possessed the minimal inhibitory concentration of 23.45 to 25.57 μg/mL for inhibition of NO production. We have previously tested β-sitosterol (<bold>1</bold>) + stigmasterol (<bold>2</bold>) (mixture) for inhibition of pro-inflammatory markers in macrophages (Patel et al., 2014[<xref rid="R19" ref-type="bibr">19</xref>]) and hence not included here. All the tested samples owned appreciable cell viability of > 84.23 % at concentration of 100 μM/100 μg/mL.</p></sec><sec><title>Effects of fraction/isolated compounds on pro-inflammatory cytokines production in macrophages</title><p>Table 3<xref ref-type="fig" rid="T3">(Tab. 3)</xref> presents the TNF-α and IL-1β inhibitory effects of LCD-F1 and isolated compounds from <italic>L. cephalotes</italic> on RAW 264.7 and J774A.1 cells. LCD-F1 inhibited TNF-α and IL-1β with an IC<sub>50</sub> ranging from 15.56 to 19.46 μg/mL and 28.51 to 31.71 μg/ mL respectively. Lupeol (<bold>3</bold>) (IC<sub>50 </sub>= 12.33 to 14.28 µM), oleanolic acid (<bold>4</bold>) (IC<sub>50 </sub>= 17.12 to 19.22 µM) and laballenic acid (<bold>5</bold>) (IC<sub>50 </sub>= 17.21 to 18.32 µM) were found to be active against TNF-α production in macrophages. In case of IL-1β inhibition, tested compounds were found to be lesser active with half maximal concentration of 32.71 to 89.21 µM as compared to TNF-α inhibition. There are reports for anti-inflammatory properties of sesquiterpenes, fatty acids and their derivatives (Keyzers and Davies-Coleman, 2005[<xref rid="R13" ref-type="bibr">13</xref>]; El-Demerdash, 2011[<xref rid="R9" ref-type="bibr">9</xref>]; Held et al., 2007[<xref rid="R12" ref-type="bibr">12</xref>]; Hamdan et al., 2013[<xref rid="R11" ref-type="bibr">11</xref>]) and hence, these components present in LCD-F1 are responsible for the inhibition of pro-inflammatory mediators (NO, TNF-α and IL-1β).</p></sec><sec><title>Inhibitory effects of LCD on pro-inflammatory cytokines in SD female rats</title><p>LCD was further evaluated in female SD rats for <italic>in vivo</italic> inhibition of pro-inflammatory cytokines. Animals were observed for any altered reaction after the subjection of LPS or/and test samples. It was found that test samples and LPS do not cause any toxic effects. As shown in Figure 3<xref ref-type="fig" rid="F3">(Fig. 3)</xref> (A and B) there is a substantial increase (<italic>p</italic> < 0.001) in the TNF-α (1623 pg/mL) and IL-1β (912 pg/ mL) levels in the LPS induced groups with respect to the normal group (< 87 pg/mL). Dexamethasone (standard drug) at a dose of 5 mg/Kg was found to suppress significantly (<italic>p</italic> < 0.001) TNF-α production by 75.8 % (394 pg/mL) and IL-1β production by 63.0 % (338 pg/mL) respectively. LCD at treated dose of 50, 100 and 400 mg/Kg manifested significant (<italic>p</italic> < 0.05 and 0.001) reduction of TNF-α levels by 1340, 1045, and 673 pg/mL and IL-1β levels by 889, 697 and 481 pg/mL respectively. Our results corroborated with the study conducted by Yadav et al., (2012[<xref rid="R23" ref-type="bibr">23</xref>]) where non-polar fraction of methanolic extract of <italic>L. cephalotes </italic>significantly reduced the increased levels of TNF-α, IL-1β and IL-6 in mouse ear tissue homogenate. Baburao et al. (2010[<xref rid="R1" ref-type="bibr">1</xref>]) also reported that at doses of 200 and 400 mg/Kg body weight <italic>p. o</italic> methanolic extract of this plant demonstrated an anti-inflammatory activity in a carrageenan induced rat paw edema model. Hence, it can be estimated that mostly the non-polar components <italic>viz</italic>. fatty acids, sesquiterpenes, steroids and triterpenes present in LCD are responsible for the anti-inflammatory effects of <italic>L. cephalotes.</italic></p><p>To summarize the results, anti-inflammatory effect of <italic>L. cephalotes</italic> is partially mediated through the suppression of pro-inflammatory mediators and hence can be utilized in part or along with available anti-inflammatory drugs for the treatment of inflammatory disorders.</p></sec></sec><sec><title>Conflict of interest</title><p>The authors declare that there is no conflict of interest.</p></sec><sec><title>Acknowledgements</title><p>NKP worked as Department of Science and Technology (DST)-Inspire Senior Research Fellow for the doctoral program. Authors are grateful to the Director, NIPER, S.A.S. 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publication-type="journal"><person-group><name><surname>Yadav</surname><given-names>KS</given-names></name><name><surname>Yadav</surname><given-names>NP</given-names></name><name><surname>Kumar</surname><given-names>N</given-names></name><name><surname>Luqman</surname><given-names>S</given-names></name></person-group><article-title>Anti-inflammatory effect of Leucas cephalotes Spreng. in 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice</article-title><source>Ann Phytomed</source><year>2012</year><volume>1</volume><fpage>62</fpage><lpage>66</lpage></element-citation></ref></ref-list></back><floats-group><fig id="T1" position="float"><label>Table 1</label><caption><title>Compounds identified in LCD-F1 of <italic>Leucas cephalotes</italic> by GC-MS</title></caption><graphic xlink:href="EXCLI-14-508-t-001"></graphic></fig><fig id="T2" position="float"><label>Table 2</label><caption><title>Cell viability and NO inhibitory effects of isolated compounds/fraction from <italic>L. cephalotes </italic></title></caption><graphic xlink:href="EXCLI-14-508-t-002"></graphic></fig><fig id="T3" position="float"><label>Table 3</label><caption><title>TNF-α and IL-1β inhibitory effects of isolated compounds/fraction from <italic>L. cephalotes </italic></title></caption><graphic xlink:href="EXCLI-14-508-t-003"></graphic></fig><fig id="F1" position="float"><label>Figure 1</label><caption><title>Compounds isolated (1-5) from DCM extract of <italic>Leucas cephalotes</italic> and identified (6-16) from LCD-F1 by GC-MS</title></caption><graphic xlink:href="EXCLI-14-508-g-001"></graphic></fig><fig id="F2" position="float"><label>Figure 2</label><caption><title>GC-MS profile for LCD-F1 of <italic>Leucas cephalotes</italic></title></caption><graphic xlink:href="EXCLI-14-508-g-002"></graphic></fig><fig id="F3" position="float"><label>Figure 3</label><caption><title>Effect of LCD on plasma levels of (A) TNF-α and (B) IL-1β in SD rats {* (<italic>p</italic> < 0.001) = <italic>vs</italic> control; #(<italic>p</italic> < 0.05), ##(<italic>p</italic> < 0.001) = <italic>vs</italic> LPS}. Values are expressed as mean ± SD; <italic>n</italic>= 5 animals. The data was analyzed by One way of ANOVA test followed by Dunnett's test. LPS: lipopolysaccharide, LCD: dichloromethane extract of <italic>L. cephalotes </italic>whole plant</title></caption><graphic xlink:href="EXCLI-14-508-g-003"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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