Research ArticleIntracellular ROS Scavenging Activity and Downregulation ofInflammatory Mediators in RAW2647 Macrophage by FreshLeaf Extracts of Pseuderanthemum palatiferum
Patcharawan Sittisart1 and Benjamart Chitsomboon2
1 School of Biology Institute of Science Suranaree University of Technology Nakhon Ratchasima 30000 Thailand2 School of Pharmacology Institute of Science Suranaree University of Technology Nakhon Ratchasima 30000 Thailand
Correspondence should be addressed to Benjamart Chitsomboon benjasutacth
Received 18 December 2013 Accepted 5 February 2014 Published 16 March 2014
Academic Editor Yoshiji Ohta
Copyright copy 2014 P Sittisart and B Chitsomboon This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
Beneficial antioxidant phytochemicals are found in many medicinal plants Pseuderanthemum palatiferum (PP) a well-knownVietnamese traditional medicinal plant in Thailand has long been used in folk medicine for curing inflammatory diseases oftenwith limited support of scientific research Therefore this study aimed to determine antioxidant and modulation of inflammatorymediators of ethanol and water extracts of PP (EEP and WEP resp) WEP had significantly higher phenolic and flavonoid levelsand DPPH radical scavenging activity than EEP However EEP exhibited greater reducing power than WEP A greater decreaseof tert-butyl hydroperoxide-induced oxidative stress in RAW2647 macrophage cells was also observed with EEP Modulationof inflammatory mediators of EEP and WEP was evaluated on LPS plus IFN-120574-stimulated RAW2647 cells EEP more potentlysuppressed LPS plus IFN-120574-induced nitric oxide (NO) production than WEP Both EEP and WEP also suppressed the expressionof iNOS and COX-2 protein levels Collectively these results suggest that PP possesses strong antioxidant and anti-inflammatoryproperties
1 Introduction
Oxidative stress is known to cause cellular damage linked tovarious degenerative processes and diseases such as agingischemic injury atherosclerosis cancer diabetes and variousinflammatory diseases [1] Macrophages are key players ininflammation and their activation is crucial in inflamma-tory processes [2] Many inflammatory stimuli includingbacterial lipopolysaccharide (LPS) and IFN-120574 can stimulatemacrophages to produce proinflammatory cytokines andsmall mediators such as nitric oxide (NO) and prostaglandinE2 (PGE
2) [3] Excess levels of NO produced by acti-
vated macrophages reflect the inflammation process andare regulated by inducible nitric oxide synthase (iNOS) [4]Overproduction of NO has been known to be associatedwith various diseases such as cancer rheumatoid arthritisseptic shock autoimmune disease and chronic inflammation[5] PGE
2 the key player in inflammatory response is
produced from arachidonic acid by prostaglandin synthase orcyclooxygenase (COX) enzymes COX exists as two isoformsCOX-1 and COX-2 COX-1 is constitutively expressed andis a housekeeping enzyme required for normal physiologicalfunctions COX-2 is considered as the inducible isoform andis primarily involved in inflammation [6] Linkage and crosstalk among NO iNOS and COX-2 during the inflammationprocess are well established NO directly enhances COX-2 activity which results in a remarkable synthesis of PGE
2
iNOS and COX-2 can work together in a variety of similarpathophysiological actions and inflammatory diseases [5 7]In additionmany inflammatory effects have been reported tobe associated with high productions of NO iNOS and COX-2 [8] Therefore an agent with inhibitory effects on excesslevels of NO iNOS and COX-2 expression would be highlybeneficial and part of an effective strategy in the treatment ofinflammatory diseases
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 309095 11 pageshttpdxdoiorg1011552014309095
2 Evidence-Based Complementary and Alternative Medicine
Over the last decade medicinal plants as potentialsources of naturally occurring antioxidants have been thefocus of intense research Moreover phytochemicals suchas flavonoids and other polyphenolics with high reactiveoxygen species (ROS) scavenging activities have been shownto exhibit multiple biological effects including antiallergicantibacterial antidiabetic anticancer and anti-inflammatoryactivities [9] As oxidative stress and inflammation are closelylinked and are implicated in many diseases [10] plants thatpossess both antioxidant and anti-inflammatory propertieshave always attracted considerable research interest Pseuder-anthemum palatiferum (Nees) Radlk (PP) a member of theAcanthaceae plant family and commonly called Hoan-Ngocis one of the most popular medicinal plants in bothThailandand Vietnam Phytochemical analysis of PP leaf extracts sug-gests many high potential antioxidant and anti-inflammatoryconstituents [11] In fact PP has been referred to as a miracleplant in folkmedicine to cure or prevent variousmaladies andinflammatory related diseases such as diarrhea sore throathypertension gastric ulcer diabetes and cancer [12 13]Nevertheless the scientific evidence to support its multiplebiological effects is still limited particularly related to anti-inflammation Although the antioxidant property of PP leafextracts has been previously shown [14 15] its intracellularROS scavenging activity has never been assessed To datethere is only one study reporting the anti-inflammatory activ-ity of PP leaf extract [13] and the mechanism responsible forthe anti-inflammation remains largely unknown This studyfurther compares antioxidant activity between ethanol andwater extracts of PP leaves using various in vitro antioxidantevaluation methods including the assessment in the cell-based DCFH-DA assay The modulation of PP leaf extractsin NO production iNOS and COX-2 expression during theinflammatory response was also investigated in the murinemacrophage-like cell line RAW2647 stimulated with LPSplus IFN-120574
2 Materials and Methods
21 Chemicals and Materials 3-(45-Dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) (+)-catechinhydrate and vitamin C were purchased from Fluka ChemieGmbH (Buchs Switzerland) 22-Diphenyl-1-picryl-hydrazyl(DPPH) penicillin G streptomycin sulfate resveratrolN-(1-naphthyl)ethylenediamine dihydrochloride (NED)sodium nitrite LPS (Escherichia coli O111B4) 2101584071015840-dichlorofluorescin-diacetate (DCFH-DA) and tert-butylhydroperoxide (tBuOOH) were purchased from Sigma-Aldrich (St Louis MO USA) Dimethyl sulfoxide (DMSO)was purchased from Amresco Inc (Solon OH USA)Quercetin dihydrate was obtained from INDOFINEChemical Company Inc (Hillsborough NJ USA)6-Hydroxy-2578-tetramethylchroman-2-carboxylic acid(Trolox) was purchased from Sigma-Aldrich Chemie GmbH(Steinheim Germany) Mouse interferon gamma (mIFN-120574)and ECL Western blotting substrate were purchased fromPierce Protein Research Products (Rockford IL USA)RPMI medium 1640 Hankrsquos balanced salt solution (HBSS)
and penicillin-streptomycin were obtained from GibcoInvitrogen (Grand Island NY USA) Fetal bovine serum(FBS) was obtained from Hyclone (Logan UT USA)Anti-iNOS and anti-tubulin mouse monoclonal antibodiesand secondary antibody goat-anti-mouse-HRP conjugatefor iNOS and tubulin were purchased from Santa CruzBiotechnology Inc (Santa Cruz CA USA) Anti-COX-2mouse polyclonal antibody and secondary antibody goat-anti-rabbit IgG-HRP conjugate for COX-2 were purchasedfrom Cayman Chemical (Ann Arbor MI USA) The mousemacrophage cell line (RAW2647 cells) was purchased fromCell Lines Service (Eppelheim Germany) All other reagentswere purchased from Sigma-Aldrich unless otherwiseindicated
22 Plant Material Fresh leaves of PP were purchased fromproducers in Yasothon province Thailand The plant wasidentified and authenticated by Dr Kongkanda ChayamaritForest Herbarium Royal Forest Department BangkokThai-land A voucher specimen (BKF 174009) was deposited atthe Forest Herbarium Royal Forest Department BangkokThailand
23 Plant Extract Preparation Fresh leaves (15 kg) were cutinto small pieces and blended in 6 L of 95 ethanol Theextract was centrifuged at 3500 g for 10min at 4∘C and thesupernatant was filtered through Whatman number 1 filterpaper The ethanolic filtrate was then concentrated usinga vacuum rotary evaporator and lyophilized to obtain theethanol extract of PP (EEP 6041 g) Forty grams of EEPwere further partitioned between hexane and water (1 1)using a separatory funnel The water fraction was collectedcentrifuged at 14000 g for 10min at 4∘C evaporated andlyophilized to obtain a water extract of PP (WEP 3271 g)The EEP and WEP were stored at minus20∘C until they wereneeded in subsequent experiments The EEP and WEP weredissolved in DMSO and water respectively when used inexperiments For cell cultures the WEP was dissolved inphosphate buffered saline (PBS)
24 Total Phenolic Content The total phenolic content of theindividual extract was determined by the method of Folin-Ciocalteu [16] Briefly 100 120583L of test solution was added to2mL of 2 Na
2CO3and mixed thoroughly After 2min
100 120583L of 50 Folin-Ciocalteu reagent was added mixedand allowed to stand at room temperature (RT) for 30minThe absorbance of extracts was measured at 750 nm bya Cecil 1000 series spectrophotometer (Cecil InstrumentsCambridge UK) against a blank consisting of only reagentsand solvents without the extract Gallic acid solutions rangingfrom 005 to 03mgmL were used to prepare a standardcurve The concentration of phenolic compounds in theextracts is expressed as mg of gallic acid equivalent (GAE)per g of dry extract
25 Total Flavonoid Content The total flavonoid contentwas determined using a colorimetric method [17] Briefly250 120583L of sample was diluted with 125mL of distilled water
Evidence-Based Complementary and Alternative Medicine 3
(DI) Then 75 120583L of 5 NaNO2solution was added to the
mixture After 6min 150 120583L of a 10 AlCl3sdot6H2O solution
was added and the mixture was allowed to stand for another5min One half mL of 1M NaOH was added and the totalvolume was brought up to 25mL with DI waterThe solutionwas thoroughly mixed and the absorbance was measuredimmediately against the prepared blank at 510 nm Catechinstandard solutions (005ndash04mgmL) were used to preparea standard curve The concentration of flavonoids in theextracts is expressed as mg of catechin equivalent (CAE) perg of dry extract
26 FRAP (Ferric Reducing Antioxidant Power) Assay Theferric reducing ability of the extracts was measured colori-metrically according to the method developed by Benzieand Strain [18] The FRAP reagent consisted of 01M acetatebuffer (pH 36) 10mM 246-tris(2-pyridyl)-135-triazine(TPTZ) solution in 40mM HCl and 20mM FeCl
3sdot6H2O
solutionThe fresh working solution was prepared by mixingthe acetate buffer the TPTZ solution and the FeCl
3sdot6H2O
solution in a 10 1 1 vvv ratio The FRAP reagent (3mL)was added to 01mL of the extract and mixed Readingswere recorded on the spectrophotometer at 593 nm andthe reaction was monitored for 10min A standard curveof 100ndash1000120583mol FeSO
4sdot7H2O was prepared Vitamin C
(10ndash90 120583gmL) Trolox (10ndash160 120583gmL) and catechin (10ndash90 120583gmL) were used as standard antioxidants The antioxi-dant power of the extracts is expressed as mmol ferrous ion(Fe2+) per g of dry extract and alsomgof vitaminC equivalent(VCE) Trolox equivalent (TRE) and catechin equivalent(CAE) per g of dry extract
27 DPPH Assay The scavenging activity of DPPH radicalswas determined as described by Sanchez-Moreno et al [19]Briefly 100 120583L of extract at different concentrations wasadded to 39mL of methanolic DPPH solution (63mM)The mixture was shaken vigorously and left to stand at RTfor 45min in the dark Samples that are able to scavengeDPPH free radicals reduce the purple DPPH radicals intothe light yellow colored product of corresponding hydrazineDPPH
2 Decreasing DPPH solution absorption (measured
spectrophotometrically at 515 nm) indicates an increase ofDPPH radical scavenging activity [20] DPPH solution plusmethanol were used as negative control and vitamin CTrolox and catechin were used as positive controls The per-cent inhibition of DPPH radicals by test samples was deter-mined by comparison with the methanol-treated controlThe free radical scavenging activity which is the percentageinhibition of free radical is calculated as follows
DPPH radical scavenging activity ()
= [119860control minus 119860 sample
119860control] times 100
(1)
where 119860 sample and 119860control are absorbances of the sampleand the control respectively The IC
50of DPPH radicals was
determined from a dose response of inhibitory curve usinglinear regression analysis
28 Cell Culture The RAW2647 macrophage cells werecultured at 37∘C 5 CO
2in an RPMI-1640 medium supple-
mented with 10 heat-inactivated FBS 100UmL penicillinand 100 120583gmL streptomycin Exponentially growing cellswere used for experiments when they reached about 80confluence
29 Cell Viability (MTT Assay) A tetrazolium dye (MTT)colorimetric assay was used to determine the viability ofRAW2647 cells as described by Chun et al [21] Briefly RAW2647 cells were plated at a density of 5 times 104 cellswell in a 96-well plate and incubated overnight at 37∘C under 5 CO
2
After incubation the cells were exposed to various concen-trations of EEP or WEP for 24 h Then MTT (05mgmL)dye solution was added in each well and further incubatedat 37∘C 5 CO
2for 4 h The media was removed and DMSO
was added to each well to dissolve formazan crystals givinga uniform dark purple color before reading at 540 nm by theBenchmark PlusMicroplate Spectrophotometer System (Bio-Rad Laboratories Inc Hercules CA USA) The percentageof cell viability was calculated by the following equation
Percent cell viability =ODtest group
ODcontrol grouptimes 100 (2)
210 Assessment of Intracellular ROS Scavenging ActivityIntracellular oxidative stresswas detected usingDCFH-DAasdescribed by Kim et al [22] with slight modification BrieflyRAW2647 cells (4 times 104 cellswell) were plated in a Costa96-well black clear bottom plate (Corning Inc Corning NYUSA) and incubated for 16ndash18 h at 37∘C and 5 CO
2 After
incubation the cells were washed with PBS twice To assessantioxidant activity the cells were preexposed to differentconcentrations of EEP WEP (50 150 or 250120583gmL) or theantioxidant positive controls catechin (250 120583M) resveratrol(20120583M) or quercetin (10 120583M) for 24 h After washing twicewith PBS the cells were exposed to 20120583M DCFH-DA inHBSS and further incubated in the dark for another 30minThe DCFH-DA was removed by washing the cells with PBStwo times Then 500120583M tBuOOH was added The unstimu-lated DCFH-DA (no tBuOOH) in the unexposed RAW2647cells served as the naive control (NA) The intensity of thefluorescence signal was detected time dependently with anexcitation wavelength of 485 nm and an emission wavelengthof 535 nm using a Gemini EM fluorescence microplate reader(Molecular Devices Sunnyvale CA USA)
211 Nitrite Assay The level of NO in the culture mediawas detected as nitrite a major stable product of NOusing Griess reagent [23] RAW2647 cells were seeded ata density of 2 times 105 cellswell in a 96-well plate The cellswere grown for 3 h to allow plate attachment prior treatingwith the antioxidant positive control vitamin C (500120583M)or various concentrations (50 100 150 200 or 250 120583gmL)of EEP or WEP After 1 h incubation the RAW2647 cellswere stimulated with 1 120583gmL LPS plus 25UmL IFN-120574 Theactivated cells were further incubated for 24 h Then 100 120583Lof supernatant was mixed with an equal volume of Griess
4 Evidence-Based Complementary and Alternative Medicine
Table 1 The percentage of recovery of crude extracts from fresh leaves of PP
Extracts Amount and source of preparation Yield (g) Percentage of recoveryEEP 1500 g of fresh leaves 6041 403 (from fresh leaves)WEP 40 g of EEP 3271 8177 (from EEP)
(99321 g of fresh leaves) 329 (from fresh leaves)
Table 2 Total phenolic and flavonoid contents and total antioxidant (FRAP) activity of EEP and WEP
Values are mean plusmn SEM (119899 = 3) and are representative of three independent experiments with similar results Different letters within the same column aresignificantly different at P lt 005 as determined by a Studentrsquos t-test
reagent (1 sulfanilamide 01 NED and 3 phosphoricacid) After 10min of incubation in the dark the absorbanceof samples was measured at 540 nm using a Microplate Spec-trophotometer System (Bio-Rad Laboratories Inc) A freshculturemediumwas used as the blank in all experimentsTheamount of nitrite in the samples was derived from a standardcurve of sodium nitrite
212 Western Blot Analysis RAW2647 cells were plated ata density of 2 times 106 cellswell in a 6-well plate After anattachment period of approximately 3 h the cells were treatedwith various concentrations (50 100 150 200 or 250120583gmL)of EEP orWEP for 1 h 50 120583gmL Trolox or 500120583Mvitamin Cwas used as antioxidant positive controls The cells were thenstimulated with 1 120583gmL LPS plus 25UmL IFN-120574 for 18 hAfter incubation the cells were washed three times with PBSand placed in 150 120583L of ice-cold lysis buffer (1mL RIPA buffersupplemented with 2mM PMSF 2 120583M leupeptin and 1 120583ME-64) for 20minThen the disrupted cells were transferred tomicrocentrifuge tubes and centrifuged at 14000 g at 4∘C for30min The supernatant was collected and the protein con-centration of cell lysate was estimated by the Lowry method[24] Cell lysate was then boiled for 5min in a 6X samplebuffer (50mM Tris-base pH 74 4 SDS 10 glycerol 42-mercaptoethanol and 005mgmL of bromophenol blue)Thirty micrograms of cellular proteins were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) using 75 and 10 polyacrylamide gels foriNOS and COX-2 respectively (125 volts 120min) Theproteins in the gel were transferred onto a nitrocellulosemembrane (Amersham Pittsburgh PA USA) at 80 volts for1 h The membrane was blocked overnight at 4∘C with 5nonfat milk in 01 Tween 20 in a PBS buffer (TPBS) Themembranes were then incubated with a 1 1000 dilution ofthe primary antibody anti-iNOS mouse monoclonal or a1 2000 dilution of the primary antibody anti-COX-2 mousepolyclonal at RT for 2 h After extensive washing with TPBSthe membranes were incubated with a 1 10000 dilution ofthe secondary antibody goat-anti-mouse-HRP conjugate foriNOS and goat-anti-rabbit IgG-HRP conjugate for COX-2 at RT for 1 h To control equal loading of total protein
in all lanes blots were also stained with primary antibodyanti-tubulin mouse monoclonal at a dilution of 1 2000 atRT for 2 h After washing the membranes were incubatedwith a 1 10000 dilution of the secondary antibody goat-anti-mouse-HRP conjugate The membranes were washedthree times for 10min each time with TPBS The blotswere incubated for 3min in ECL Western blotting substrateand exposed to film The relative expression of proteins wasquantified densitometrically using the software imageJ andcalculated according to the reference band of tubulin
213 Statistical Analysis All statistical analyses were con-ducted using GraphPad software (GraphPad Prism 5 USA)The data from the total phenolic and flavonoid contents aswell as FRAP value results were analyzed by a Studentrsquos 119905-test to determine the statistical significance between twogroups DPPH MTT and nitrite assays were analyzed byone-way analysis of variance (ANOVA) with a post hocTukeyrsquos analysis to determine differences between treatmentand control groups [25] The data from intracellular ROSscavenging were analyzed by two-way ANOVA followed byBonfferonnirsquos post hoc test [26]
3 Results
31 The Percentage of Recovery of Crude Extracts from FreshLeaves of PP The percentages of recovery of crude extractsfrom fresh leaves of PP are shown in Table 1 EEP exhibited apercentage of recovery of 403 while WEP had percentageof recovery of 329 based on the original weight of freshleaves WEP was prepared from the water fraction of EEPthat was partitioned with hexane and water (1 1 vv) with apercentage of recovery of 8177 based on EEP
32 Phenolic and Flavonoid Contents WEP had a signifi-cantly higher level (119875 lt 005) of total phenolic and flavonoidcontent than that of EEP (Table 2) and more than half of thephenolics in WEP and EEP are flavonoids
33 Ferric Reducing Antioxidant Power EEP and WEP wereanalyzed for their reducing ability along with three standard
Evidence-Based Complementary and Alternative Medicine 5
80
100
60
40
20
0
0 5 10 15 20 25 30 35
Inhi
bitio
n (
)
Concentration (120583gmL)
WEPEEPCatechin
Vitamin CTrolox
Figure 1 DPPH radical scavenging activity of PP leaf extracts (EEPand WEP) and positive controls (vitamin C Trolox and catechin)Values are means plusmn SEM (119899 = 3) and are representative of threeindependent experiments with similar results
antioxidants vitamin C Trolox and catechin The results ofFRAP values in terms of ferrous ion (Fe2+) and vitamin CTrolox and catechin equivalents are shown in Table 2 EEPexhibited a higher degree of electron donating capacity thanWEP as suggested by the significantly higher FRAP values(119875 lt 005) of EEP when compared with WEP
34 DPPH Free Radical Scavenging Activity The free radicalscavenging capacities of EEP andWEP are shown in Figure 1The results show that both EEP and WEP exhibit the abilityto scavenge DPPH free radicals The scavenging activityagainst DPPH radicals of WEP (IC
50= 2155 plusmn 006 120583gmL)
is significantly greater (119875 lt 0001) than EEP (IC50
=2345 plusmn 012120583gmL) by 19 plusmn 015 but the scavengingcapacity of these is not as effective as the other positiveantioxidant controls 125 120583gmL EEP and 25 120583gmL WEPscavenged the DPPH radicals by 2927 plusmn 020 and 612 plusmn015 respectively and the scavenging capacities of bothare more pronounced at higher concentrations The highestconcentration (325120583gmL) of EEP andWEP could scavengethe DPPH radicals by 6596 plusmn 021 and 7319 plusmn 009respectively In the present study the scavenging abilities ofvitamin C (IC
50= 394 plusmn 001 120583gmL) and catechin (IC
50=
355 plusmn 001 120583gmL) were similar and both are significantlyhigher (119875 lt 0001) than Trolox (IC
50= 590 plusmn 027 120583gmL)
35 Effect of EEP and WEP on RAW2647 Cell Viability Thecell viability of RAW2647 cells exposed to EEP or WEP wasdetermined by MTT assay The cells were incubated for 24 hwith various concentrations of EEP (005 025 05 10 or150mgmL) or WEP (010 050 150 or 450mgmL) Asshown in Figure 2 both EEP andWEP displayed low toxicitytowards RAW2647 cells as evidenced by an apparent lack ofeffect on cell viability until the concentration of each extract
reached 15mgmL At 15mgmL EEP and WEP decreasedthe viability of RAW2647 cells by 3414 plusmn 969 and 2158 plusmn166 (119875 lt 005) respectively However the cytotoxiceffect is more pronounced at higher concentrations WEP at45mgmL decreased the cell viability by as much as 5421 plusmn174 (119875 lt 005) The effect of EEP and WEP on RAW2647cell viability was also confirmed by trypan blue exclusion andpropidium iodide staining methods which exhibited similarresults (data not shown)Therefore a nontoxic concentrationrange of 0ndash025mgmL of both EEP and WEP was selectedfor RAW2647 cell treatment in the subsequent studies
36 EEP and WEP as Intracellular ROS Scavengers Thedirect scavenging effect of EEP and WEP on intracellularfree radical stress was investigated in RAW2647 cells usingthe DCFH-DA assay The increment of DCF fluorescenceemission following ROS-mediated oxidation of DCFH wasfollowed for 240min As shown in Figure 3(a) standardantioxidant positive controls catechin (250 120583M) resveratrol(20120583M) and quercetin (10 120583M) could scavenge ROS signif-icantly (119875 lt 005) throughout the incubation time whencompared to the vehicle control (VH)With as little as 30minof incubation catechin resveratrol and quercetin showedconsiderable radical scavenging activity EEP (Figure 3(b))and WEP (Figure 3(c)) decreased the DCF fluorescent emis-sion in a dose- and time-dependent manner Again with aslittle as 30min of incubation both EEP and WEP at lowconcentration (50120583gmL) showed similar radical scavengingactivity as the antioxidant controls Various concentrations ofEEP significantly decreased (119875 lt 005) the DCF fluorescentemission throughout the incubation time when comparedto the VH control At high concentration (150 120583gmL) EEPexhibited a strong scavenging activity as suggested by thecapability to lower fluorescent intensity to below basal levelof the unstimulated DCFH-DA control at 180ndash240min Inaddition the highest concentration of EEP (250120583gmL)significantly lowered (119875 lt 005) DCF fluorescent intensityto below the basal level at all time points Similarly 150 and250 120583gmL of WEP also significantly decreased (119875 lt 005)the DCF fluorescent emission throughout the incubationtime compared to the tBuOOH control However the lowestconcentration ofWEP (50120583gmL) significantly reduced (119875 lt005) the DCF fluorescent emission until 210min only
37 NO Suppression by EEP and WEP in LPS Plus IFN-120574-Activated RAW2647 Cells RAW2647 cells were pretreatedwith antioxidants vitamin C EEP or WEP for 1 h thenstimulated with LPS plus IFN-120574 and measured for NOproduction using the Griess assay As shown in Figure 4unstimulated RAW2647 cells (NA) secreted basal levels ofNO while the production of NO was increased to about43 120583M in LPS plus IFN-120574-activated RAW2647 cells Theantioxidant control 500 120583M vitamin C decreased the NOproduction by almost 35 Pretreatment of RAW2647 cellswith EEP or WEP significantly suppressed (119875 lt 005) theinduction of NO in a dose-related manner (Figures 4(a) and4(b)) and the suppression was observed in all EEP- andWEP-treated groups These results also clearly indicate that
6 Evidence-Based Complementary and Alternative Medicine
80
60
40
20
0
b b b b
aab
Cel
l via
bilit
y (
)120
100
Control 005 025 050 150100
EEP (mgmL)
(a)
80
60
40
20
0
b
a
ccd
d
Cel
l via
bilit
y (
)
120
100
Control 010 050 150 450
WEP (mgmL)
(b)
Figure 2 Effect of EEP andWEP on cell viability of RAW2647 cellsThe effect of EEP (a) andWEP (b) on cell viability was assessed byMTTValues are expressed as means plusmn SEM (119899 = 3) and are representative of three independent experiments with similar results Bars marked withdifferent letters are significantly different at 119875 lt 005 as determined by one-way ANOVA
EEP is a stronger suppressant of NO induction than WEPConcentrations of 50 120583gmL of EEP and 150 and 200 120583gmLofWEPwere required to exhibit the NO suppression with thesame efficiency as 500 120583M (8806120583gmL) vitamin C
38 Suppression of iNOS and COX-2 Protein Expression byEEP and WEP in LPS Plus IFN-120574-Activated RAW2647 CellsTo determine if suppression of NO production by EEP orWEPwas related to changes in iNOS aswell as COX-2 proteinlevels Western blotting analysis was performed RAW2647cells were pretreated with antioxidants Trolox (50120583gmL)vitamin C (500120583M) or PP extracts (EEP or WEP) at 50ndash250120583gmL for 1 h prior activation with LPS (1120583gmL) plusIFN-120574 (25UmL) for 18 h Total proteins were extracted andanalyzed for the expression of iNOS and COX-2 by Westernblotting LPS plus IFN-120574 induced increases in iNOS (Figures5(a) and 5(b)) and COX-2 (Figures 5(c) and 5(d)) expressioncompared to the unstimulated cultures Antioxidant controls(Trolox and vitamin C) decreased LPS plus IFN-120574-inducediNOS and COX-2 protein levels The data also suggestedthat the suppression by 500120583M (8806120583gmL) vitamin Cis more pronounced than 50 120583gmL Trolox Compared tothe corresponding controls both EEP and WEP produced adose-dependent suppression of iNOS level in LPS plus IFN-120574-activated RAW2647 cells (Figures 5(a) and 5(b)) suggestingthat the suppression of NO production by EEP and WEP ismediated by decreasing the expression of iNOS In agreementwith the result of NO suppression 50ndash200 120583gmL EEP wasprobably more efficient than WEP in iNOS suppressionThe iNOS expression was almost completely eliminated at200120583gmL EEP and was barely observed at 250 120583gmLWEPThe inflammatory modulation of EEP and WEP was alsofurther supported by the dose-dependent suppression of theCOX-2 level by both EEP and WEP (Figures 5(c) and 5(d))in the activated RAW2647 cells Notably EEP and WEPexhibited higher suppression of iNOS than COX-2
4 Discussion
It is well known that major phytochemicals of plant leafextracts possessing antioxidant activity are flavonoids andother phenolic compounds Researchers have found thatflavonoids from PP leaves display antioxidant activity andall ethyl acetate chloroform and 119899-butanol-soluble fractionsof PP contain flavonoids [11 15] In addition Nguyen andEun [14] found phenolics and flavonoids in extracts of PPleaves when assessed with Folin-Ciocalteu and aluminumtrichloride PP leaf extracts also have antioxidant activitieswhen evaluated with DPPH and FRAP assays Similarlythe present study also showed that both EEP and WEPcontain high levels of flavonoids and phenolics and exhibitantioxidant activity The most frequently used antioxidantstandards for food samples (vitamin C Trolox catechinresveratrol and quercetin) were used as positive antioxidantcontrols in the present study
This study revealed that DPPH radical scavenging capac-ity of WEP is greater than that of EEP (Figure 1) In contrastEEP has higher ferric reducing power than WEP (Table 2)Such contradictory results between DPPH and FRAP assaysare not unusual Though both assays are based on a singleelectron transfer reaction [27] their characteristics sensitiv-ities mechanisms of the reaction and endpoints are totallydifferent For instance the DPPH method is based on thefree radical scavenging activity while FRAP measures thecapability of reducing Fe3+ to Fe2+ Depending on whatspecific phytochemical constituents present in the extractare providing the antioxidant activity their discrete chemicalstructures positions numbers and types of substitutions caninfluence their redox properties and hence their antioxidantpotentials [28]
Though both DPPH and FRAP assays are frequently usedfor assessing antioxidant capacity they have some drawbacksIn the DPPH assay interfering compounds may have sig-nificant absorption at the same measured wavelength In
Evidence-Based Complementary and Alternative Medicine 7
Figure 3 Cellular radical scavenging activity in tBuOOH-activated RAW2647 cells Intracellular ROS level generated in cells was measuredby the DCFH-DA RAW2647 cells were pretreated with indicated concentrations of antioxidants (a) EEP (b) or WEP (c) for 24 h priorto take-up of 20 120583M DCFH-DA for 30min Results are mean plusmn SEM (119899 = 4) and are representative of three independent experiments withsimilar results Pointsmarked with different letters are significantly different at119875 lt 005when compared at the same time point as determinedby two-way ANOVA
addition theDPPH radical is not present in living organismsFor the FRAPmethod compounds with low redox potentialwhich probably do not serve as antioxidants in vivo still canreduce the Fe3+ Interfering compounds may also absorb atthe same wavelength and the assay is also performed at anonphysiological pH [29] Therefore antioxidant activitiesof EEP and WEP were also evaluated by the cell-basedassay using an intracellular fluorescent probe DCFH-DAWhen the nonfluorescent DCFH-DA is taken up into cellsits diacetate moiety will be hydrolyzed by cellular esterasesto generate the more polar DCFH which is trapped insidethe cells In the presence of ROS intracellular DCFH isfurther oxidized to form the fluorescent DCF product [30]
The macrophage cell line RAW2647 is usually the cell ofchoice in studying ROS-mediated cellular events since itcan generate high amounts of ROS following an oxidantchallenge Catechin resveratrol and quercetin at the levelof concentration used in this study have been shown andoptimized to exhibit a strong suppression of intracellular ROSgeneration [22 31 32] Therefore the present study selectedthese compounds as antioxidant positive controls for theDCFH-DA assay The present study demonstrated that allantioxidant standards 250120583M catechin 20120583M resveratroland 10 120583M quercetin exerted a strong inhibition of ROSgeneration induced by tBuOOH over a period of 30 to240min In addition to extracellular antioxidant capacity
8 Evidence-Based Complementary and Alternative Medicine
45
40
35
30
25
20
15
10
5
0
NA
VH 50
150
250
e
d
c
bb
a
e
d
bcNitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
EEP (120583gmL)
(a)
b
e
cc
bc
d
f
a
45
40
35
30
25
20
15
10
5
0
NA 50
150
250
Nitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
WEP (120583gmL)
(b)
Figure 4 EEP (a) and WEP (b) suppressed LPS plus IFN-120574-induced nitrite production in RAW2647 cells RAW2647 cells were incubatedfor 24 hwith LPS (1120583gmL) plus IFN-120574 (25UmL) in the presence or absence of indicated concentrations of vitaminC (500120583M) EEP orWEPAccumulated nitrite in the culturemediumwas determined by the Griess reactionThe values aremeansplusmn SEM (119899 = 3) and are representativeof three independent experiments with similar results Bars marked with different letters are significantly different at 119875 lt 005 as determinedby one-way ANOVA
EEP and WEP also served as intracellular ROS scavengersand subsequently decreased the oxidation of DCFH (Figures3(b) and 3(c)) Both EEP and WEP were as efficient as theantioxidant standards in scavenging ROS Notably EEP was abetter reducer of DCF fluorescence thanWEPThe reductionof DCF fluorescence by EEP and WEP is not due to directcytotoxicity as the range of concentration used in the studieshad no effect on RAW2647 cell viability (Figure 2)
Although the current study shows that an ethanol extractfrom PP leaves has in vivo anti-inflammatory activities[13] its mechanism of anti-inflammation is still unrevealedInflammatory disorders are characterized among otherevents by the production of significant amounts of freeradicals nitrogen reactive species and pro-inflammatorycytokines [10] High NO concentration combines with super-oxides to form peroxynitrite ions (OONOminus) which areresponsible for cell and tissue damage from inflammation[33] Therefore we investigated inflammatory effects of EEPand WEP on the suppression of NO production in LPS plusIFN-120574-activated RAW2647 cells At the concentration rangeof 50ndash250120583gmL both EEP and WEP dose-dependentlysuppressed NO production and the suppression was morepronounced in EEP than WEP (Figure 4) These resultsagreed with the observation that EEP was also a betterscavenger of intracellular ROS than WEP (Figures 3(b) and3(c))
As enhanced NO production by LPS and IFN-120574-stimulated RAW2647 cells mainly occurs via increasedintracellular content of iNOS [3 4] the effect of EEP andWEP on iNOS expression was investigatedThe present studyclearly indicates that the suppressive effect of EEP and WEPon NO production was mediated through the inhibition of
iNOS expression (Figures 5(a) and 5(b)) In agreement withthe study of NO suppression the suppressive effect of EEP(50ndash200120583gmL) on iNOS was more remarkable than that ofWEP
In addition to iNOS induction LPS and IFN-120574 alsoefficiently enhance COX-2 expression in RAW2647 cells [35] An increased level of COX-2 expression is also knownto account for the excessive production of PGE
2in most
if not all inflammatory cells and tissues [34] This studyshows that both EEP andWEP can exhibit anti-inflammatoryactivity by reducing high COX-2 protein levels in a dose-related manner (Figures 5(c) and 5(d)) Thus EEP and WEPmight play important roles in attenuating inflammationand cellular damage through their extra- and intracellularROS scavenging activity and downregulation of NO iNOSand COX-2 Concordantly Khumpook et al [13] recentlyreported the in vivo anti-inflammatory activity of PP leavesas evidenced by decreased lipid peroxidation and NO levelin concomitance with increased superoxide dismutase in thecotton-induced chronic inflammation in Albino rats uponexposure to an ethanol extract of PP leaves for 17 days
In fact several medicinal plant extracts with naturalantioxidant properties together with suppressive effects onNO iNOS andorCOX-2 expression inRAW2647 have beenreported to display a wide spectrum of bioactivities Theseactivities include anti-inflammation such as curcumin fromCurcuma longa resveratrol from grape skins red wines andother plants and a mixture of 120573-sitosterol and stigmasterolfrom Andrographis paniculata [8 35 36] Previous investi-gators demonstrated that pretreatment of RAW2647 withflavonoids such as apigenin genistein and kaempferol sup-pressed LPS-stimulated expression of NO iNOS and COX-2
Evidence-Based Complementary and Alternative Medicine 9
FoldiNOS
Tubulin
003 086 058 030 061 038 033 024 015
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
EEP (120583gmL)
LPSIFN-120574
(a)
Fold
Tubulin
iNOS
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
WEP (120583gmL)
LPSIFN-120574
008 111 083 052 073 063 048 036 008
(b)
COX-2
Fold
Tubulin
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
006 093 076 074 087 079 065 044 030
EEP (120583gmL)
LPSIFN-120574
(c)
Fold
Tubulin
COX-2
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
001 113 066 063 091 078 067 064 050
WEP (120583gmL)
LPSIFN-120574
(d)
Figure 5 Effect of EEP on LPS plus IFN-120574-induced iNOS (a) and COX-2 (c) andWEP on LPS plus IFN-120574-induced iNOS (b) and COX-2 (d)protein levels in RAW2647 cells The relative expression of proteins was quantified densitometrically using ImageJ software and normalizedto tubulin reference bands Data are representative of at least two independent experiments
protein production [37] Major chemical constituents of PPleaves consist of 120573-sitosterol stigmasterol kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucoside[11] All aforementioned compounds have been shown topossess anti-inflammatory properties Both kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucosidemay be metabolized into kaempferol and apigenin whichalso have antioxidant and anti-inflammatory activities Thusit is possible that phenolic and flavonoid compounds inboth EEP andWEP provide substantial antioxidant and anti-inflammatory activities
In summary the cytoprotective effects of EEP and WEPis due to their abilities to decrease ROS generation and NOradical production in cells In addition both EEP and WEPexert anti-inflammatory effects through the suppression ofNO release and decrease the protein expression of iNOSand COX-2 Thus PP leaves possess high potential forfurther exploration in the research development of anti-inflammatory medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was financially supported by a Royal GoldenJubilee Scholarship Grant (PhD02082548) and by the
National Research Council of Thailand (NRCT) throughSuranaree University of Technology (SUT-104-53-36-09)
References
[1] B Halliwell and J M C Gutteridge Free Radicals in BiologyandMedicine OxfordUniversity Press NewYork NYUSA 3rdedition 1999
[2] N Fujiwara and K Kobayashi ldquoMacrophages in inflammationrdquoCurrent Drug TargetsmdashInflammation and Allergy vol 4 no 3pp 281ndash286 2005
[3] S I Jang Y-J Kim W-Y Lee et al ldquoScoparone from Artemisiacapillaris inhibits the release of inflammatory mediators inRAW 2647 cells upon stimulation cells by interferon-120574 plusLPSrdquo Archives of Pharmacal Research vol 28 no 2 pp 203ndash208 2005
[4] R Korhonen A Lahti H Kankaanranta and E MoilanenldquoNitric oxide production and signaling in inflammationrdquo Cur-rent Drug TargetsmdashInflammation and Allergy vol 4 no 4 pp471ndash479 2005
[5] E Karpuzoglu and S A Ahmed ldquoEstrogen regulation of nitricoxide and inducible nitric oxide synthase (iNOS) in immunecells implications for immunity autoimmune diseases andapoptosisrdquo Nitric Oxide vol 15 no 3 pp 177ndash186 2006
[6] S G Harris J Padilla L Koumas D Ray and R PPhipps ldquoProstaglandins as modulators of immunityrdquo Trends inImmunology vol 23 no 3 pp 144ndash150 2002
[7] I-N Hsieh A S-Y Chang C-M Teng C-C Chen andC-R Yang ldquoAciculatin inhibits lipopolysaccharide-mediated
10 Evidence-Based Complementary and Alternative Medicine
inducible nitric oxide synthase and cyclooxygenase-2 expres-sion via suppressing NF-120581B and JNKp38 MAPK activationpathwaysrdquo Journal of Biomedical Science vol 18 no 1 article28 2011
[8] J B Calixto M F Otuki and A R S Santos ldquoAnti-inflammatory compounds of plant originmdashpart I action onarachidonic acid pathway nitric oxide and nuclear factor 120581 B(NF-120581B)rdquo Planta Medica vol 69 no 11 pp 973ndash983 2003
[9] K B Pandey and S I Rizvi ldquoCurrent understanding of dietarypolyphenols and their role in health and diseaserdquo CurrentNutrition and Food Science vol 5 no 4 pp 249ndash263 2009
[10] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer How are theylinkedrdquo Free Radical Biology and Medicine vol 49 no 11 pp1603ndash1616 2010
[11] P M Giang H V Bao and P T Son ldquoPhytochemical studyon Pseuderanthemum palatiferum (Nees) Radlk AcanthaceaerdquoJournal of Chemistry vol 41 no 2 pp 115ndash118 2003
[12] H K Dieu C B Loc S Yamasaki and Y Hirata ldquoThe ethnob-otanical and botanical study on Pseuderanthemum palatiferumas a new medicinal plant in the Mekong Delta of VietnamrdquoJapan Agricultural Research Quarterly vol 39 no 3 pp 191ndash1962005
[13] T Khumpook S Chomdej S Saenphet D Amornlerdpi-son and K Saenphet ldquoAnti-inflammatory activity of ethanolextract from the leaves of Pseuderanthemum palatiferum (Nees)RadlkrdquoChiangMai Journal of Science vol 40 no 3 pp 321ndash3312013
[14] Q-V Nguyen and J-B Eun ldquoAntioxidant activity of solventextracts from Vietnamese medicinal plantsrdquo Journal of Medic-inal Plant Research vol 5 no 13 pp 2798ndash2811 2011
[15] P M Giang H V Bao and P T Son ldquoStudy on anti-oxidativeactivities and preliminary investigation on antibacterial anti-fungal of extracted fraction rich in flavonoids from leaves ofPseuderanthemum palatiferum (Nees) Radlkrdquo TC Duoc Hocvol 9 no 9 pp 9ndash12 2005 (In Vietnamese with Englishsummary)
[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998
[17] M Liu X Q Li C Weber C Y Lee J Brown and R HLiu ldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002
[18] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[19] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoFreeradical scavenging capacity and inhibition of lipid oxidation ofwines grape juices and related polyphenolic constituentsrdquo FoodResearch International vol 32 no 6 pp 407ndash412 1999
[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958
[21] S-C Chun S Y Jee S G Lee S J Park J R Lee and SC Kim ldquoAnti-inflammatory activity of the methanol extractof Moutan Cortex in LPS-activated Raw2647 cellsrdquo Evidence-Based Complementary and Alternative Medicine vol 4 no 3pp 327ndash333 2007
[22] G-N Kim Y-I Kwon and H-D Jang ldquoProtective mech-anism of quercetin and rutin on 22rsquo-azobis(2-amidino-propane)dihydrochloride or Cu2+-induced oxidative stress inHepG2 cellsrdquoToxicology in Vitro vol 25 no 1 pp 138ndash144 2011
[23] M G B de Oliveira R B Marques M F de Santana et al ldquo120572-Terpineol reducesmechanical hypernociception and inflamma-tory responserdquoBasic ampClinical PharmacologyampToxicology vol111 no 2 pp 120ndash125 2012
[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[25] A K L Nascimento R FMelo-Silveira N Dantas-Santos et alldquoAntioxidant and antiproliferative activities of leaf extracts fromPlukenetia volubilis Linneo (Euphorbiaceae)rdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID950272 10 pages 2013
[26] A Muthuraman N Singh and A S Jaggi ldquoProtective effectof Acorus calamus L in rat model of vincristine inducedpainful neuropathy an evidence of anti-inflammatory and anti-oxidative activityrdquo Food and Chemical Toxicology vol 49 no 10pp 2557ndash2563 2011
[27] A Bunea DO Rugina AM Pintea Z Sconta C I Bunea andC Socaciu ldquoComparative polyphenolic content and antioxidantactivities of some wild and cultivated blueberries from Roma-niardquoNotulae Botanicae Horti Agrobotanici Cluj-Napoca vol 39no 2 pp 70ndash76 2011
[28] K E Heim A R Tagliaferro and D J Bobilya ldquoFlavonoidantioxidants chemistry metabolism and structure-activityrelationshipsrdquo Journal of Nutritional Biochemistry vol 13 no10 pp 572ndash584 2002
[29] J Perez-Jimenez S Arranz M Tabernero et al ldquoUpdatedmethodology to determine antioxidant capacity in plant foodsoils and beverages extraction measurement and expression ofresultsrdquo Food Research International vol 41 no 3 pp 274ndash2852008
[30] K L Wolfe and R H Lui ldquoCellular antioxidant activity(CAA) assay for assessing antioxidants foods and dietarysupplementsrdquo Journal of Agricultural and Food Chemistry vol55 no 22 pp 8896ndash8907 2007
[31] M Okawara H Katsuki E Kurimoto H Shibata T Kumeand A Akaike ldquoResveratrol protects dopaminergic neuronsin midbrain slice culture from multiple insultsrdquo BiochemicalPharmacology vol 73 no 4 pp 550ndash560 2007
[32] H Nishikawa KWakano and S Kitani ldquoInhibition of NADPHoxidase subunits translocation by tea catechin EGCG in mastcellrdquo Biochemical and Biophysical Research Communicationsvol 362 no 2 pp 504ndash509 2007
[33] J Fang T Seki and H Maeda ldquoTherapeutic strategies by mod-ulating oxygen stress in cancer and inflammationrdquo AdvancedDrug Delivery Reviews vol 61 no 4 pp 290ndash302 2009
[34] K Subbaramaiah and A J Dannenberg ldquoCyclooxygenase 2 amolecular target for cancer prevention and treatmentrdquo Trendsin Pharmacological Sciences vol 24 no 2 pp 96ndash102 2003
[35] Y-J Surh K-S ChunH-H Cha et al ldquoMolecularmechanismsunderlying chemopreventive activities of anti-inflammatoryphytochemicals down-regulation of COX-2 and iNOS throughsuppression of NF-120581B activationrdquoMutation Research vol 480-481 pp 243ndash268 2001
[36] W-W Chao Y-H Kuo and B-F Lin ldquoAnti-inflammatoryactivity of new compounds from Andrographis paniculata byNF-120581B transactivation inhibitionrdquo Journal of Agricultural andFood Chemistry vol 58 no 4 pp 2505ndash2512 2010
Evidence-Based Complementary and Alternative Medicine 11
[37] Y-C Liang Y-T Huang S-H Tsai S-Y Lin-Shiau C-FChen and J-K Lin ldquoSuppression of inducible cyclooxygenaseand inducible nitric oxide synthase by apigenin and relatedflavonoids in mouse macrophagesrdquo Carcinogenesis vol 20 no10 pp 1945ndash1952 1999
2 Evidence-Based Complementary and Alternative Medicine
Over the last decade medicinal plants as potentialsources of naturally occurring antioxidants have been thefocus of intense research Moreover phytochemicals suchas flavonoids and other polyphenolics with high reactiveoxygen species (ROS) scavenging activities have been shownto exhibit multiple biological effects including antiallergicantibacterial antidiabetic anticancer and anti-inflammatoryactivities [9] As oxidative stress and inflammation are closelylinked and are implicated in many diseases [10] plants thatpossess both antioxidant and anti-inflammatory propertieshave always attracted considerable research interest Pseuder-anthemum palatiferum (Nees) Radlk (PP) a member of theAcanthaceae plant family and commonly called Hoan-Ngocis one of the most popular medicinal plants in bothThailandand Vietnam Phytochemical analysis of PP leaf extracts sug-gests many high potential antioxidant and anti-inflammatoryconstituents [11] In fact PP has been referred to as a miracleplant in folkmedicine to cure or prevent variousmaladies andinflammatory related diseases such as diarrhea sore throathypertension gastric ulcer diabetes and cancer [12 13]Nevertheless the scientific evidence to support its multiplebiological effects is still limited particularly related to anti-inflammation Although the antioxidant property of PP leafextracts has been previously shown [14 15] its intracellularROS scavenging activity has never been assessed To datethere is only one study reporting the anti-inflammatory activ-ity of PP leaf extract [13] and the mechanism responsible forthe anti-inflammation remains largely unknown This studyfurther compares antioxidant activity between ethanol andwater extracts of PP leaves using various in vitro antioxidantevaluation methods including the assessment in the cell-based DCFH-DA assay The modulation of PP leaf extractsin NO production iNOS and COX-2 expression during theinflammatory response was also investigated in the murinemacrophage-like cell line RAW2647 stimulated with LPSplus IFN-120574
2 Materials and Methods
21 Chemicals and Materials 3-(45-Dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) (+)-catechinhydrate and vitamin C were purchased from Fluka ChemieGmbH (Buchs Switzerland) 22-Diphenyl-1-picryl-hydrazyl(DPPH) penicillin G streptomycin sulfate resveratrolN-(1-naphthyl)ethylenediamine dihydrochloride (NED)sodium nitrite LPS (Escherichia coli O111B4) 2101584071015840-dichlorofluorescin-diacetate (DCFH-DA) and tert-butylhydroperoxide (tBuOOH) were purchased from Sigma-Aldrich (St Louis MO USA) Dimethyl sulfoxide (DMSO)was purchased from Amresco Inc (Solon OH USA)Quercetin dihydrate was obtained from INDOFINEChemical Company Inc (Hillsborough NJ USA)6-Hydroxy-2578-tetramethylchroman-2-carboxylic acid(Trolox) was purchased from Sigma-Aldrich Chemie GmbH(Steinheim Germany) Mouse interferon gamma (mIFN-120574)and ECL Western blotting substrate were purchased fromPierce Protein Research Products (Rockford IL USA)RPMI medium 1640 Hankrsquos balanced salt solution (HBSS)
and penicillin-streptomycin were obtained from GibcoInvitrogen (Grand Island NY USA) Fetal bovine serum(FBS) was obtained from Hyclone (Logan UT USA)Anti-iNOS and anti-tubulin mouse monoclonal antibodiesand secondary antibody goat-anti-mouse-HRP conjugatefor iNOS and tubulin were purchased from Santa CruzBiotechnology Inc (Santa Cruz CA USA) Anti-COX-2mouse polyclonal antibody and secondary antibody goat-anti-rabbit IgG-HRP conjugate for COX-2 were purchasedfrom Cayman Chemical (Ann Arbor MI USA) The mousemacrophage cell line (RAW2647 cells) was purchased fromCell Lines Service (Eppelheim Germany) All other reagentswere purchased from Sigma-Aldrich unless otherwiseindicated
22 Plant Material Fresh leaves of PP were purchased fromproducers in Yasothon province Thailand The plant wasidentified and authenticated by Dr Kongkanda ChayamaritForest Herbarium Royal Forest Department BangkokThai-land A voucher specimen (BKF 174009) was deposited atthe Forest Herbarium Royal Forest Department BangkokThailand
23 Plant Extract Preparation Fresh leaves (15 kg) were cutinto small pieces and blended in 6 L of 95 ethanol Theextract was centrifuged at 3500 g for 10min at 4∘C and thesupernatant was filtered through Whatman number 1 filterpaper The ethanolic filtrate was then concentrated usinga vacuum rotary evaporator and lyophilized to obtain theethanol extract of PP (EEP 6041 g) Forty grams of EEPwere further partitioned between hexane and water (1 1)using a separatory funnel The water fraction was collectedcentrifuged at 14000 g for 10min at 4∘C evaporated andlyophilized to obtain a water extract of PP (WEP 3271 g)The EEP and WEP were stored at minus20∘C until they wereneeded in subsequent experiments The EEP and WEP weredissolved in DMSO and water respectively when used inexperiments For cell cultures the WEP was dissolved inphosphate buffered saline (PBS)
24 Total Phenolic Content The total phenolic content of theindividual extract was determined by the method of Folin-Ciocalteu [16] Briefly 100 120583L of test solution was added to2mL of 2 Na
2CO3and mixed thoroughly After 2min
100 120583L of 50 Folin-Ciocalteu reagent was added mixedand allowed to stand at room temperature (RT) for 30minThe absorbance of extracts was measured at 750 nm bya Cecil 1000 series spectrophotometer (Cecil InstrumentsCambridge UK) against a blank consisting of only reagentsand solvents without the extract Gallic acid solutions rangingfrom 005 to 03mgmL were used to prepare a standardcurve The concentration of phenolic compounds in theextracts is expressed as mg of gallic acid equivalent (GAE)per g of dry extract
25 Total Flavonoid Content The total flavonoid contentwas determined using a colorimetric method [17] Briefly250 120583L of sample was diluted with 125mL of distilled water
Evidence-Based Complementary and Alternative Medicine 3
(DI) Then 75 120583L of 5 NaNO2solution was added to the
mixture After 6min 150 120583L of a 10 AlCl3sdot6H2O solution
was added and the mixture was allowed to stand for another5min One half mL of 1M NaOH was added and the totalvolume was brought up to 25mL with DI waterThe solutionwas thoroughly mixed and the absorbance was measuredimmediately against the prepared blank at 510 nm Catechinstandard solutions (005ndash04mgmL) were used to preparea standard curve The concentration of flavonoids in theextracts is expressed as mg of catechin equivalent (CAE) perg of dry extract
26 FRAP (Ferric Reducing Antioxidant Power) Assay Theferric reducing ability of the extracts was measured colori-metrically according to the method developed by Benzieand Strain [18] The FRAP reagent consisted of 01M acetatebuffer (pH 36) 10mM 246-tris(2-pyridyl)-135-triazine(TPTZ) solution in 40mM HCl and 20mM FeCl
3sdot6H2O
solutionThe fresh working solution was prepared by mixingthe acetate buffer the TPTZ solution and the FeCl
3sdot6H2O
solution in a 10 1 1 vvv ratio The FRAP reagent (3mL)was added to 01mL of the extract and mixed Readingswere recorded on the spectrophotometer at 593 nm andthe reaction was monitored for 10min A standard curveof 100ndash1000120583mol FeSO
4sdot7H2O was prepared Vitamin C
(10ndash90 120583gmL) Trolox (10ndash160 120583gmL) and catechin (10ndash90 120583gmL) were used as standard antioxidants The antioxi-dant power of the extracts is expressed as mmol ferrous ion(Fe2+) per g of dry extract and alsomgof vitaminC equivalent(VCE) Trolox equivalent (TRE) and catechin equivalent(CAE) per g of dry extract
27 DPPH Assay The scavenging activity of DPPH radicalswas determined as described by Sanchez-Moreno et al [19]Briefly 100 120583L of extract at different concentrations wasadded to 39mL of methanolic DPPH solution (63mM)The mixture was shaken vigorously and left to stand at RTfor 45min in the dark Samples that are able to scavengeDPPH free radicals reduce the purple DPPH radicals intothe light yellow colored product of corresponding hydrazineDPPH
2 Decreasing DPPH solution absorption (measured
spectrophotometrically at 515 nm) indicates an increase ofDPPH radical scavenging activity [20] DPPH solution plusmethanol were used as negative control and vitamin CTrolox and catechin were used as positive controls The per-cent inhibition of DPPH radicals by test samples was deter-mined by comparison with the methanol-treated controlThe free radical scavenging activity which is the percentageinhibition of free radical is calculated as follows
DPPH radical scavenging activity ()
= [119860control minus 119860 sample
119860control] times 100
(1)
where 119860 sample and 119860control are absorbances of the sampleand the control respectively The IC
50of DPPH radicals was
determined from a dose response of inhibitory curve usinglinear regression analysis
28 Cell Culture The RAW2647 macrophage cells werecultured at 37∘C 5 CO
2in an RPMI-1640 medium supple-
mented with 10 heat-inactivated FBS 100UmL penicillinand 100 120583gmL streptomycin Exponentially growing cellswere used for experiments when they reached about 80confluence
29 Cell Viability (MTT Assay) A tetrazolium dye (MTT)colorimetric assay was used to determine the viability ofRAW2647 cells as described by Chun et al [21] Briefly RAW2647 cells were plated at a density of 5 times 104 cellswell in a 96-well plate and incubated overnight at 37∘C under 5 CO
2
After incubation the cells were exposed to various concen-trations of EEP or WEP for 24 h Then MTT (05mgmL)dye solution was added in each well and further incubatedat 37∘C 5 CO
2for 4 h The media was removed and DMSO
was added to each well to dissolve formazan crystals givinga uniform dark purple color before reading at 540 nm by theBenchmark PlusMicroplate Spectrophotometer System (Bio-Rad Laboratories Inc Hercules CA USA) The percentageof cell viability was calculated by the following equation
Percent cell viability =ODtest group
ODcontrol grouptimes 100 (2)
210 Assessment of Intracellular ROS Scavenging ActivityIntracellular oxidative stresswas detected usingDCFH-DAasdescribed by Kim et al [22] with slight modification BrieflyRAW2647 cells (4 times 104 cellswell) were plated in a Costa96-well black clear bottom plate (Corning Inc Corning NYUSA) and incubated for 16ndash18 h at 37∘C and 5 CO
2 After
incubation the cells were washed with PBS twice To assessantioxidant activity the cells were preexposed to differentconcentrations of EEP WEP (50 150 or 250120583gmL) or theantioxidant positive controls catechin (250 120583M) resveratrol(20120583M) or quercetin (10 120583M) for 24 h After washing twicewith PBS the cells were exposed to 20120583M DCFH-DA inHBSS and further incubated in the dark for another 30minThe DCFH-DA was removed by washing the cells with PBStwo times Then 500120583M tBuOOH was added The unstimu-lated DCFH-DA (no tBuOOH) in the unexposed RAW2647cells served as the naive control (NA) The intensity of thefluorescence signal was detected time dependently with anexcitation wavelength of 485 nm and an emission wavelengthof 535 nm using a Gemini EM fluorescence microplate reader(Molecular Devices Sunnyvale CA USA)
211 Nitrite Assay The level of NO in the culture mediawas detected as nitrite a major stable product of NOusing Griess reagent [23] RAW2647 cells were seeded ata density of 2 times 105 cellswell in a 96-well plate The cellswere grown for 3 h to allow plate attachment prior treatingwith the antioxidant positive control vitamin C (500120583M)or various concentrations (50 100 150 200 or 250 120583gmL)of EEP or WEP After 1 h incubation the RAW2647 cellswere stimulated with 1 120583gmL LPS plus 25UmL IFN-120574 Theactivated cells were further incubated for 24 h Then 100 120583Lof supernatant was mixed with an equal volume of Griess
4 Evidence-Based Complementary and Alternative Medicine
Table 1 The percentage of recovery of crude extracts from fresh leaves of PP
Extracts Amount and source of preparation Yield (g) Percentage of recoveryEEP 1500 g of fresh leaves 6041 403 (from fresh leaves)WEP 40 g of EEP 3271 8177 (from EEP)
(99321 g of fresh leaves) 329 (from fresh leaves)
Table 2 Total phenolic and flavonoid contents and total antioxidant (FRAP) activity of EEP and WEP
Values are mean plusmn SEM (119899 = 3) and are representative of three independent experiments with similar results Different letters within the same column aresignificantly different at P lt 005 as determined by a Studentrsquos t-test
reagent (1 sulfanilamide 01 NED and 3 phosphoricacid) After 10min of incubation in the dark the absorbanceof samples was measured at 540 nm using a Microplate Spec-trophotometer System (Bio-Rad Laboratories Inc) A freshculturemediumwas used as the blank in all experimentsTheamount of nitrite in the samples was derived from a standardcurve of sodium nitrite
212 Western Blot Analysis RAW2647 cells were plated ata density of 2 times 106 cellswell in a 6-well plate After anattachment period of approximately 3 h the cells were treatedwith various concentrations (50 100 150 200 or 250120583gmL)of EEP orWEP for 1 h 50 120583gmL Trolox or 500120583Mvitamin Cwas used as antioxidant positive controls The cells were thenstimulated with 1 120583gmL LPS plus 25UmL IFN-120574 for 18 hAfter incubation the cells were washed three times with PBSand placed in 150 120583L of ice-cold lysis buffer (1mL RIPA buffersupplemented with 2mM PMSF 2 120583M leupeptin and 1 120583ME-64) for 20minThen the disrupted cells were transferred tomicrocentrifuge tubes and centrifuged at 14000 g at 4∘C for30min The supernatant was collected and the protein con-centration of cell lysate was estimated by the Lowry method[24] Cell lysate was then boiled for 5min in a 6X samplebuffer (50mM Tris-base pH 74 4 SDS 10 glycerol 42-mercaptoethanol and 005mgmL of bromophenol blue)Thirty micrograms of cellular proteins were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) using 75 and 10 polyacrylamide gels foriNOS and COX-2 respectively (125 volts 120min) Theproteins in the gel were transferred onto a nitrocellulosemembrane (Amersham Pittsburgh PA USA) at 80 volts for1 h The membrane was blocked overnight at 4∘C with 5nonfat milk in 01 Tween 20 in a PBS buffer (TPBS) Themembranes were then incubated with a 1 1000 dilution ofthe primary antibody anti-iNOS mouse monoclonal or a1 2000 dilution of the primary antibody anti-COX-2 mousepolyclonal at RT for 2 h After extensive washing with TPBSthe membranes were incubated with a 1 10000 dilution ofthe secondary antibody goat-anti-mouse-HRP conjugate foriNOS and goat-anti-rabbit IgG-HRP conjugate for COX-2 at RT for 1 h To control equal loading of total protein
in all lanes blots were also stained with primary antibodyanti-tubulin mouse monoclonal at a dilution of 1 2000 atRT for 2 h After washing the membranes were incubatedwith a 1 10000 dilution of the secondary antibody goat-anti-mouse-HRP conjugate The membranes were washedthree times for 10min each time with TPBS The blotswere incubated for 3min in ECL Western blotting substrateand exposed to film The relative expression of proteins wasquantified densitometrically using the software imageJ andcalculated according to the reference band of tubulin
213 Statistical Analysis All statistical analyses were con-ducted using GraphPad software (GraphPad Prism 5 USA)The data from the total phenolic and flavonoid contents aswell as FRAP value results were analyzed by a Studentrsquos 119905-test to determine the statistical significance between twogroups DPPH MTT and nitrite assays were analyzed byone-way analysis of variance (ANOVA) with a post hocTukeyrsquos analysis to determine differences between treatmentand control groups [25] The data from intracellular ROSscavenging were analyzed by two-way ANOVA followed byBonfferonnirsquos post hoc test [26]
3 Results
31 The Percentage of Recovery of Crude Extracts from FreshLeaves of PP The percentages of recovery of crude extractsfrom fresh leaves of PP are shown in Table 1 EEP exhibited apercentage of recovery of 403 while WEP had percentageof recovery of 329 based on the original weight of freshleaves WEP was prepared from the water fraction of EEPthat was partitioned with hexane and water (1 1 vv) with apercentage of recovery of 8177 based on EEP
32 Phenolic and Flavonoid Contents WEP had a signifi-cantly higher level (119875 lt 005) of total phenolic and flavonoidcontent than that of EEP (Table 2) and more than half of thephenolics in WEP and EEP are flavonoids
33 Ferric Reducing Antioxidant Power EEP and WEP wereanalyzed for their reducing ability along with three standard
Evidence-Based Complementary and Alternative Medicine 5
80
100
60
40
20
0
0 5 10 15 20 25 30 35
Inhi
bitio
n (
)
Concentration (120583gmL)
WEPEEPCatechin
Vitamin CTrolox
Figure 1 DPPH radical scavenging activity of PP leaf extracts (EEPand WEP) and positive controls (vitamin C Trolox and catechin)Values are means plusmn SEM (119899 = 3) and are representative of threeindependent experiments with similar results
antioxidants vitamin C Trolox and catechin The results ofFRAP values in terms of ferrous ion (Fe2+) and vitamin CTrolox and catechin equivalents are shown in Table 2 EEPexhibited a higher degree of electron donating capacity thanWEP as suggested by the significantly higher FRAP values(119875 lt 005) of EEP when compared with WEP
34 DPPH Free Radical Scavenging Activity The free radicalscavenging capacities of EEP andWEP are shown in Figure 1The results show that both EEP and WEP exhibit the abilityto scavenge DPPH free radicals The scavenging activityagainst DPPH radicals of WEP (IC
50= 2155 plusmn 006 120583gmL)
is significantly greater (119875 lt 0001) than EEP (IC50
=2345 plusmn 012120583gmL) by 19 plusmn 015 but the scavengingcapacity of these is not as effective as the other positiveantioxidant controls 125 120583gmL EEP and 25 120583gmL WEPscavenged the DPPH radicals by 2927 plusmn 020 and 612 plusmn015 respectively and the scavenging capacities of bothare more pronounced at higher concentrations The highestconcentration (325120583gmL) of EEP andWEP could scavengethe DPPH radicals by 6596 plusmn 021 and 7319 plusmn 009respectively In the present study the scavenging abilities ofvitamin C (IC
50= 394 plusmn 001 120583gmL) and catechin (IC
50=
355 plusmn 001 120583gmL) were similar and both are significantlyhigher (119875 lt 0001) than Trolox (IC
50= 590 plusmn 027 120583gmL)
35 Effect of EEP and WEP on RAW2647 Cell Viability Thecell viability of RAW2647 cells exposed to EEP or WEP wasdetermined by MTT assay The cells were incubated for 24 hwith various concentrations of EEP (005 025 05 10 or150mgmL) or WEP (010 050 150 or 450mgmL) Asshown in Figure 2 both EEP andWEP displayed low toxicitytowards RAW2647 cells as evidenced by an apparent lack ofeffect on cell viability until the concentration of each extract
reached 15mgmL At 15mgmL EEP and WEP decreasedthe viability of RAW2647 cells by 3414 plusmn 969 and 2158 plusmn166 (119875 lt 005) respectively However the cytotoxiceffect is more pronounced at higher concentrations WEP at45mgmL decreased the cell viability by as much as 5421 plusmn174 (119875 lt 005) The effect of EEP and WEP on RAW2647cell viability was also confirmed by trypan blue exclusion andpropidium iodide staining methods which exhibited similarresults (data not shown)Therefore a nontoxic concentrationrange of 0ndash025mgmL of both EEP and WEP was selectedfor RAW2647 cell treatment in the subsequent studies
36 EEP and WEP as Intracellular ROS Scavengers Thedirect scavenging effect of EEP and WEP on intracellularfree radical stress was investigated in RAW2647 cells usingthe DCFH-DA assay The increment of DCF fluorescenceemission following ROS-mediated oxidation of DCFH wasfollowed for 240min As shown in Figure 3(a) standardantioxidant positive controls catechin (250 120583M) resveratrol(20120583M) and quercetin (10 120583M) could scavenge ROS signif-icantly (119875 lt 005) throughout the incubation time whencompared to the vehicle control (VH)With as little as 30minof incubation catechin resveratrol and quercetin showedconsiderable radical scavenging activity EEP (Figure 3(b))and WEP (Figure 3(c)) decreased the DCF fluorescent emis-sion in a dose- and time-dependent manner Again with aslittle as 30min of incubation both EEP and WEP at lowconcentration (50120583gmL) showed similar radical scavengingactivity as the antioxidant controls Various concentrations ofEEP significantly decreased (119875 lt 005) the DCF fluorescentemission throughout the incubation time when comparedto the VH control At high concentration (150 120583gmL) EEPexhibited a strong scavenging activity as suggested by thecapability to lower fluorescent intensity to below basal levelof the unstimulated DCFH-DA control at 180ndash240min Inaddition the highest concentration of EEP (250120583gmL)significantly lowered (119875 lt 005) DCF fluorescent intensityto below the basal level at all time points Similarly 150 and250 120583gmL of WEP also significantly decreased (119875 lt 005)the DCF fluorescent emission throughout the incubationtime compared to the tBuOOH control However the lowestconcentration ofWEP (50120583gmL) significantly reduced (119875 lt005) the DCF fluorescent emission until 210min only
37 NO Suppression by EEP and WEP in LPS Plus IFN-120574-Activated RAW2647 Cells RAW2647 cells were pretreatedwith antioxidants vitamin C EEP or WEP for 1 h thenstimulated with LPS plus IFN-120574 and measured for NOproduction using the Griess assay As shown in Figure 4unstimulated RAW2647 cells (NA) secreted basal levels ofNO while the production of NO was increased to about43 120583M in LPS plus IFN-120574-activated RAW2647 cells Theantioxidant control 500 120583M vitamin C decreased the NOproduction by almost 35 Pretreatment of RAW2647 cellswith EEP or WEP significantly suppressed (119875 lt 005) theinduction of NO in a dose-related manner (Figures 4(a) and4(b)) and the suppression was observed in all EEP- andWEP-treated groups These results also clearly indicate that
6 Evidence-Based Complementary and Alternative Medicine
80
60
40
20
0
b b b b
aab
Cel
l via
bilit
y (
)120
100
Control 005 025 050 150100
EEP (mgmL)
(a)
80
60
40
20
0
b
a
ccd
d
Cel
l via
bilit
y (
)
120
100
Control 010 050 150 450
WEP (mgmL)
(b)
Figure 2 Effect of EEP andWEP on cell viability of RAW2647 cellsThe effect of EEP (a) andWEP (b) on cell viability was assessed byMTTValues are expressed as means plusmn SEM (119899 = 3) and are representative of three independent experiments with similar results Bars marked withdifferent letters are significantly different at 119875 lt 005 as determined by one-way ANOVA
EEP is a stronger suppressant of NO induction than WEPConcentrations of 50 120583gmL of EEP and 150 and 200 120583gmLofWEPwere required to exhibit the NO suppression with thesame efficiency as 500 120583M (8806120583gmL) vitamin C
38 Suppression of iNOS and COX-2 Protein Expression byEEP and WEP in LPS Plus IFN-120574-Activated RAW2647 CellsTo determine if suppression of NO production by EEP orWEPwas related to changes in iNOS aswell as COX-2 proteinlevels Western blotting analysis was performed RAW2647cells were pretreated with antioxidants Trolox (50120583gmL)vitamin C (500120583M) or PP extracts (EEP or WEP) at 50ndash250120583gmL for 1 h prior activation with LPS (1120583gmL) plusIFN-120574 (25UmL) for 18 h Total proteins were extracted andanalyzed for the expression of iNOS and COX-2 by Westernblotting LPS plus IFN-120574 induced increases in iNOS (Figures5(a) and 5(b)) and COX-2 (Figures 5(c) and 5(d)) expressioncompared to the unstimulated cultures Antioxidant controls(Trolox and vitamin C) decreased LPS plus IFN-120574-inducediNOS and COX-2 protein levels The data also suggestedthat the suppression by 500120583M (8806120583gmL) vitamin Cis more pronounced than 50 120583gmL Trolox Compared tothe corresponding controls both EEP and WEP produced adose-dependent suppression of iNOS level in LPS plus IFN-120574-activated RAW2647 cells (Figures 5(a) and 5(b)) suggestingthat the suppression of NO production by EEP and WEP ismediated by decreasing the expression of iNOS In agreementwith the result of NO suppression 50ndash200 120583gmL EEP wasprobably more efficient than WEP in iNOS suppressionThe iNOS expression was almost completely eliminated at200120583gmL EEP and was barely observed at 250 120583gmLWEPThe inflammatory modulation of EEP and WEP was alsofurther supported by the dose-dependent suppression of theCOX-2 level by both EEP and WEP (Figures 5(c) and 5(d))in the activated RAW2647 cells Notably EEP and WEPexhibited higher suppression of iNOS than COX-2
4 Discussion
It is well known that major phytochemicals of plant leafextracts possessing antioxidant activity are flavonoids andother phenolic compounds Researchers have found thatflavonoids from PP leaves display antioxidant activity andall ethyl acetate chloroform and 119899-butanol-soluble fractionsof PP contain flavonoids [11 15] In addition Nguyen andEun [14] found phenolics and flavonoids in extracts of PPleaves when assessed with Folin-Ciocalteu and aluminumtrichloride PP leaf extracts also have antioxidant activitieswhen evaluated with DPPH and FRAP assays Similarlythe present study also showed that both EEP and WEPcontain high levels of flavonoids and phenolics and exhibitantioxidant activity The most frequently used antioxidantstandards for food samples (vitamin C Trolox catechinresveratrol and quercetin) were used as positive antioxidantcontrols in the present study
This study revealed that DPPH radical scavenging capac-ity of WEP is greater than that of EEP (Figure 1) In contrastEEP has higher ferric reducing power than WEP (Table 2)Such contradictory results between DPPH and FRAP assaysare not unusual Though both assays are based on a singleelectron transfer reaction [27] their characteristics sensitiv-ities mechanisms of the reaction and endpoints are totallydifferent For instance the DPPH method is based on thefree radical scavenging activity while FRAP measures thecapability of reducing Fe3+ to Fe2+ Depending on whatspecific phytochemical constituents present in the extractare providing the antioxidant activity their discrete chemicalstructures positions numbers and types of substitutions caninfluence their redox properties and hence their antioxidantpotentials [28]
Though both DPPH and FRAP assays are frequently usedfor assessing antioxidant capacity they have some drawbacksIn the DPPH assay interfering compounds may have sig-nificant absorption at the same measured wavelength In
Evidence-Based Complementary and Alternative Medicine 7
Figure 3 Cellular radical scavenging activity in tBuOOH-activated RAW2647 cells Intracellular ROS level generated in cells was measuredby the DCFH-DA RAW2647 cells were pretreated with indicated concentrations of antioxidants (a) EEP (b) or WEP (c) for 24 h priorto take-up of 20 120583M DCFH-DA for 30min Results are mean plusmn SEM (119899 = 4) and are representative of three independent experiments withsimilar results Pointsmarked with different letters are significantly different at119875 lt 005when compared at the same time point as determinedby two-way ANOVA
addition theDPPH radical is not present in living organismsFor the FRAPmethod compounds with low redox potentialwhich probably do not serve as antioxidants in vivo still canreduce the Fe3+ Interfering compounds may also absorb atthe same wavelength and the assay is also performed at anonphysiological pH [29] Therefore antioxidant activitiesof EEP and WEP were also evaluated by the cell-basedassay using an intracellular fluorescent probe DCFH-DAWhen the nonfluorescent DCFH-DA is taken up into cellsits diacetate moiety will be hydrolyzed by cellular esterasesto generate the more polar DCFH which is trapped insidethe cells In the presence of ROS intracellular DCFH isfurther oxidized to form the fluorescent DCF product [30]
The macrophage cell line RAW2647 is usually the cell ofchoice in studying ROS-mediated cellular events since itcan generate high amounts of ROS following an oxidantchallenge Catechin resveratrol and quercetin at the levelof concentration used in this study have been shown andoptimized to exhibit a strong suppression of intracellular ROSgeneration [22 31 32] Therefore the present study selectedthese compounds as antioxidant positive controls for theDCFH-DA assay The present study demonstrated that allantioxidant standards 250120583M catechin 20120583M resveratroland 10 120583M quercetin exerted a strong inhibition of ROSgeneration induced by tBuOOH over a period of 30 to240min In addition to extracellular antioxidant capacity
8 Evidence-Based Complementary and Alternative Medicine
45
40
35
30
25
20
15
10
5
0
NA
VH 50
150
250
e
d
c
bb
a
e
d
bcNitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
EEP (120583gmL)
(a)
b
e
cc
bc
d
f
a
45
40
35
30
25
20
15
10
5
0
NA 50
150
250
Nitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
WEP (120583gmL)
(b)
Figure 4 EEP (a) and WEP (b) suppressed LPS plus IFN-120574-induced nitrite production in RAW2647 cells RAW2647 cells were incubatedfor 24 hwith LPS (1120583gmL) plus IFN-120574 (25UmL) in the presence or absence of indicated concentrations of vitaminC (500120583M) EEP orWEPAccumulated nitrite in the culturemediumwas determined by the Griess reactionThe values aremeansplusmn SEM (119899 = 3) and are representativeof three independent experiments with similar results Bars marked with different letters are significantly different at 119875 lt 005 as determinedby one-way ANOVA
EEP and WEP also served as intracellular ROS scavengersand subsequently decreased the oxidation of DCFH (Figures3(b) and 3(c)) Both EEP and WEP were as efficient as theantioxidant standards in scavenging ROS Notably EEP was abetter reducer of DCF fluorescence thanWEPThe reductionof DCF fluorescence by EEP and WEP is not due to directcytotoxicity as the range of concentration used in the studieshad no effect on RAW2647 cell viability (Figure 2)
Although the current study shows that an ethanol extractfrom PP leaves has in vivo anti-inflammatory activities[13] its mechanism of anti-inflammation is still unrevealedInflammatory disorders are characterized among otherevents by the production of significant amounts of freeradicals nitrogen reactive species and pro-inflammatorycytokines [10] High NO concentration combines with super-oxides to form peroxynitrite ions (OONOminus) which areresponsible for cell and tissue damage from inflammation[33] Therefore we investigated inflammatory effects of EEPand WEP on the suppression of NO production in LPS plusIFN-120574-activated RAW2647 cells At the concentration rangeof 50ndash250120583gmL both EEP and WEP dose-dependentlysuppressed NO production and the suppression was morepronounced in EEP than WEP (Figure 4) These resultsagreed with the observation that EEP was also a betterscavenger of intracellular ROS than WEP (Figures 3(b) and3(c))
As enhanced NO production by LPS and IFN-120574-stimulated RAW2647 cells mainly occurs via increasedintracellular content of iNOS [3 4] the effect of EEP andWEP on iNOS expression was investigatedThe present studyclearly indicates that the suppressive effect of EEP and WEPon NO production was mediated through the inhibition of
iNOS expression (Figures 5(a) and 5(b)) In agreement withthe study of NO suppression the suppressive effect of EEP(50ndash200120583gmL) on iNOS was more remarkable than that ofWEP
In addition to iNOS induction LPS and IFN-120574 alsoefficiently enhance COX-2 expression in RAW2647 cells [35] An increased level of COX-2 expression is also knownto account for the excessive production of PGE
2in most
if not all inflammatory cells and tissues [34] This studyshows that both EEP andWEP can exhibit anti-inflammatoryactivity by reducing high COX-2 protein levels in a dose-related manner (Figures 5(c) and 5(d)) Thus EEP and WEPmight play important roles in attenuating inflammationand cellular damage through their extra- and intracellularROS scavenging activity and downregulation of NO iNOSand COX-2 Concordantly Khumpook et al [13] recentlyreported the in vivo anti-inflammatory activity of PP leavesas evidenced by decreased lipid peroxidation and NO levelin concomitance with increased superoxide dismutase in thecotton-induced chronic inflammation in Albino rats uponexposure to an ethanol extract of PP leaves for 17 days
In fact several medicinal plant extracts with naturalantioxidant properties together with suppressive effects onNO iNOS andorCOX-2 expression inRAW2647 have beenreported to display a wide spectrum of bioactivities Theseactivities include anti-inflammation such as curcumin fromCurcuma longa resveratrol from grape skins red wines andother plants and a mixture of 120573-sitosterol and stigmasterolfrom Andrographis paniculata [8 35 36] Previous investi-gators demonstrated that pretreatment of RAW2647 withflavonoids such as apigenin genistein and kaempferol sup-pressed LPS-stimulated expression of NO iNOS and COX-2
Evidence-Based Complementary and Alternative Medicine 9
FoldiNOS
Tubulin
003 086 058 030 061 038 033 024 015
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
EEP (120583gmL)
LPSIFN-120574
(a)
Fold
Tubulin
iNOS
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
WEP (120583gmL)
LPSIFN-120574
008 111 083 052 073 063 048 036 008
(b)
COX-2
Fold
Tubulin
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
006 093 076 074 087 079 065 044 030
EEP (120583gmL)
LPSIFN-120574
(c)
Fold
Tubulin
COX-2
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
001 113 066 063 091 078 067 064 050
WEP (120583gmL)
LPSIFN-120574
(d)
Figure 5 Effect of EEP on LPS plus IFN-120574-induced iNOS (a) and COX-2 (c) andWEP on LPS plus IFN-120574-induced iNOS (b) and COX-2 (d)protein levels in RAW2647 cells The relative expression of proteins was quantified densitometrically using ImageJ software and normalizedto tubulin reference bands Data are representative of at least two independent experiments
protein production [37] Major chemical constituents of PPleaves consist of 120573-sitosterol stigmasterol kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucoside[11] All aforementioned compounds have been shown topossess anti-inflammatory properties Both kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucosidemay be metabolized into kaempferol and apigenin whichalso have antioxidant and anti-inflammatory activities Thusit is possible that phenolic and flavonoid compounds inboth EEP andWEP provide substantial antioxidant and anti-inflammatory activities
In summary the cytoprotective effects of EEP and WEPis due to their abilities to decrease ROS generation and NOradical production in cells In addition both EEP and WEPexert anti-inflammatory effects through the suppression ofNO release and decrease the protein expression of iNOSand COX-2 Thus PP leaves possess high potential forfurther exploration in the research development of anti-inflammatory medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was financially supported by a Royal GoldenJubilee Scholarship Grant (PhD02082548) and by the
National Research Council of Thailand (NRCT) throughSuranaree University of Technology (SUT-104-53-36-09)
References
[1] B Halliwell and J M C Gutteridge Free Radicals in BiologyandMedicine OxfordUniversity Press NewYork NYUSA 3rdedition 1999
[2] N Fujiwara and K Kobayashi ldquoMacrophages in inflammationrdquoCurrent Drug TargetsmdashInflammation and Allergy vol 4 no 3pp 281ndash286 2005
[3] S I Jang Y-J Kim W-Y Lee et al ldquoScoparone from Artemisiacapillaris inhibits the release of inflammatory mediators inRAW 2647 cells upon stimulation cells by interferon-120574 plusLPSrdquo Archives of Pharmacal Research vol 28 no 2 pp 203ndash208 2005
[4] R Korhonen A Lahti H Kankaanranta and E MoilanenldquoNitric oxide production and signaling in inflammationrdquo Cur-rent Drug TargetsmdashInflammation and Allergy vol 4 no 4 pp471ndash479 2005
[5] E Karpuzoglu and S A Ahmed ldquoEstrogen regulation of nitricoxide and inducible nitric oxide synthase (iNOS) in immunecells implications for immunity autoimmune diseases andapoptosisrdquo Nitric Oxide vol 15 no 3 pp 177ndash186 2006
[6] S G Harris J Padilla L Koumas D Ray and R PPhipps ldquoProstaglandins as modulators of immunityrdquo Trends inImmunology vol 23 no 3 pp 144ndash150 2002
[7] I-N Hsieh A S-Y Chang C-M Teng C-C Chen andC-R Yang ldquoAciculatin inhibits lipopolysaccharide-mediated
10 Evidence-Based Complementary and Alternative Medicine
inducible nitric oxide synthase and cyclooxygenase-2 expres-sion via suppressing NF-120581B and JNKp38 MAPK activationpathwaysrdquo Journal of Biomedical Science vol 18 no 1 article28 2011
[8] J B Calixto M F Otuki and A R S Santos ldquoAnti-inflammatory compounds of plant originmdashpart I action onarachidonic acid pathway nitric oxide and nuclear factor 120581 B(NF-120581B)rdquo Planta Medica vol 69 no 11 pp 973ndash983 2003
[9] K B Pandey and S I Rizvi ldquoCurrent understanding of dietarypolyphenols and their role in health and diseaserdquo CurrentNutrition and Food Science vol 5 no 4 pp 249ndash263 2009
[10] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer How are theylinkedrdquo Free Radical Biology and Medicine vol 49 no 11 pp1603ndash1616 2010
[11] P M Giang H V Bao and P T Son ldquoPhytochemical studyon Pseuderanthemum palatiferum (Nees) Radlk AcanthaceaerdquoJournal of Chemistry vol 41 no 2 pp 115ndash118 2003
[12] H K Dieu C B Loc S Yamasaki and Y Hirata ldquoThe ethnob-otanical and botanical study on Pseuderanthemum palatiferumas a new medicinal plant in the Mekong Delta of VietnamrdquoJapan Agricultural Research Quarterly vol 39 no 3 pp 191ndash1962005
[13] T Khumpook S Chomdej S Saenphet D Amornlerdpi-son and K Saenphet ldquoAnti-inflammatory activity of ethanolextract from the leaves of Pseuderanthemum palatiferum (Nees)RadlkrdquoChiangMai Journal of Science vol 40 no 3 pp 321ndash3312013
[14] Q-V Nguyen and J-B Eun ldquoAntioxidant activity of solventextracts from Vietnamese medicinal plantsrdquo Journal of Medic-inal Plant Research vol 5 no 13 pp 2798ndash2811 2011
[15] P M Giang H V Bao and P T Son ldquoStudy on anti-oxidativeactivities and preliminary investigation on antibacterial anti-fungal of extracted fraction rich in flavonoids from leaves ofPseuderanthemum palatiferum (Nees) Radlkrdquo TC Duoc Hocvol 9 no 9 pp 9ndash12 2005 (In Vietnamese with Englishsummary)
[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998
[17] M Liu X Q Li C Weber C Y Lee J Brown and R HLiu ldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002
[18] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[19] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoFreeradical scavenging capacity and inhibition of lipid oxidation ofwines grape juices and related polyphenolic constituentsrdquo FoodResearch International vol 32 no 6 pp 407ndash412 1999
[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958
[21] S-C Chun S Y Jee S G Lee S J Park J R Lee and SC Kim ldquoAnti-inflammatory activity of the methanol extractof Moutan Cortex in LPS-activated Raw2647 cellsrdquo Evidence-Based Complementary and Alternative Medicine vol 4 no 3pp 327ndash333 2007
[22] G-N Kim Y-I Kwon and H-D Jang ldquoProtective mech-anism of quercetin and rutin on 22rsquo-azobis(2-amidino-propane)dihydrochloride or Cu2+-induced oxidative stress inHepG2 cellsrdquoToxicology in Vitro vol 25 no 1 pp 138ndash144 2011
[23] M G B de Oliveira R B Marques M F de Santana et al ldquo120572-Terpineol reducesmechanical hypernociception and inflamma-tory responserdquoBasic ampClinical PharmacologyampToxicology vol111 no 2 pp 120ndash125 2012
[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[25] A K L Nascimento R FMelo-Silveira N Dantas-Santos et alldquoAntioxidant and antiproliferative activities of leaf extracts fromPlukenetia volubilis Linneo (Euphorbiaceae)rdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID950272 10 pages 2013
[26] A Muthuraman N Singh and A S Jaggi ldquoProtective effectof Acorus calamus L in rat model of vincristine inducedpainful neuropathy an evidence of anti-inflammatory and anti-oxidative activityrdquo Food and Chemical Toxicology vol 49 no 10pp 2557ndash2563 2011
[27] A Bunea DO Rugina AM Pintea Z Sconta C I Bunea andC Socaciu ldquoComparative polyphenolic content and antioxidantactivities of some wild and cultivated blueberries from Roma-niardquoNotulae Botanicae Horti Agrobotanici Cluj-Napoca vol 39no 2 pp 70ndash76 2011
[28] K E Heim A R Tagliaferro and D J Bobilya ldquoFlavonoidantioxidants chemistry metabolism and structure-activityrelationshipsrdquo Journal of Nutritional Biochemistry vol 13 no10 pp 572ndash584 2002
[29] J Perez-Jimenez S Arranz M Tabernero et al ldquoUpdatedmethodology to determine antioxidant capacity in plant foodsoils and beverages extraction measurement and expression ofresultsrdquo Food Research International vol 41 no 3 pp 274ndash2852008
[30] K L Wolfe and R H Lui ldquoCellular antioxidant activity(CAA) assay for assessing antioxidants foods and dietarysupplementsrdquo Journal of Agricultural and Food Chemistry vol55 no 22 pp 8896ndash8907 2007
[31] M Okawara H Katsuki E Kurimoto H Shibata T Kumeand A Akaike ldquoResveratrol protects dopaminergic neuronsin midbrain slice culture from multiple insultsrdquo BiochemicalPharmacology vol 73 no 4 pp 550ndash560 2007
[32] H Nishikawa KWakano and S Kitani ldquoInhibition of NADPHoxidase subunits translocation by tea catechin EGCG in mastcellrdquo Biochemical and Biophysical Research Communicationsvol 362 no 2 pp 504ndash509 2007
[33] J Fang T Seki and H Maeda ldquoTherapeutic strategies by mod-ulating oxygen stress in cancer and inflammationrdquo AdvancedDrug Delivery Reviews vol 61 no 4 pp 290ndash302 2009
[34] K Subbaramaiah and A J Dannenberg ldquoCyclooxygenase 2 amolecular target for cancer prevention and treatmentrdquo Trendsin Pharmacological Sciences vol 24 no 2 pp 96ndash102 2003
[35] Y-J Surh K-S ChunH-H Cha et al ldquoMolecularmechanismsunderlying chemopreventive activities of anti-inflammatoryphytochemicals down-regulation of COX-2 and iNOS throughsuppression of NF-120581B activationrdquoMutation Research vol 480-481 pp 243ndash268 2001
[36] W-W Chao Y-H Kuo and B-F Lin ldquoAnti-inflammatoryactivity of new compounds from Andrographis paniculata byNF-120581B transactivation inhibitionrdquo Journal of Agricultural andFood Chemistry vol 58 no 4 pp 2505ndash2512 2010
Evidence-Based Complementary and Alternative Medicine 11
[37] Y-C Liang Y-T Huang S-H Tsai S-Y Lin-Shiau C-FChen and J-K Lin ldquoSuppression of inducible cyclooxygenaseand inducible nitric oxide synthase by apigenin and relatedflavonoids in mouse macrophagesrdquo Carcinogenesis vol 20 no10 pp 1945ndash1952 1999
Evidence-Based Complementary and Alternative Medicine 3
(DI) Then 75 120583L of 5 NaNO2solution was added to the
mixture After 6min 150 120583L of a 10 AlCl3sdot6H2O solution
was added and the mixture was allowed to stand for another5min One half mL of 1M NaOH was added and the totalvolume was brought up to 25mL with DI waterThe solutionwas thoroughly mixed and the absorbance was measuredimmediately against the prepared blank at 510 nm Catechinstandard solutions (005ndash04mgmL) were used to preparea standard curve The concentration of flavonoids in theextracts is expressed as mg of catechin equivalent (CAE) perg of dry extract
26 FRAP (Ferric Reducing Antioxidant Power) Assay Theferric reducing ability of the extracts was measured colori-metrically according to the method developed by Benzieand Strain [18] The FRAP reagent consisted of 01M acetatebuffer (pH 36) 10mM 246-tris(2-pyridyl)-135-triazine(TPTZ) solution in 40mM HCl and 20mM FeCl
3sdot6H2O
solutionThe fresh working solution was prepared by mixingthe acetate buffer the TPTZ solution and the FeCl
3sdot6H2O
solution in a 10 1 1 vvv ratio The FRAP reagent (3mL)was added to 01mL of the extract and mixed Readingswere recorded on the spectrophotometer at 593 nm andthe reaction was monitored for 10min A standard curveof 100ndash1000120583mol FeSO
4sdot7H2O was prepared Vitamin C
(10ndash90 120583gmL) Trolox (10ndash160 120583gmL) and catechin (10ndash90 120583gmL) were used as standard antioxidants The antioxi-dant power of the extracts is expressed as mmol ferrous ion(Fe2+) per g of dry extract and alsomgof vitaminC equivalent(VCE) Trolox equivalent (TRE) and catechin equivalent(CAE) per g of dry extract
27 DPPH Assay The scavenging activity of DPPH radicalswas determined as described by Sanchez-Moreno et al [19]Briefly 100 120583L of extract at different concentrations wasadded to 39mL of methanolic DPPH solution (63mM)The mixture was shaken vigorously and left to stand at RTfor 45min in the dark Samples that are able to scavengeDPPH free radicals reduce the purple DPPH radicals intothe light yellow colored product of corresponding hydrazineDPPH
2 Decreasing DPPH solution absorption (measured
spectrophotometrically at 515 nm) indicates an increase ofDPPH radical scavenging activity [20] DPPH solution plusmethanol were used as negative control and vitamin CTrolox and catechin were used as positive controls The per-cent inhibition of DPPH radicals by test samples was deter-mined by comparison with the methanol-treated controlThe free radical scavenging activity which is the percentageinhibition of free radical is calculated as follows
DPPH radical scavenging activity ()
= [119860control minus 119860 sample
119860control] times 100
(1)
where 119860 sample and 119860control are absorbances of the sampleand the control respectively The IC
50of DPPH radicals was
determined from a dose response of inhibitory curve usinglinear regression analysis
28 Cell Culture The RAW2647 macrophage cells werecultured at 37∘C 5 CO
2in an RPMI-1640 medium supple-
mented with 10 heat-inactivated FBS 100UmL penicillinand 100 120583gmL streptomycin Exponentially growing cellswere used for experiments when they reached about 80confluence
29 Cell Viability (MTT Assay) A tetrazolium dye (MTT)colorimetric assay was used to determine the viability ofRAW2647 cells as described by Chun et al [21] Briefly RAW2647 cells were plated at a density of 5 times 104 cellswell in a 96-well plate and incubated overnight at 37∘C under 5 CO
2
After incubation the cells were exposed to various concen-trations of EEP or WEP for 24 h Then MTT (05mgmL)dye solution was added in each well and further incubatedat 37∘C 5 CO
2for 4 h The media was removed and DMSO
was added to each well to dissolve formazan crystals givinga uniform dark purple color before reading at 540 nm by theBenchmark PlusMicroplate Spectrophotometer System (Bio-Rad Laboratories Inc Hercules CA USA) The percentageof cell viability was calculated by the following equation
Percent cell viability =ODtest group
ODcontrol grouptimes 100 (2)
210 Assessment of Intracellular ROS Scavenging ActivityIntracellular oxidative stresswas detected usingDCFH-DAasdescribed by Kim et al [22] with slight modification BrieflyRAW2647 cells (4 times 104 cellswell) were plated in a Costa96-well black clear bottom plate (Corning Inc Corning NYUSA) and incubated for 16ndash18 h at 37∘C and 5 CO
2 After
incubation the cells were washed with PBS twice To assessantioxidant activity the cells were preexposed to differentconcentrations of EEP WEP (50 150 or 250120583gmL) or theantioxidant positive controls catechin (250 120583M) resveratrol(20120583M) or quercetin (10 120583M) for 24 h After washing twicewith PBS the cells were exposed to 20120583M DCFH-DA inHBSS and further incubated in the dark for another 30minThe DCFH-DA was removed by washing the cells with PBStwo times Then 500120583M tBuOOH was added The unstimu-lated DCFH-DA (no tBuOOH) in the unexposed RAW2647cells served as the naive control (NA) The intensity of thefluorescence signal was detected time dependently with anexcitation wavelength of 485 nm and an emission wavelengthof 535 nm using a Gemini EM fluorescence microplate reader(Molecular Devices Sunnyvale CA USA)
211 Nitrite Assay The level of NO in the culture mediawas detected as nitrite a major stable product of NOusing Griess reagent [23] RAW2647 cells were seeded ata density of 2 times 105 cellswell in a 96-well plate The cellswere grown for 3 h to allow plate attachment prior treatingwith the antioxidant positive control vitamin C (500120583M)or various concentrations (50 100 150 200 or 250 120583gmL)of EEP or WEP After 1 h incubation the RAW2647 cellswere stimulated with 1 120583gmL LPS plus 25UmL IFN-120574 Theactivated cells were further incubated for 24 h Then 100 120583Lof supernatant was mixed with an equal volume of Griess
4 Evidence-Based Complementary and Alternative Medicine
Table 1 The percentage of recovery of crude extracts from fresh leaves of PP
Extracts Amount and source of preparation Yield (g) Percentage of recoveryEEP 1500 g of fresh leaves 6041 403 (from fresh leaves)WEP 40 g of EEP 3271 8177 (from EEP)
(99321 g of fresh leaves) 329 (from fresh leaves)
Table 2 Total phenolic and flavonoid contents and total antioxidant (FRAP) activity of EEP and WEP
Values are mean plusmn SEM (119899 = 3) and are representative of three independent experiments with similar results Different letters within the same column aresignificantly different at P lt 005 as determined by a Studentrsquos t-test
reagent (1 sulfanilamide 01 NED and 3 phosphoricacid) After 10min of incubation in the dark the absorbanceof samples was measured at 540 nm using a Microplate Spec-trophotometer System (Bio-Rad Laboratories Inc) A freshculturemediumwas used as the blank in all experimentsTheamount of nitrite in the samples was derived from a standardcurve of sodium nitrite
212 Western Blot Analysis RAW2647 cells were plated ata density of 2 times 106 cellswell in a 6-well plate After anattachment period of approximately 3 h the cells were treatedwith various concentrations (50 100 150 200 or 250120583gmL)of EEP orWEP for 1 h 50 120583gmL Trolox or 500120583Mvitamin Cwas used as antioxidant positive controls The cells were thenstimulated with 1 120583gmL LPS plus 25UmL IFN-120574 for 18 hAfter incubation the cells were washed three times with PBSand placed in 150 120583L of ice-cold lysis buffer (1mL RIPA buffersupplemented with 2mM PMSF 2 120583M leupeptin and 1 120583ME-64) for 20minThen the disrupted cells were transferred tomicrocentrifuge tubes and centrifuged at 14000 g at 4∘C for30min The supernatant was collected and the protein con-centration of cell lysate was estimated by the Lowry method[24] Cell lysate was then boiled for 5min in a 6X samplebuffer (50mM Tris-base pH 74 4 SDS 10 glycerol 42-mercaptoethanol and 005mgmL of bromophenol blue)Thirty micrograms of cellular proteins were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) using 75 and 10 polyacrylamide gels foriNOS and COX-2 respectively (125 volts 120min) Theproteins in the gel were transferred onto a nitrocellulosemembrane (Amersham Pittsburgh PA USA) at 80 volts for1 h The membrane was blocked overnight at 4∘C with 5nonfat milk in 01 Tween 20 in a PBS buffer (TPBS) Themembranes were then incubated with a 1 1000 dilution ofthe primary antibody anti-iNOS mouse monoclonal or a1 2000 dilution of the primary antibody anti-COX-2 mousepolyclonal at RT for 2 h After extensive washing with TPBSthe membranes were incubated with a 1 10000 dilution ofthe secondary antibody goat-anti-mouse-HRP conjugate foriNOS and goat-anti-rabbit IgG-HRP conjugate for COX-2 at RT for 1 h To control equal loading of total protein
in all lanes blots were also stained with primary antibodyanti-tubulin mouse monoclonal at a dilution of 1 2000 atRT for 2 h After washing the membranes were incubatedwith a 1 10000 dilution of the secondary antibody goat-anti-mouse-HRP conjugate The membranes were washedthree times for 10min each time with TPBS The blotswere incubated for 3min in ECL Western blotting substrateand exposed to film The relative expression of proteins wasquantified densitometrically using the software imageJ andcalculated according to the reference band of tubulin
213 Statistical Analysis All statistical analyses were con-ducted using GraphPad software (GraphPad Prism 5 USA)The data from the total phenolic and flavonoid contents aswell as FRAP value results were analyzed by a Studentrsquos 119905-test to determine the statistical significance between twogroups DPPH MTT and nitrite assays were analyzed byone-way analysis of variance (ANOVA) with a post hocTukeyrsquos analysis to determine differences between treatmentand control groups [25] The data from intracellular ROSscavenging were analyzed by two-way ANOVA followed byBonfferonnirsquos post hoc test [26]
3 Results
31 The Percentage of Recovery of Crude Extracts from FreshLeaves of PP The percentages of recovery of crude extractsfrom fresh leaves of PP are shown in Table 1 EEP exhibited apercentage of recovery of 403 while WEP had percentageof recovery of 329 based on the original weight of freshleaves WEP was prepared from the water fraction of EEPthat was partitioned with hexane and water (1 1 vv) with apercentage of recovery of 8177 based on EEP
32 Phenolic and Flavonoid Contents WEP had a signifi-cantly higher level (119875 lt 005) of total phenolic and flavonoidcontent than that of EEP (Table 2) and more than half of thephenolics in WEP and EEP are flavonoids
33 Ferric Reducing Antioxidant Power EEP and WEP wereanalyzed for their reducing ability along with three standard
Evidence-Based Complementary and Alternative Medicine 5
80
100
60
40
20
0
0 5 10 15 20 25 30 35
Inhi
bitio
n (
)
Concentration (120583gmL)
WEPEEPCatechin
Vitamin CTrolox
Figure 1 DPPH radical scavenging activity of PP leaf extracts (EEPand WEP) and positive controls (vitamin C Trolox and catechin)Values are means plusmn SEM (119899 = 3) and are representative of threeindependent experiments with similar results
antioxidants vitamin C Trolox and catechin The results ofFRAP values in terms of ferrous ion (Fe2+) and vitamin CTrolox and catechin equivalents are shown in Table 2 EEPexhibited a higher degree of electron donating capacity thanWEP as suggested by the significantly higher FRAP values(119875 lt 005) of EEP when compared with WEP
34 DPPH Free Radical Scavenging Activity The free radicalscavenging capacities of EEP andWEP are shown in Figure 1The results show that both EEP and WEP exhibit the abilityto scavenge DPPH free radicals The scavenging activityagainst DPPH radicals of WEP (IC
50= 2155 plusmn 006 120583gmL)
is significantly greater (119875 lt 0001) than EEP (IC50
=2345 plusmn 012120583gmL) by 19 plusmn 015 but the scavengingcapacity of these is not as effective as the other positiveantioxidant controls 125 120583gmL EEP and 25 120583gmL WEPscavenged the DPPH radicals by 2927 plusmn 020 and 612 plusmn015 respectively and the scavenging capacities of bothare more pronounced at higher concentrations The highestconcentration (325120583gmL) of EEP andWEP could scavengethe DPPH radicals by 6596 plusmn 021 and 7319 plusmn 009respectively In the present study the scavenging abilities ofvitamin C (IC
50= 394 plusmn 001 120583gmL) and catechin (IC
50=
355 plusmn 001 120583gmL) were similar and both are significantlyhigher (119875 lt 0001) than Trolox (IC
50= 590 plusmn 027 120583gmL)
35 Effect of EEP and WEP on RAW2647 Cell Viability Thecell viability of RAW2647 cells exposed to EEP or WEP wasdetermined by MTT assay The cells were incubated for 24 hwith various concentrations of EEP (005 025 05 10 or150mgmL) or WEP (010 050 150 or 450mgmL) Asshown in Figure 2 both EEP andWEP displayed low toxicitytowards RAW2647 cells as evidenced by an apparent lack ofeffect on cell viability until the concentration of each extract
reached 15mgmL At 15mgmL EEP and WEP decreasedthe viability of RAW2647 cells by 3414 plusmn 969 and 2158 plusmn166 (119875 lt 005) respectively However the cytotoxiceffect is more pronounced at higher concentrations WEP at45mgmL decreased the cell viability by as much as 5421 plusmn174 (119875 lt 005) The effect of EEP and WEP on RAW2647cell viability was also confirmed by trypan blue exclusion andpropidium iodide staining methods which exhibited similarresults (data not shown)Therefore a nontoxic concentrationrange of 0ndash025mgmL of both EEP and WEP was selectedfor RAW2647 cell treatment in the subsequent studies
36 EEP and WEP as Intracellular ROS Scavengers Thedirect scavenging effect of EEP and WEP on intracellularfree radical stress was investigated in RAW2647 cells usingthe DCFH-DA assay The increment of DCF fluorescenceemission following ROS-mediated oxidation of DCFH wasfollowed for 240min As shown in Figure 3(a) standardantioxidant positive controls catechin (250 120583M) resveratrol(20120583M) and quercetin (10 120583M) could scavenge ROS signif-icantly (119875 lt 005) throughout the incubation time whencompared to the vehicle control (VH)With as little as 30minof incubation catechin resveratrol and quercetin showedconsiderable radical scavenging activity EEP (Figure 3(b))and WEP (Figure 3(c)) decreased the DCF fluorescent emis-sion in a dose- and time-dependent manner Again with aslittle as 30min of incubation both EEP and WEP at lowconcentration (50120583gmL) showed similar radical scavengingactivity as the antioxidant controls Various concentrations ofEEP significantly decreased (119875 lt 005) the DCF fluorescentemission throughout the incubation time when comparedto the VH control At high concentration (150 120583gmL) EEPexhibited a strong scavenging activity as suggested by thecapability to lower fluorescent intensity to below basal levelof the unstimulated DCFH-DA control at 180ndash240min Inaddition the highest concentration of EEP (250120583gmL)significantly lowered (119875 lt 005) DCF fluorescent intensityto below the basal level at all time points Similarly 150 and250 120583gmL of WEP also significantly decreased (119875 lt 005)the DCF fluorescent emission throughout the incubationtime compared to the tBuOOH control However the lowestconcentration ofWEP (50120583gmL) significantly reduced (119875 lt005) the DCF fluorescent emission until 210min only
37 NO Suppression by EEP and WEP in LPS Plus IFN-120574-Activated RAW2647 Cells RAW2647 cells were pretreatedwith antioxidants vitamin C EEP or WEP for 1 h thenstimulated with LPS plus IFN-120574 and measured for NOproduction using the Griess assay As shown in Figure 4unstimulated RAW2647 cells (NA) secreted basal levels ofNO while the production of NO was increased to about43 120583M in LPS plus IFN-120574-activated RAW2647 cells Theantioxidant control 500 120583M vitamin C decreased the NOproduction by almost 35 Pretreatment of RAW2647 cellswith EEP or WEP significantly suppressed (119875 lt 005) theinduction of NO in a dose-related manner (Figures 4(a) and4(b)) and the suppression was observed in all EEP- andWEP-treated groups These results also clearly indicate that
6 Evidence-Based Complementary and Alternative Medicine
80
60
40
20
0
b b b b
aab
Cel
l via
bilit
y (
)120
100
Control 005 025 050 150100
EEP (mgmL)
(a)
80
60
40
20
0
b
a
ccd
d
Cel
l via
bilit
y (
)
120
100
Control 010 050 150 450
WEP (mgmL)
(b)
Figure 2 Effect of EEP andWEP on cell viability of RAW2647 cellsThe effect of EEP (a) andWEP (b) on cell viability was assessed byMTTValues are expressed as means plusmn SEM (119899 = 3) and are representative of three independent experiments with similar results Bars marked withdifferent letters are significantly different at 119875 lt 005 as determined by one-way ANOVA
EEP is a stronger suppressant of NO induction than WEPConcentrations of 50 120583gmL of EEP and 150 and 200 120583gmLofWEPwere required to exhibit the NO suppression with thesame efficiency as 500 120583M (8806120583gmL) vitamin C
38 Suppression of iNOS and COX-2 Protein Expression byEEP and WEP in LPS Plus IFN-120574-Activated RAW2647 CellsTo determine if suppression of NO production by EEP orWEPwas related to changes in iNOS aswell as COX-2 proteinlevels Western blotting analysis was performed RAW2647cells were pretreated with antioxidants Trolox (50120583gmL)vitamin C (500120583M) or PP extracts (EEP or WEP) at 50ndash250120583gmL for 1 h prior activation with LPS (1120583gmL) plusIFN-120574 (25UmL) for 18 h Total proteins were extracted andanalyzed for the expression of iNOS and COX-2 by Westernblotting LPS plus IFN-120574 induced increases in iNOS (Figures5(a) and 5(b)) and COX-2 (Figures 5(c) and 5(d)) expressioncompared to the unstimulated cultures Antioxidant controls(Trolox and vitamin C) decreased LPS plus IFN-120574-inducediNOS and COX-2 protein levels The data also suggestedthat the suppression by 500120583M (8806120583gmL) vitamin Cis more pronounced than 50 120583gmL Trolox Compared tothe corresponding controls both EEP and WEP produced adose-dependent suppression of iNOS level in LPS plus IFN-120574-activated RAW2647 cells (Figures 5(a) and 5(b)) suggestingthat the suppression of NO production by EEP and WEP ismediated by decreasing the expression of iNOS In agreementwith the result of NO suppression 50ndash200 120583gmL EEP wasprobably more efficient than WEP in iNOS suppressionThe iNOS expression was almost completely eliminated at200120583gmL EEP and was barely observed at 250 120583gmLWEPThe inflammatory modulation of EEP and WEP was alsofurther supported by the dose-dependent suppression of theCOX-2 level by both EEP and WEP (Figures 5(c) and 5(d))in the activated RAW2647 cells Notably EEP and WEPexhibited higher suppression of iNOS than COX-2
4 Discussion
It is well known that major phytochemicals of plant leafextracts possessing antioxidant activity are flavonoids andother phenolic compounds Researchers have found thatflavonoids from PP leaves display antioxidant activity andall ethyl acetate chloroform and 119899-butanol-soluble fractionsof PP contain flavonoids [11 15] In addition Nguyen andEun [14] found phenolics and flavonoids in extracts of PPleaves when assessed with Folin-Ciocalteu and aluminumtrichloride PP leaf extracts also have antioxidant activitieswhen evaluated with DPPH and FRAP assays Similarlythe present study also showed that both EEP and WEPcontain high levels of flavonoids and phenolics and exhibitantioxidant activity The most frequently used antioxidantstandards for food samples (vitamin C Trolox catechinresveratrol and quercetin) were used as positive antioxidantcontrols in the present study
This study revealed that DPPH radical scavenging capac-ity of WEP is greater than that of EEP (Figure 1) In contrastEEP has higher ferric reducing power than WEP (Table 2)Such contradictory results between DPPH and FRAP assaysare not unusual Though both assays are based on a singleelectron transfer reaction [27] their characteristics sensitiv-ities mechanisms of the reaction and endpoints are totallydifferent For instance the DPPH method is based on thefree radical scavenging activity while FRAP measures thecapability of reducing Fe3+ to Fe2+ Depending on whatspecific phytochemical constituents present in the extractare providing the antioxidant activity their discrete chemicalstructures positions numbers and types of substitutions caninfluence their redox properties and hence their antioxidantpotentials [28]
Though both DPPH and FRAP assays are frequently usedfor assessing antioxidant capacity they have some drawbacksIn the DPPH assay interfering compounds may have sig-nificant absorption at the same measured wavelength In
Evidence-Based Complementary and Alternative Medicine 7
Figure 3 Cellular radical scavenging activity in tBuOOH-activated RAW2647 cells Intracellular ROS level generated in cells was measuredby the DCFH-DA RAW2647 cells were pretreated with indicated concentrations of antioxidants (a) EEP (b) or WEP (c) for 24 h priorto take-up of 20 120583M DCFH-DA for 30min Results are mean plusmn SEM (119899 = 4) and are representative of three independent experiments withsimilar results Pointsmarked with different letters are significantly different at119875 lt 005when compared at the same time point as determinedby two-way ANOVA
addition theDPPH radical is not present in living organismsFor the FRAPmethod compounds with low redox potentialwhich probably do not serve as antioxidants in vivo still canreduce the Fe3+ Interfering compounds may also absorb atthe same wavelength and the assay is also performed at anonphysiological pH [29] Therefore antioxidant activitiesof EEP and WEP were also evaluated by the cell-basedassay using an intracellular fluorescent probe DCFH-DAWhen the nonfluorescent DCFH-DA is taken up into cellsits diacetate moiety will be hydrolyzed by cellular esterasesto generate the more polar DCFH which is trapped insidethe cells In the presence of ROS intracellular DCFH isfurther oxidized to form the fluorescent DCF product [30]
The macrophage cell line RAW2647 is usually the cell ofchoice in studying ROS-mediated cellular events since itcan generate high amounts of ROS following an oxidantchallenge Catechin resveratrol and quercetin at the levelof concentration used in this study have been shown andoptimized to exhibit a strong suppression of intracellular ROSgeneration [22 31 32] Therefore the present study selectedthese compounds as antioxidant positive controls for theDCFH-DA assay The present study demonstrated that allantioxidant standards 250120583M catechin 20120583M resveratroland 10 120583M quercetin exerted a strong inhibition of ROSgeneration induced by tBuOOH over a period of 30 to240min In addition to extracellular antioxidant capacity
8 Evidence-Based Complementary and Alternative Medicine
45
40
35
30
25
20
15
10
5
0
NA
VH 50
150
250
e
d
c
bb
a
e
d
bcNitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
EEP (120583gmL)
(a)
b
e
cc
bc
d
f
a
45
40
35
30
25
20
15
10
5
0
NA 50
150
250
Nitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
WEP (120583gmL)
(b)
Figure 4 EEP (a) and WEP (b) suppressed LPS plus IFN-120574-induced nitrite production in RAW2647 cells RAW2647 cells were incubatedfor 24 hwith LPS (1120583gmL) plus IFN-120574 (25UmL) in the presence or absence of indicated concentrations of vitaminC (500120583M) EEP orWEPAccumulated nitrite in the culturemediumwas determined by the Griess reactionThe values aremeansplusmn SEM (119899 = 3) and are representativeof three independent experiments with similar results Bars marked with different letters are significantly different at 119875 lt 005 as determinedby one-way ANOVA
EEP and WEP also served as intracellular ROS scavengersand subsequently decreased the oxidation of DCFH (Figures3(b) and 3(c)) Both EEP and WEP were as efficient as theantioxidant standards in scavenging ROS Notably EEP was abetter reducer of DCF fluorescence thanWEPThe reductionof DCF fluorescence by EEP and WEP is not due to directcytotoxicity as the range of concentration used in the studieshad no effect on RAW2647 cell viability (Figure 2)
Although the current study shows that an ethanol extractfrom PP leaves has in vivo anti-inflammatory activities[13] its mechanism of anti-inflammation is still unrevealedInflammatory disorders are characterized among otherevents by the production of significant amounts of freeradicals nitrogen reactive species and pro-inflammatorycytokines [10] High NO concentration combines with super-oxides to form peroxynitrite ions (OONOminus) which areresponsible for cell and tissue damage from inflammation[33] Therefore we investigated inflammatory effects of EEPand WEP on the suppression of NO production in LPS plusIFN-120574-activated RAW2647 cells At the concentration rangeof 50ndash250120583gmL both EEP and WEP dose-dependentlysuppressed NO production and the suppression was morepronounced in EEP than WEP (Figure 4) These resultsagreed with the observation that EEP was also a betterscavenger of intracellular ROS than WEP (Figures 3(b) and3(c))
As enhanced NO production by LPS and IFN-120574-stimulated RAW2647 cells mainly occurs via increasedintracellular content of iNOS [3 4] the effect of EEP andWEP on iNOS expression was investigatedThe present studyclearly indicates that the suppressive effect of EEP and WEPon NO production was mediated through the inhibition of
iNOS expression (Figures 5(a) and 5(b)) In agreement withthe study of NO suppression the suppressive effect of EEP(50ndash200120583gmL) on iNOS was more remarkable than that ofWEP
In addition to iNOS induction LPS and IFN-120574 alsoefficiently enhance COX-2 expression in RAW2647 cells [35] An increased level of COX-2 expression is also knownto account for the excessive production of PGE
2in most
if not all inflammatory cells and tissues [34] This studyshows that both EEP andWEP can exhibit anti-inflammatoryactivity by reducing high COX-2 protein levels in a dose-related manner (Figures 5(c) and 5(d)) Thus EEP and WEPmight play important roles in attenuating inflammationand cellular damage through their extra- and intracellularROS scavenging activity and downregulation of NO iNOSand COX-2 Concordantly Khumpook et al [13] recentlyreported the in vivo anti-inflammatory activity of PP leavesas evidenced by decreased lipid peroxidation and NO levelin concomitance with increased superoxide dismutase in thecotton-induced chronic inflammation in Albino rats uponexposure to an ethanol extract of PP leaves for 17 days
In fact several medicinal plant extracts with naturalantioxidant properties together with suppressive effects onNO iNOS andorCOX-2 expression inRAW2647 have beenreported to display a wide spectrum of bioactivities Theseactivities include anti-inflammation such as curcumin fromCurcuma longa resveratrol from grape skins red wines andother plants and a mixture of 120573-sitosterol and stigmasterolfrom Andrographis paniculata [8 35 36] Previous investi-gators demonstrated that pretreatment of RAW2647 withflavonoids such as apigenin genistein and kaempferol sup-pressed LPS-stimulated expression of NO iNOS and COX-2
Evidence-Based Complementary and Alternative Medicine 9
FoldiNOS
Tubulin
003 086 058 030 061 038 033 024 015
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
EEP (120583gmL)
LPSIFN-120574
(a)
Fold
Tubulin
iNOS
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
WEP (120583gmL)
LPSIFN-120574
008 111 083 052 073 063 048 036 008
(b)
COX-2
Fold
Tubulin
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
006 093 076 074 087 079 065 044 030
EEP (120583gmL)
LPSIFN-120574
(c)
Fold
Tubulin
COX-2
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
001 113 066 063 091 078 067 064 050
WEP (120583gmL)
LPSIFN-120574
(d)
Figure 5 Effect of EEP on LPS plus IFN-120574-induced iNOS (a) and COX-2 (c) andWEP on LPS plus IFN-120574-induced iNOS (b) and COX-2 (d)protein levels in RAW2647 cells The relative expression of proteins was quantified densitometrically using ImageJ software and normalizedto tubulin reference bands Data are representative of at least two independent experiments
protein production [37] Major chemical constituents of PPleaves consist of 120573-sitosterol stigmasterol kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucoside[11] All aforementioned compounds have been shown topossess anti-inflammatory properties Both kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucosidemay be metabolized into kaempferol and apigenin whichalso have antioxidant and anti-inflammatory activities Thusit is possible that phenolic and flavonoid compounds inboth EEP andWEP provide substantial antioxidant and anti-inflammatory activities
In summary the cytoprotective effects of EEP and WEPis due to their abilities to decrease ROS generation and NOradical production in cells In addition both EEP and WEPexert anti-inflammatory effects through the suppression ofNO release and decrease the protein expression of iNOSand COX-2 Thus PP leaves possess high potential forfurther exploration in the research development of anti-inflammatory medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was financially supported by a Royal GoldenJubilee Scholarship Grant (PhD02082548) and by the
National Research Council of Thailand (NRCT) throughSuranaree University of Technology (SUT-104-53-36-09)
References
[1] B Halliwell and J M C Gutteridge Free Radicals in BiologyandMedicine OxfordUniversity Press NewYork NYUSA 3rdedition 1999
[2] N Fujiwara and K Kobayashi ldquoMacrophages in inflammationrdquoCurrent Drug TargetsmdashInflammation and Allergy vol 4 no 3pp 281ndash286 2005
[3] S I Jang Y-J Kim W-Y Lee et al ldquoScoparone from Artemisiacapillaris inhibits the release of inflammatory mediators inRAW 2647 cells upon stimulation cells by interferon-120574 plusLPSrdquo Archives of Pharmacal Research vol 28 no 2 pp 203ndash208 2005
[4] R Korhonen A Lahti H Kankaanranta and E MoilanenldquoNitric oxide production and signaling in inflammationrdquo Cur-rent Drug TargetsmdashInflammation and Allergy vol 4 no 4 pp471ndash479 2005
[5] E Karpuzoglu and S A Ahmed ldquoEstrogen regulation of nitricoxide and inducible nitric oxide synthase (iNOS) in immunecells implications for immunity autoimmune diseases andapoptosisrdquo Nitric Oxide vol 15 no 3 pp 177ndash186 2006
[6] S G Harris J Padilla L Koumas D Ray and R PPhipps ldquoProstaglandins as modulators of immunityrdquo Trends inImmunology vol 23 no 3 pp 144ndash150 2002
[7] I-N Hsieh A S-Y Chang C-M Teng C-C Chen andC-R Yang ldquoAciculatin inhibits lipopolysaccharide-mediated
10 Evidence-Based Complementary and Alternative Medicine
inducible nitric oxide synthase and cyclooxygenase-2 expres-sion via suppressing NF-120581B and JNKp38 MAPK activationpathwaysrdquo Journal of Biomedical Science vol 18 no 1 article28 2011
[8] J B Calixto M F Otuki and A R S Santos ldquoAnti-inflammatory compounds of plant originmdashpart I action onarachidonic acid pathway nitric oxide and nuclear factor 120581 B(NF-120581B)rdquo Planta Medica vol 69 no 11 pp 973ndash983 2003
[9] K B Pandey and S I Rizvi ldquoCurrent understanding of dietarypolyphenols and their role in health and diseaserdquo CurrentNutrition and Food Science vol 5 no 4 pp 249ndash263 2009
[10] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer How are theylinkedrdquo Free Radical Biology and Medicine vol 49 no 11 pp1603ndash1616 2010
[11] P M Giang H V Bao and P T Son ldquoPhytochemical studyon Pseuderanthemum palatiferum (Nees) Radlk AcanthaceaerdquoJournal of Chemistry vol 41 no 2 pp 115ndash118 2003
[12] H K Dieu C B Loc S Yamasaki and Y Hirata ldquoThe ethnob-otanical and botanical study on Pseuderanthemum palatiferumas a new medicinal plant in the Mekong Delta of VietnamrdquoJapan Agricultural Research Quarterly vol 39 no 3 pp 191ndash1962005
[13] T Khumpook S Chomdej S Saenphet D Amornlerdpi-son and K Saenphet ldquoAnti-inflammatory activity of ethanolextract from the leaves of Pseuderanthemum palatiferum (Nees)RadlkrdquoChiangMai Journal of Science vol 40 no 3 pp 321ndash3312013
[14] Q-V Nguyen and J-B Eun ldquoAntioxidant activity of solventextracts from Vietnamese medicinal plantsrdquo Journal of Medic-inal Plant Research vol 5 no 13 pp 2798ndash2811 2011
[15] P M Giang H V Bao and P T Son ldquoStudy on anti-oxidativeactivities and preliminary investigation on antibacterial anti-fungal of extracted fraction rich in flavonoids from leaves ofPseuderanthemum palatiferum (Nees) Radlkrdquo TC Duoc Hocvol 9 no 9 pp 9ndash12 2005 (In Vietnamese with Englishsummary)
[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998
[17] M Liu X Q Li C Weber C Y Lee J Brown and R HLiu ldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002
[18] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[19] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoFreeradical scavenging capacity and inhibition of lipid oxidation ofwines grape juices and related polyphenolic constituentsrdquo FoodResearch International vol 32 no 6 pp 407ndash412 1999
[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958
[21] S-C Chun S Y Jee S G Lee S J Park J R Lee and SC Kim ldquoAnti-inflammatory activity of the methanol extractof Moutan Cortex in LPS-activated Raw2647 cellsrdquo Evidence-Based Complementary and Alternative Medicine vol 4 no 3pp 327ndash333 2007
[22] G-N Kim Y-I Kwon and H-D Jang ldquoProtective mech-anism of quercetin and rutin on 22rsquo-azobis(2-amidino-propane)dihydrochloride or Cu2+-induced oxidative stress inHepG2 cellsrdquoToxicology in Vitro vol 25 no 1 pp 138ndash144 2011
[23] M G B de Oliveira R B Marques M F de Santana et al ldquo120572-Terpineol reducesmechanical hypernociception and inflamma-tory responserdquoBasic ampClinical PharmacologyampToxicology vol111 no 2 pp 120ndash125 2012
[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[25] A K L Nascimento R FMelo-Silveira N Dantas-Santos et alldquoAntioxidant and antiproliferative activities of leaf extracts fromPlukenetia volubilis Linneo (Euphorbiaceae)rdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID950272 10 pages 2013
[26] A Muthuraman N Singh and A S Jaggi ldquoProtective effectof Acorus calamus L in rat model of vincristine inducedpainful neuropathy an evidence of anti-inflammatory and anti-oxidative activityrdquo Food and Chemical Toxicology vol 49 no 10pp 2557ndash2563 2011
[27] A Bunea DO Rugina AM Pintea Z Sconta C I Bunea andC Socaciu ldquoComparative polyphenolic content and antioxidantactivities of some wild and cultivated blueberries from Roma-niardquoNotulae Botanicae Horti Agrobotanici Cluj-Napoca vol 39no 2 pp 70ndash76 2011
[28] K E Heim A R Tagliaferro and D J Bobilya ldquoFlavonoidantioxidants chemistry metabolism and structure-activityrelationshipsrdquo Journal of Nutritional Biochemistry vol 13 no10 pp 572ndash584 2002
[29] J Perez-Jimenez S Arranz M Tabernero et al ldquoUpdatedmethodology to determine antioxidant capacity in plant foodsoils and beverages extraction measurement and expression ofresultsrdquo Food Research International vol 41 no 3 pp 274ndash2852008
[30] K L Wolfe and R H Lui ldquoCellular antioxidant activity(CAA) assay for assessing antioxidants foods and dietarysupplementsrdquo Journal of Agricultural and Food Chemistry vol55 no 22 pp 8896ndash8907 2007
[31] M Okawara H Katsuki E Kurimoto H Shibata T Kumeand A Akaike ldquoResveratrol protects dopaminergic neuronsin midbrain slice culture from multiple insultsrdquo BiochemicalPharmacology vol 73 no 4 pp 550ndash560 2007
[32] H Nishikawa KWakano and S Kitani ldquoInhibition of NADPHoxidase subunits translocation by tea catechin EGCG in mastcellrdquo Biochemical and Biophysical Research Communicationsvol 362 no 2 pp 504ndash509 2007
[33] J Fang T Seki and H Maeda ldquoTherapeutic strategies by mod-ulating oxygen stress in cancer and inflammationrdquo AdvancedDrug Delivery Reviews vol 61 no 4 pp 290ndash302 2009
[34] K Subbaramaiah and A J Dannenberg ldquoCyclooxygenase 2 amolecular target for cancer prevention and treatmentrdquo Trendsin Pharmacological Sciences vol 24 no 2 pp 96ndash102 2003
[35] Y-J Surh K-S ChunH-H Cha et al ldquoMolecularmechanismsunderlying chemopreventive activities of anti-inflammatoryphytochemicals down-regulation of COX-2 and iNOS throughsuppression of NF-120581B activationrdquoMutation Research vol 480-481 pp 243ndash268 2001
[36] W-W Chao Y-H Kuo and B-F Lin ldquoAnti-inflammatoryactivity of new compounds from Andrographis paniculata byNF-120581B transactivation inhibitionrdquo Journal of Agricultural andFood Chemistry vol 58 no 4 pp 2505ndash2512 2010
Evidence-Based Complementary and Alternative Medicine 11
[37] Y-C Liang Y-T Huang S-H Tsai S-Y Lin-Shiau C-FChen and J-K Lin ldquoSuppression of inducible cyclooxygenaseand inducible nitric oxide synthase by apigenin and relatedflavonoids in mouse macrophagesrdquo Carcinogenesis vol 20 no10 pp 1945ndash1952 1999
4 Evidence-Based Complementary and Alternative Medicine
Table 1 The percentage of recovery of crude extracts from fresh leaves of PP
Extracts Amount and source of preparation Yield (g) Percentage of recoveryEEP 1500 g of fresh leaves 6041 403 (from fresh leaves)WEP 40 g of EEP 3271 8177 (from EEP)
(99321 g of fresh leaves) 329 (from fresh leaves)
Table 2 Total phenolic and flavonoid contents and total antioxidant (FRAP) activity of EEP and WEP
Values are mean plusmn SEM (119899 = 3) and are representative of three independent experiments with similar results Different letters within the same column aresignificantly different at P lt 005 as determined by a Studentrsquos t-test
reagent (1 sulfanilamide 01 NED and 3 phosphoricacid) After 10min of incubation in the dark the absorbanceof samples was measured at 540 nm using a Microplate Spec-trophotometer System (Bio-Rad Laboratories Inc) A freshculturemediumwas used as the blank in all experimentsTheamount of nitrite in the samples was derived from a standardcurve of sodium nitrite
212 Western Blot Analysis RAW2647 cells were plated ata density of 2 times 106 cellswell in a 6-well plate After anattachment period of approximately 3 h the cells were treatedwith various concentrations (50 100 150 200 or 250120583gmL)of EEP orWEP for 1 h 50 120583gmL Trolox or 500120583Mvitamin Cwas used as antioxidant positive controls The cells were thenstimulated with 1 120583gmL LPS plus 25UmL IFN-120574 for 18 hAfter incubation the cells were washed three times with PBSand placed in 150 120583L of ice-cold lysis buffer (1mL RIPA buffersupplemented with 2mM PMSF 2 120583M leupeptin and 1 120583ME-64) for 20minThen the disrupted cells were transferred tomicrocentrifuge tubes and centrifuged at 14000 g at 4∘C for30min The supernatant was collected and the protein con-centration of cell lysate was estimated by the Lowry method[24] Cell lysate was then boiled for 5min in a 6X samplebuffer (50mM Tris-base pH 74 4 SDS 10 glycerol 42-mercaptoethanol and 005mgmL of bromophenol blue)Thirty micrograms of cellular proteins were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) using 75 and 10 polyacrylamide gels foriNOS and COX-2 respectively (125 volts 120min) Theproteins in the gel were transferred onto a nitrocellulosemembrane (Amersham Pittsburgh PA USA) at 80 volts for1 h The membrane was blocked overnight at 4∘C with 5nonfat milk in 01 Tween 20 in a PBS buffer (TPBS) Themembranes were then incubated with a 1 1000 dilution ofthe primary antibody anti-iNOS mouse monoclonal or a1 2000 dilution of the primary antibody anti-COX-2 mousepolyclonal at RT for 2 h After extensive washing with TPBSthe membranes were incubated with a 1 10000 dilution ofthe secondary antibody goat-anti-mouse-HRP conjugate foriNOS and goat-anti-rabbit IgG-HRP conjugate for COX-2 at RT for 1 h To control equal loading of total protein
in all lanes blots were also stained with primary antibodyanti-tubulin mouse monoclonal at a dilution of 1 2000 atRT for 2 h After washing the membranes were incubatedwith a 1 10000 dilution of the secondary antibody goat-anti-mouse-HRP conjugate The membranes were washedthree times for 10min each time with TPBS The blotswere incubated for 3min in ECL Western blotting substrateand exposed to film The relative expression of proteins wasquantified densitometrically using the software imageJ andcalculated according to the reference band of tubulin
213 Statistical Analysis All statistical analyses were con-ducted using GraphPad software (GraphPad Prism 5 USA)The data from the total phenolic and flavonoid contents aswell as FRAP value results were analyzed by a Studentrsquos 119905-test to determine the statistical significance between twogroups DPPH MTT and nitrite assays were analyzed byone-way analysis of variance (ANOVA) with a post hocTukeyrsquos analysis to determine differences between treatmentand control groups [25] The data from intracellular ROSscavenging were analyzed by two-way ANOVA followed byBonfferonnirsquos post hoc test [26]
3 Results
31 The Percentage of Recovery of Crude Extracts from FreshLeaves of PP The percentages of recovery of crude extractsfrom fresh leaves of PP are shown in Table 1 EEP exhibited apercentage of recovery of 403 while WEP had percentageof recovery of 329 based on the original weight of freshleaves WEP was prepared from the water fraction of EEPthat was partitioned with hexane and water (1 1 vv) with apercentage of recovery of 8177 based on EEP
32 Phenolic and Flavonoid Contents WEP had a signifi-cantly higher level (119875 lt 005) of total phenolic and flavonoidcontent than that of EEP (Table 2) and more than half of thephenolics in WEP and EEP are flavonoids
33 Ferric Reducing Antioxidant Power EEP and WEP wereanalyzed for their reducing ability along with three standard
Evidence-Based Complementary and Alternative Medicine 5
80
100
60
40
20
0
0 5 10 15 20 25 30 35
Inhi
bitio
n (
)
Concentration (120583gmL)
WEPEEPCatechin
Vitamin CTrolox
Figure 1 DPPH radical scavenging activity of PP leaf extracts (EEPand WEP) and positive controls (vitamin C Trolox and catechin)Values are means plusmn SEM (119899 = 3) and are representative of threeindependent experiments with similar results
antioxidants vitamin C Trolox and catechin The results ofFRAP values in terms of ferrous ion (Fe2+) and vitamin CTrolox and catechin equivalents are shown in Table 2 EEPexhibited a higher degree of electron donating capacity thanWEP as suggested by the significantly higher FRAP values(119875 lt 005) of EEP when compared with WEP
34 DPPH Free Radical Scavenging Activity The free radicalscavenging capacities of EEP andWEP are shown in Figure 1The results show that both EEP and WEP exhibit the abilityto scavenge DPPH free radicals The scavenging activityagainst DPPH radicals of WEP (IC
50= 2155 plusmn 006 120583gmL)
is significantly greater (119875 lt 0001) than EEP (IC50
=2345 plusmn 012120583gmL) by 19 plusmn 015 but the scavengingcapacity of these is not as effective as the other positiveantioxidant controls 125 120583gmL EEP and 25 120583gmL WEPscavenged the DPPH radicals by 2927 plusmn 020 and 612 plusmn015 respectively and the scavenging capacities of bothare more pronounced at higher concentrations The highestconcentration (325120583gmL) of EEP andWEP could scavengethe DPPH radicals by 6596 plusmn 021 and 7319 plusmn 009respectively In the present study the scavenging abilities ofvitamin C (IC
50= 394 plusmn 001 120583gmL) and catechin (IC
50=
355 plusmn 001 120583gmL) were similar and both are significantlyhigher (119875 lt 0001) than Trolox (IC
50= 590 plusmn 027 120583gmL)
35 Effect of EEP and WEP on RAW2647 Cell Viability Thecell viability of RAW2647 cells exposed to EEP or WEP wasdetermined by MTT assay The cells were incubated for 24 hwith various concentrations of EEP (005 025 05 10 or150mgmL) or WEP (010 050 150 or 450mgmL) Asshown in Figure 2 both EEP andWEP displayed low toxicitytowards RAW2647 cells as evidenced by an apparent lack ofeffect on cell viability until the concentration of each extract
reached 15mgmL At 15mgmL EEP and WEP decreasedthe viability of RAW2647 cells by 3414 plusmn 969 and 2158 plusmn166 (119875 lt 005) respectively However the cytotoxiceffect is more pronounced at higher concentrations WEP at45mgmL decreased the cell viability by as much as 5421 plusmn174 (119875 lt 005) The effect of EEP and WEP on RAW2647cell viability was also confirmed by trypan blue exclusion andpropidium iodide staining methods which exhibited similarresults (data not shown)Therefore a nontoxic concentrationrange of 0ndash025mgmL of both EEP and WEP was selectedfor RAW2647 cell treatment in the subsequent studies
36 EEP and WEP as Intracellular ROS Scavengers Thedirect scavenging effect of EEP and WEP on intracellularfree radical stress was investigated in RAW2647 cells usingthe DCFH-DA assay The increment of DCF fluorescenceemission following ROS-mediated oxidation of DCFH wasfollowed for 240min As shown in Figure 3(a) standardantioxidant positive controls catechin (250 120583M) resveratrol(20120583M) and quercetin (10 120583M) could scavenge ROS signif-icantly (119875 lt 005) throughout the incubation time whencompared to the vehicle control (VH)With as little as 30minof incubation catechin resveratrol and quercetin showedconsiderable radical scavenging activity EEP (Figure 3(b))and WEP (Figure 3(c)) decreased the DCF fluorescent emis-sion in a dose- and time-dependent manner Again with aslittle as 30min of incubation both EEP and WEP at lowconcentration (50120583gmL) showed similar radical scavengingactivity as the antioxidant controls Various concentrations ofEEP significantly decreased (119875 lt 005) the DCF fluorescentemission throughout the incubation time when comparedto the VH control At high concentration (150 120583gmL) EEPexhibited a strong scavenging activity as suggested by thecapability to lower fluorescent intensity to below basal levelof the unstimulated DCFH-DA control at 180ndash240min Inaddition the highest concentration of EEP (250120583gmL)significantly lowered (119875 lt 005) DCF fluorescent intensityto below the basal level at all time points Similarly 150 and250 120583gmL of WEP also significantly decreased (119875 lt 005)the DCF fluorescent emission throughout the incubationtime compared to the tBuOOH control However the lowestconcentration ofWEP (50120583gmL) significantly reduced (119875 lt005) the DCF fluorescent emission until 210min only
37 NO Suppression by EEP and WEP in LPS Plus IFN-120574-Activated RAW2647 Cells RAW2647 cells were pretreatedwith antioxidants vitamin C EEP or WEP for 1 h thenstimulated with LPS plus IFN-120574 and measured for NOproduction using the Griess assay As shown in Figure 4unstimulated RAW2647 cells (NA) secreted basal levels ofNO while the production of NO was increased to about43 120583M in LPS plus IFN-120574-activated RAW2647 cells Theantioxidant control 500 120583M vitamin C decreased the NOproduction by almost 35 Pretreatment of RAW2647 cellswith EEP or WEP significantly suppressed (119875 lt 005) theinduction of NO in a dose-related manner (Figures 4(a) and4(b)) and the suppression was observed in all EEP- andWEP-treated groups These results also clearly indicate that
6 Evidence-Based Complementary and Alternative Medicine
80
60
40
20
0
b b b b
aab
Cel
l via
bilit
y (
)120
100
Control 005 025 050 150100
EEP (mgmL)
(a)
80
60
40
20
0
b
a
ccd
d
Cel
l via
bilit
y (
)
120
100
Control 010 050 150 450
WEP (mgmL)
(b)
Figure 2 Effect of EEP andWEP on cell viability of RAW2647 cellsThe effect of EEP (a) andWEP (b) on cell viability was assessed byMTTValues are expressed as means plusmn SEM (119899 = 3) and are representative of three independent experiments with similar results Bars marked withdifferent letters are significantly different at 119875 lt 005 as determined by one-way ANOVA
EEP is a stronger suppressant of NO induction than WEPConcentrations of 50 120583gmL of EEP and 150 and 200 120583gmLofWEPwere required to exhibit the NO suppression with thesame efficiency as 500 120583M (8806120583gmL) vitamin C
38 Suppression of iNOS and COX-2 Protein Expression byEEP and WEP in LPS Plus IFN-120574-Activated RAW2647 CellsTo determine if suppression of NO production by EEP orWEPwas related to changes in iNOS aswell as COX-2 proteinlevels Western blotting analysis was performed RAW2647cells were pretreated with antioxidants Trolox (50120583gmL)vitamin C (500120583M) or PP extracts (EEP or WEP) at 50ndash250120583gmL for 1 h prior activation with LPS (1120583gmL) plusIFN-120574 (25UmL) for 18 h Total proteins were extracted andanalyzed for the expression of iNOS and COX-2 by Westernblotting LPS plus IFN-120574 induced increases in iNOS (Figures5(a) and 5(b)) and COX-2 (Figures 5(c) and 5(d)) expressioncompared to the unstimulated cultures Antioxidant controls(Trolox and vitamin C) decreased LPS plus IFN-120574-inducediNOS and COX-2 protein levels The data also suggestedthat the suppression by 500120583M (8806120583gmL) vitamin Cis more pronounced than 50 120583gmL Trolox Compared tothe corresponding controls both EEP and WEP produced adose-dependent suppression of iNOS level in LPS plus IFN-120574-activated RAW2647 cells (Figures 5(a) and 5(b)) suggestingthat the suppression of NO production by EEP and WEP ismediated by decreasing the expression of iNOS In agreementwith the result of NO suppression 50ndash200 120583gmL EEP wasprobably more efficient than WEP in iNOS suppressionThe iNOS expression was almost completely eliminated at200120583gmL EEP and was barely observed at 250 120583gmLWEPThe inflammatory modulation of EEP and WEP was alsofurther supported by the dose-dependent suppression of theCOX-2 level by both EEP and WEP (Figures 5(c) and 5(d))in the activated RAW2647 cells Notably EEP and WEPexhibited higher suppression of iNOS than COX-2
4 Discussion
It is well known that major phytochemicals of plant leafextracts possessing antioxidant activity are flavonoids andother phenolic compounds Researchers have found thatflavonoids from PP leaves display antioxidant activity andall ethyl acetate chloroform and 119899-butanol-soluble fractionsof PP contain flavonoids [11 15] In addition Nguyen andEun [14] found phenolics and flavonoids in extracts of PPleaves when assessed with Folin-Ciocalteu and aluminumtrichloride PP leaf extracts also have antioxidant activitieswhen evaluated with DPPH and FRAP assays Similarlythe present study also showed that both EEP and WEPcontain high levels of flavonoids and phenolics and exhibitantioxidant activity The most frequently used antioxidantstandards for food samples (vitamin C Trolox catechinresveratrol and quercetin) were used as positive antioxidantcontrols in the present study
This study revealed that DPPH radical scavenging capac-ity of WEP is greater than that of EEP (Figure 1) In contrastEEP has higher ferric reducing power than WEP (Table 2)Such contradictory results between DPPH and FRAP assaysare not unusual Though both assays are based on a singleelectron transfer reaction [27] their characteristics sensitiv-ities mechanisms of the reaction and endpoints are totallydifferent For instance the DPPH method is based on thefree radical scavenging activity while FRAP measures thecapability of reducing Fe3+ to Fe2+ Depending on whatspecific phytochemical constituents present in the extractare providing the antioxidant activity their discrete chemicalstructures positions numbers and types of substitutions caninfluence their redox properties and hence their antioxidantpotentials [28]
Though both DPPH and FRAP assays are frequently usedfor assessing antioxidant capacity they have some drawbacksIn the DPPH assay interfering compounds may have sig-nificant absorption at the same measured wavelength In
Evidence-Based Complementary and Alternative Medicine 7
Figure 3 Cellular radical scavenging activity in tBuOOH-activated RAW2647 cells Intracellular ROS level generated in cells was measuredby the DCFH-DA RAW2647 cells were pretreated with indicated concentrations of antioxidants (a) EEP (b) or WEP (c) for 24 h priorto take-up of 20 120583M DCFH-DA for 30min Results are mean plusmn SEM (119899 = 4) and are representative of three independent experiments withsimilar results Pointsmarked with different letters are significantly different at119875 lt 005when compared at the same time point as determinedby two-way ANOVA
addition theDPPH radical is not present in living organismsFor the FRAPmethod compounds with low redox potentialwhich probably do not serve as antioxidants in vivo still canreduce the Fe3+ Interfering compounds may also absorb atthe same wavelength and the assay is also performed at anonphysiological pH [29] Therefore antioxidant activitiesof EEP and WEP were also evaluated by the cell-basedassay using an intracellular fluorescent probe DCFH-DAWhen the nonfluorescent DCFH-DA is taken up into cellsits diacetate moiety will be hydrolyzed by cellular esterasesto generate the more polar DCFH which is trapped insidethe cells In the presence of ROS intracellular DCFH isfurther oxidized to form the fluorescent DCF product [30]
The macrophage cell line RAW2647 is usually the cell ofchoice in studying ROS-mediated cellular events since itcan generate high amounts of ROS following an oxidantchallenge Catechin resveratrol and quercetin at the levelof concentration used in this study have been shown andoptimized to exhibit a strong suppression of intracellular ROSgeneration [22 31 32] Therefore the present study selectedthese compounds as antioxidant positive controls for theDCFH-DA assay The present study demonstrated that allantioxidant standards 250120583M catechin 20120583M resveratroland 10 120583M quercetin exerted a strong inhibition of ROSgeneration induced by tBuOOH over a period of 30 to240min In addition to extracellular antioxidant capacity
8 Evidence-Based Complementary and Alternative Medicine
45
40
35
30
25
20
15
10
5
0
NA
VH 50
150
250
e
d
c
bb
a
e
d
bcNitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
EEP (120583gmL)
(a)
b
e
cc
bc
d
f
a
45
40
35
30
25
20
15
10
5
0
NA 50
150
250
Nitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
WEP (120583gmL)
(b)
Figure 4 EEP (a) and WEP (b) suppressed LPS plus IFN-120574-induced nitrite production in RAW2647 cells RAW2647 cells were incubatedfor 24 hwith LPS (1120583gmL) plus IFN-120574 (25UmL) in the presence or absence of indicated concentrations of vitaminC (500120583M) EEP orWEPAccumulated nitrite in the culturemediumwas determined by the Griess reactionThe values aremeansplusmn SEM (119899 = 3) and are representativeof three independent experiments with similar results Bars marked with different letters are significantly different at 119875 lt 005 as determinedby one-way ANOVA
EEP and WEP also served as intracellular ROS scavengersand subsequently decreased the oxidation of DCFH (Figures3(b) and 3(c)) Both EEP and WEP were as efficient as theantioxidant standards in scavenging ROS Notably EEP was abetter reducer of DCF fluorescence thanWEPThe reductionof DCF fluorescence by EEP and WEP is not due to directcytotoxicity as the range of concentration used in the studieshad no effect on RAW2647 cell viability (Figure 2)
Although the current study shows that an ethanol extractfrom PP leaves has in vivo anti-inflammatory activities[13] its mechanism of anti-inflammation is still unrevealedInflammatory disorders are characterized among otherevents by the production of significant amounts of freeradicals nitrogen reactive species and pro-inflammatorycytokines [10] High NO concentration combines with super-oxides to form peroxynitrite ions (OONOminus) which areresponsible for cell and tissue damage from inflammation[33] Therefore we investigated inflammatory effects of EEPand WEP on the suppression of NO production in LPS plusIFN-120574-activated RAW2647 cells At the concentration rangeof 50ndash250120583gmL both EEP and WEP dose-dependentlysuppressed NO production and the suppression was morepronounced in EEP than WEP (Figure 4) These resultsagreed with the observation that EEP was also a betterscavenger of intracellular ROS than WEP (Figures 3(b) and3(c))
As enhanced NO production by LPS and IFN-120574-stimulated RAW2647 cells mainly occurs via increasedintracellular content of iNOS [3 4] the effect of EEP andWEP on iNOS expression was investigatedThe present studyclearly indicates that the suppressive effect of EEP and WEPon NO production was mediated through the inhibition of
iNOS expression (Figures 5(a) and 5(b)) In agreement withthe study of NO suppression the suppressive effect of EEP(50ndash200120583gmL) on iNOS was more remarkable than that ofWEP
In addition to iNOS induction LPS and IFN-120574 alsoefficiently enhance COX-2 expression in RAW2647 cells [35] An increased level of COX-2 expression is also knownto account for the excessive production of PGE
2in most
if not all inflammatory cells and tissues [34] This studyshows that both EEP andWEP can exhibit anti-inflammatoryactivity by reducing high COX-2 protein levels in a dose-related manner (Figures 5(c) and 5(d)) Thus EEP and WEPmight play important roles in attenuating inflammationand cellular damage through their extra- and intracellularROS scavenging activity and downregulation of NO iNOSand COX-2 Concordantly Khumpook et al [13] recentlyreported the in vivo anti-inflammatory activity of PP leavesas evidenced by decreased lipid peroxidation and NO levelin concomitance with increased superoxide dismutase in thecotton-induced chronic inflammation in Albino rats uponexposure to an ethanol extract of PP leaves for 17 days
In fact several medicinal plant extracts with naturalantioxidant properties together with suppressive effects onNO iNOS andorCOX-2 expression inRAW2647 have beenreported to display a wide spectrum of bioactivities Theseactivities include anti-inflammation such as curcumin fromCurcuma longa resveratrol from grape skins red wines andother plants and a mixture of 120573-sitosterol and stigmasterolfrom Andrographis paniculata [8 35 36] Previous investi-gators demonstrated that pretreatment of RAW2647 withflavonoids such as apigenin genistein and kaempferol sup-pressed LPS-stimulated expression of NO iNOS and COX-2
Evidence-Based Complementary and Alternative Medicine 9
FoldiNOS
Tubulin
003 086 058 030 061 038 033 024 015
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
EEP (120583gmL)
LPSIFN-120574
(a)
Fold
Tubulin
iNOS
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
WEP (120583gmL)
LPSIFN-120574
008 111 083 052 073 063 048 036 008
(b)
COX-2
Fold
Tubulin
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
006 093 076 074 087 079 065 044 030
EEP (120583gmL)
LPSIFN-120574
(c)
Fold
Tubulin
COX-2
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
001 113 066 063 091 078 067 064 050
WEP (120583gmL)
LPSIFN-120574
(d)
Figure 5 Effect of EEP on LPS plus IFN-120574-induced iNOS (a) and COX-2 (c) andWEP on LPS plus IFN-120574-induced iNOS (b) and COX-2 (d)protein levels in RAW2647 cells The relative expression of proteins was quantified densitometrically using ImageJ software and normalizedto tubulin reference bands Data are representative of at least two independent experiments
protein production [37] Major chemical constituents of PPleaves consist of 120573-sitosterol stigmasterol kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucoside[11] All aforementioned compounds have been shown topossess anti-inflammatory properties Both kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucosidemay be metabolized into kaempferol and apigenin whichalso have antioxidant and anti-inflammatory activities Thusit is possible that phenolic and flavonoid compounds inboth EEP andWEP provide substantial antioxidant and anti-inflammatory activities
In summary the cytoprotective effects of EEP and WEPis due to their abilities to decrease ROS generation and NOradical production in cells In addition both EEP and WEPexert anti-inflammatory effects through the suppression ofNO release and decrease the protein expression of iNOSand COX-2 Thus PP leaves possess high potential forfurther exploration in the research development of anti-inflammatory medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was financially supported by a Royal GoldenJubilee Scholarship Grant (PhD02082548) and by the
National Research Council of Thailand (NRCT) throughSuranaree University of Technology (SUT-104-53-36-09)
References
[1] B Halliwell and J M C Gutteridge Free Radicals in BiologyandMedicine OxfordUniversity Press NewYork NYUSA 3rdedition 1999
[2] N Fujiwara and K Kobayashi ldquoMacrophages in inflammationrdquoCurrent Drug TargetsmdashInflammation and Allergy vol 4 no 3pp 281ndash286 2005
[3] S I Jang Y-J Kim W-Y Lee et al ldquoScoparone from Artemisiacapillaris inhibits the release of inflammatory mediators inRAW 2647 cells upon stimulation cells by interferon-120574 plusLPSrdquo Archives of Pharmacal Research vol 28 no 2 pp 203ndash208 2005
[4] R Korhonen A Lahti H Kankaanranta and E MoilanenldquoNitric oxide production and signaling in inflammationrdquo Cur-rent Drug TargetsmdashInflammation and Allergy vol 4 no 4 pp471ndash479 2005
[5] E Karpuzoglu and S A Ahmed ldquoEstrogen regulation of nitricoxide and inducible nitric oxide synthase (iNOS) in immunecells implications for immunity autoimmune diseases andapoptosisrdquo Nitric Oxide vol 15 no 3 pp 177ndash186 2006
[6] S G Harris J Padilla L Koumas D Ray and R PPhipps ldquoProstaglandins as modulators of immunityrdquo Trends inImmunology vol 23 no 3 pp 144ndash150 2002
[7] I-N Hsieh A S-Y Chang C-M Teng C-C Chen andC-R Yang ldquoAciculatin inhibits lipopolysaccharide-mediated
10 Evidence-Based Complementary and Alternative Medicine
inducible nitric oxide synthase and cyclooxygenase-2 expres-sion via suppressing NF-120581B and JNKp38 MAPK activationpathwaysrdquo Journal of Biomedical Science vol 18 no 1 article28 2011
[8] J B Calixto M F Otuki and A R S Santos ldquoAnti-inflammatory compounds of plant originmdashpart I action onarachidonic acid pathway nitric oxide and nuclear factor 120581 B(NF-120581B)rdquo Planta Medica vol 69 no 11 pp 973ndash983 2003
[9] K B Pandey and S I Rizvi ldquoCurrent understanding of dietarypolyphenols and their role in health and diseaserdquo CurrentNutrition and Food Science vol 5 no 4 pp 249ndash263 2009
[10] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer How are theylinkedrdquo Free Radical Biology and Medicine vol 49 no 11 pp1603ndash1616 2010
[11] P M Giang H V Bao and P T Son ldquoPhytochemical studyon Pseuderanthemum palatiferum (Nees) Radlk AcanthaceaerdquoJournal of Chemistry vol 41 no 2 pp 115ndash118 2003
[12] H K Dieu C B Loc S Yamasaki and Y Hirata ldquoThe ethnob-otanical and botanical study on Pseuderanthemum palatiferumas a new medicinal plant in the Mekong Delta of VietnamrdquoJapan Agricultural Research Quarterly vol 39 no 3 pp 191ndash1962005
[13] T Khumpook S Chomdej S Saenphet D Amornlerdpi-son and K Saenphet ldquoAnti-inflammatory activity of ethanolextract from the leaves of Pseuderanthemum palatiferum (Nees)RadlkrdquoChiangMai Journal of Science vol 40 no 3 pp 321ndash3312013
[14] Q-V Nguyen and J-B Eun ldquoAntioxidant activity of solventextracts from Vietnamese medicinal plantsrdquo Journal of Medic-inal Plant Research vol 5 no 13 pp 2798ndash2811 2011
[15] P M Giang H V Bao and P T Son ldquoStudy on anti-oxidativeactivities and preliminary investigation on antibacterial anti-fungal of extracted fraction rich in flavonoids from leaves ofPseuderanthemum palatiferum (Nees) Radlkrdquo TC Duoc Hocvol 9 no 9 pp 9ndash12 2005 (In Vietnamese with Englishsummary)
[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998
[17] M Liu X Q Li C Weber C Y Lee J Brown and R HLiu ldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002
[18] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[19] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoFreeradical scavenging capacity and inhibition of lipid oxidation ofwines grape juices and related polyphenolic constituentsrdquo FoodResearch International vol 32 no 6 pp 407ndash412 1999
[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958
[21] S-C Chun S Y Jee S G Lee S J Park J R Lee and SC Kim ldquoAnti-inflammatory activity of the methanol extractof Moutan Cortex in LPS-activated Raw2647 cellsrdquo Evidence-Based Complementary and Alternative Medicine vol 4 no 3pp 327ndash333 2007
[22] G-N Kim Y-I Kwon and H-D Jang ldquoProtective mech-anism of quercetin and rutin on 22rsquo-azobis(2-amidino-propane)dihydrochloride or Cu2+-induced oxidative stress inHepG2 cellsrdquoToxicology in Vitro vol 25 no 1 pp 138ndash144 2011
[23] M G B de Oliveira R B Marques M F de Santana et al ldquo120572-Terpineol reducesmechanical hypernociception and inflamma-tory responserdquoBasic ampClinical PharmacologyampToxicology vol111 no 2 pp 120ndash125 2012
[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[25] A K L Nascimento R FMelo-Silveira N Dantas-Santos et alldquoAntioxidant and antiproliferative activities of leaf extracts fromPlukenetia volubilis Linneo (Euphorbiaceae)rdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID950272 10 pages 2013
[26] A Muthuraman N Singh and A S Jaggi ldquoProtective effectof Acorus calamus L in rat model of vincristine inducedpainful neuropathy an evidence of anti-inflammatory and anti-oxidative activityrdquo Food and Chemical Toxicology vol 49 no 10pp 2557ndash2563 2011
[27] A Bunea DO Rugina AM Pintea Z Sconta C I Bunea andC Socaciu ldquoComparative polyphenolic content and antioxidantactivities of some wild and cultivated blueberries from Roma-niardquoNotulae Botanicae Horti Agrobotanici Cluj-Napoca vol 39no 2 pp 70ndash76 2011
[28] K E Heim A R Tagliaferro and D J Bobilya ldquoFlavonoidantioxidants chemistry metabolism and structure-activityrelationshipsrdquo Journal of Nutritional Biochemistry vol 13 no10 pp 572ndash584 2002
[29] J Perez-Jimenez S Arranz M Tabernero et al ldquoUpdatedmethodology to determine antioxidant capacity in plant foodsoils and beverages extraction measurement and expression ofresultsrdquo Food Research International vol 41 no 3 pp 274ndash2852008
[30] K L Wolfe and R H Lui ldquoCellular antioxidant activity(CAA) assay for assessing antioxidants foods and dietarysupplementsrdquo Journal of Agricultural and Food Chemistry vol55 no 22 pp 8896ndash8907 2007
[31] M Okawara H Katsuki E Kurimoto H Shibata T Kumeand A Akaike ldquoResveratrol protects dopaminergic neuronsin midbrain slice culture from multiple insultsrdquo BiochemicalPharmacology vol 73 no 4 pp 550ndash560 2007
[32] H Nishikawa KWakano and S Kitani ldquoInhibition of NADPHoxidase subunits translocation by tea catechin EGCG in mastcellrdquo Biochemical and Biophysical Research Communicationsvol 362 no 2 pp 504ndash509 2007
[33] J Fang T Seki and H Maeda ldquoTherapeutic strategies by mod-ulating oxygen stress in cancer and inflammationrdquo AdvancedDrug Delivery Reviews vol 61 no 4 pp 290ndash302 2009
[34] K Subbaramaiah and A J Dannenberg ldquoCyclooxygenase 2 amolecular target for cancer prevention and treatmentrdquo Trendsin Pharmacological Sciences vol 24 no 2 pp 96ndash102 2003
[35] Y-J Surh K-S ChunH-H Cha et al ldquoMolecularmechanismsunderlying chemopreventive activities of anti-inflammatoryphytochemicals down-regulation of COX-2 and iNOS throughsuppression of NF-120581B activationrdquoMutation Research vol 480-481 pp 243ndash268 2001
[36] W-W Chao Y-H Kuo and B-F Lin ldquoAnti-inflammatoryactivity of new compounds from Andrographis paniculata byNF-120581B transactivation inhibitionrdquo Journal of Agricultural andFood Chemistry vol 58 no 4 pp 2505ndash2512 2010
Evidence-Based Complementary and Alternative Medicine 11
[37] Y-C Liang Y-T Huang S-H Tsai S-Y Lin-Shiau C-FChen and J-K Lin ldquoSuppression of inducible cyclooxygenaseand inducible nitric oxide synthase by apigenin and relatedflavonoids in mouse macrophagesrdquo Carcinogenesis vol 20 no10 pp 1945ndash1952 1999
Evidence-Based Complementary and Alternative Medicine 5
80
100
60
40
20
0
0 5 10 15 20 25 30 35
Inhi
bitio
n (
)
Concentration (120583gmL)
WEPEEPCatechin
Vitamin CTrolox
Figure 1 DPPH radical scavenging activity of PP leaf extracts (EEPand WEP) and positive controls (vitamin C Trolox and catechin)Values are means plusmn SEM (119899 = 3) and are representative of threeindependent experiments with similar results
antioxidants vitamin C Trolox and catechin The results ofFRAP values in terms of ferrous ion (Fe2+) and vitamin CTrolox and catechin equivalents are shown in Table 2 EEPexhibited a higher degree of electron donating capacity thanWEP as suggested by the significantly higher FRAP values(119875 lt 005) of EEP when compared with WEP
34 DPPH Free Radical Scavenging Activity The free radicalscavenging capacities of EEP andWEP are shown in Figure 1The results show that both EEP and WEP exhibit the abilityto scavenge DPPH free radicals The scavenging activityagainst DPPH radicals of WEP (IC
50= 2155 plusmn 006 120583gmL)
is significantly greater (119875 lt 0001) than EEP (IC50
=2345 plusmn 012120583gmL) by 19 plusmn 015 but the scavengingcapacity of these is not as effective as the other positiveantioxidant controls 125 120583gmL EEP and 25 120583gmL WEPscavenged the DPPH radicals by 2927 plusmn 020 and 612 plusmn015 respectively and the scavenging capacities of bothare more pronounced at higher concentrations The highestconcentration (325120583gmL) of EEP andWEP could scavengethe DPPH radicals by 6596 plusmn 021 and 7319 plusmn 009respectively In the present study the scavenging abilities ofvitamin C (IC
50= 394 plusmn 001 120583gmL) and catechin (IC
50=
355 plusmn 001 120583gmL) were similar and both are significantlyhigher (119875 lt 0001) than Trolox (IC
50= 590 plusmn 027 120583gmL)
35 Effect of EEP and WEP on RAW2647 Cell Viability Thecell viability of RAW2647 cells exposed to EEP or WEP wasdetermined by MTT assay The cells were incubated for 24 hwith various concentrations of EEP (005 025 05 10 or150mgmL) or WEP (010 050 150 or 450mgmL) Asshown in Figure 2 both EEP andWEP displayed low toxicitytowards RAW2647 cells as evidenced by an apparent lack ofeffect on cell viability until the concentration of each extract
reached 15mgmL At 15mgmL EEP and WEP decreasedthe viability of RAW2647 cells by 3414 plusmn 969 and 2158 plusmn166 (119875 lt 005) respectively However the cytotoxiceffect is more pronounced at higher concentrations WEP at45mgmL decreased the cell viability by as much as 5421 plusmn174 (119875 lt 005) The effect of EEP and WEP on RAW2647cell viability was also confirmed by trypan blue exclusion andpropidium iodide staining methods which exhibited similarresults (data not shown)Therefore a nontoxic concentrationrange of 0ndash025mgmL of both EEP and WEP was selectedfor RAW2647 cell treatment in the subsequent studies
36 EEP and WEP as Intracellular ROS Scavengers Thedirect scavenging effect of EEP and WEP on intracellularfree radical stress was investigated in RAW2647 cells usingthe DCFH-DA assay The increment of DCF fluorescenceemission following ROS-mediated oxidation of DCFH wasfollowed for 240min As shown in Figure 3(a) standardantioxidant positive controls catechin (250 120583M) resveratrol(20120583M) and quercetin (10 120583M) could scavenge ROS signif-icantly (119875 lt 005) throughout the incubation time whencompared to the vehicle control (VH)With as little as 30minof incubation catechin resveratrol and quercetin showedconsiderable radical scavenging activity EEP (Figure 3(b))and WEP (Figure 3(c)) decreased the DCF fluorescent emis-sion in a dose- and time-dependent manner Again with aslittle as 30min of incubation both EEP and WEP at lowconcentration (50120583gmL) showed similar radical scavengingactivity as the antioxidant controls Various concentrations ofEEP significantly decreased (119875 lt 005) the DCF fluorescentemission throughout the incubation time when comparedto the VH control At high concentration (150 120583gmL) EEPexhibited a strong scavenging activity as suggested by thecapability to lower fluorescent intensity to below basal levelof the unstimulated DCFH-DA control at 180ndash240min Inaddition the highest concentration of EEP (250120583gmL)significantly lowered (119875 lt 005) DCF fluorescent intensityto below the basal level at all time points Similarly 150 and250 120583gmL of WEP also significantly decreased (119875 lt 005)the DCF fluorescent emission throughout the incubationtime compared to the tBuOOH control However the lowestconcentration ofWEP (50120583gmL) significantly reduced (119875 lt005) the DCF fluorescent emission until 210min only
37 NO Suppression by EEP and WEP in LPS Plus IFN-120574-Activated RAW2647 Cells RAW2647 cells were pretreatedwith antioxidants vitamin C EEP or WEP for 1 h thenstimulated with LPS plus IFN-120574 and measured for NOproduction using the Griess assay As shown in Figure 4unstimulated RAW2647 cells (NA) secreted basal levels ofNO while the production of NO was increased to about43 120583M in LPS plus IFN-120574-activated RAW2647 cells Theantioxidant control 500 120583M vitamin C decreased the NOproduction by almost 35 Pretreatment of RAW2647 cellswith EEP or WEP significantly suppressed (119875 lt 005) theinduction of NO in a dose-related manner (Figures 4(a) and4(b)) and the suppression was observed in all EEP- andWEP-treated groups These results also clearly indicate that
6 Evidence-Based Complementary and Alternative Medicine
80
60
40
20
0
b b b b
aab
Cel
l via
bilit
y (
)120
100
Control 005 025 050 150100
EEP (mgmL)
(a)
80
60
40
20
0
b
a
ccd
d
Cel
l via
bilit
y (
)
120
100
Control 010 050 150 450
WEP (mgmL)
(b)
Figure 2 Effect of EEP andWEP on cell viability of RAW2647 cellsThe effect of EEP (a) andWEP (b) on cell viability was assessed byMTTValues are expressed as means plusmn SEM (119899 = 3) and are representative of three independent experiments with similar results Bars marked withdifferent letters are significantly different at 119875 lt 005 as determined by one-way ANOVA
EEP is a stronger suppressant of NO induction than WEPConcentrations of 50 120583gmL of EEP and 150 and 200 120583gmLofWEPwere required to exhibit the NO suppression with thesame efficiency as 500 120583M (8806120583gmL) vitamin C
38 Suppression of iNOS and COX-2 Protein Expression byEEP and WEP in LPS Plus IFN-120574-Activated RAW2647 CellsTo determine if suppression of NO production by EEP orWEPwas related to changes in iNOS aswell as COX-2 proteinlevels Western blotting analysis was performed RAW2647cells were pretreated with antioxidants Trolox (50120583gmL)vitamin C (500120583M) or PP extracts (EEP or WEP) at 50ndash250120583gmL for 1 h prior activation with LPS (1120583gmL) plusIFN-120574 (25UmL) for 18 h Total proteins were extracted andanalyzed for the expression of iNOS and COX-2 by Westernblotting LPS plus IFN-120574 induced increases in iNOS (Figures5(a) and 5(b)) and COX-2 (Figures 5(c) and 5(d)) expressioncompared to the unstimulated cultures Antioxidant controls(Trolox and vitamin C) decreased LPS plus IFN-120574-inducediNOS and COX-2 protein levels The data also suggestedthat the suppression by 500120583M (8806120583gmL) vitamin Cis more pronounced than 50 120583gmL Trolox Compared tothe corresponding controls both EEP and WEP produced adose-dependent suppression of iNOS level in LPS plus IFN-120574-activated RAW2647 cells (Figures 5(a) and 5(b)) suggestingthat the suppression of NO production by EEP and WEP ismediated by decreasing the expression of iNOS In agreementwith the result of NO suppression 50ndash200 120583gmL EEP wasprobably more efficient than WEP in iNOS suppressionThe iNOS expression was almost completely eliminated at200120583gmL EEP and was barely observed at 250 120583gmLWEPThe inflammatory modulation of EEP and WEP was alsofurther supported by the dose-dependent suppression of theCOX-2 level by both EEP and WEP (Figures 5(c) and 5(d))in the activated RAW2647 cells Notably EEP and WEPexhibited higher suppression of iNOS than COX-2
4 Discussion
It is well known that major phytochemicals of plant leafextracts possessing antioxidant activity are flavonoids andother phenolic compounds Researchers have found thatflavonoids from PP leaves display antioxidant activity andall ethyl acetate chloroform and 119899-butanol-soluble fractionsof PP contain flavonoids [11 15] In addition Nguyen andEun [14] found phenolics and flavonoids in extracts of PPleaves when assessed with Folin-Ciocalteu and aluminumtrichloride PP leaf extracts also have antioxidant activitieswhen evaluated with DPPH and FRAP assays Similarlythe present study also showed that both EEP and WEPcontain high levels of flavonoids and phenolics and exhibitantioxidant activity The most frequently used antioxidantstandards for food samples (vitamin C Trolox catechinresveratrol and quercetin) were used as positive antioxidantcontrols in the present study
This study revealed that DPPH radical scavenging capac-ity of WEP is greater than that of EEP (Figure 1) In contrastEEP has higher ferric reducing power than WEP (Table 2)Such contradictory results between DPPH and FRAP assaysare not unusual Though both assays are based on a singleelectron transfer reaction [27] their characteristics sensitiv-ities mechanisms of the reaction and endpoints are totallydifferent For instance the DPPH method is based on thefree radical scavenging activity while FRAP measures thecapability of reducing Fe3+ to Fe2+ Depending on whatspecific phytochemical constituents present in the extractare providing the antioxidant activity their discrete chemicalstructures positions numbers and types of substitutions caninfluence their redox properties and hence their antioxidantpotentials [28]
Though both DPPH and FRAP assays are frequently usedfor assessing antioxidant capacity they have some drawbacksIn the DPPH assay interfering compounds may have sig-nificant absorption at the same measured wavelength In
Evidence-Based Complementary and Alternative Medicine 7
Figure 3 Cellular radical scavenging activity in tBuOOH-activated RAW2647 cells Intracellular ROS level generated in cells was measuredby the DCFH-DA RAW2647 cells were pretreated with indicated concentrations of antioxidants (a) EEP (b) or WEP (c) for 24 h priorto take-up of 20 120583M DCFH-DA for 30min Results are mean plusmn SEM (119899 = 4) and are representative of three independent experiments withsimilar results Pointsmarked with different letters are significantly different at119875 lt 005when compared at the same time point as determinedby two-way ANOVA
addition theDPPH radical is not present in living organismsFor the FRAPmethod compounds with low redox potentialwhich probably do not serve as antioxidants in vivo still canreduce the Fe3+ Interfering compounds may also absorb atthe same wavelength and the assay is also performed at anonphysiological pH [29] Therefore antioxidant activitiesof EEP and WEP were also evaluated by the cell-basedassay using an intracellular fluorescent probe DCFH-DAWhen the nonfluorescent DCFH-DA is taken up into cellsits diacetate moiety will be hydrolyzed by cellular esterasesto generate the more polar DCFH which is trapped insidethe cells In the presence of ROS intracellular DCFH isfurther oxidized to form the fluorescent DCF product [30]
The macrophage cell line RAW2647 is usually the cell ofchoice in studying ROS-mediated cellular events since itcan generate high amounts of ROS following an oxidantchallenge Catechin resveratrol and quercetin at the levelof concentration used in this study have been shown andoptimized to exhibit a strong suppression of intracellular ROSgeneration [22 31 32] Therefore the present study selectedthese compounds as antioxidant positive controls for theDCFH-DA assay The present study demonstrated that allantioxidant standards 250120583M catechin 20120583M resveratroland 10 120583M quercetin exerted a strong inhibition of ROSgeneration induced by tBuOOH over a period of 30 to240min In addition to extracellular antioxidant capacity
8 Evidence-Based Complementary and Alternative Medicine
45
40
35
30
25
20
15
10
5
0
NA
VH 50
150
250
e
d
c
bb
a
e
d
bcNitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
EEP (120583gmL)
(a)
b
e
cc
bc
d
f
a
45
40
35
30
25
20
15
10
5
0
NA 50
150
250
Nitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
WEP (120583gmL)
(b)
Figure 4 EEP (a) and WEP (b) suppressed LPS plus IFN-120574-induced nitrite production in RAW2647 cells RAW2647 cells were incubatedfor 24 hwith LPS (1120583gmL) plus IFN-120574 (25UmL) in the presence or absence of indicated concentrations of vitaminC (500120583M) EEP orWEPAccumulated nitrite in the culturemediumwas determined by the Griess reactionThe values aremeansplusmn SEM (119899 = 3) and are representativeof three independent experiments with similar results Bars marked with different letters are significantly different at 119875 lt 005 as determinedby one-way ANOVA
EEP and WEP also served as intracellular ROS scavengersand subsequently decreased the oxidation of DCFH (Figures3(b) and 3(c)) Both EEP and WEP were as efficient as theantioxidant standards in scavenging ROS Notably EEP was abetter reducer of DCF fluorescence thanWEPThe reductionof DCF fluorescence by EEP and WEP is not due to directcytotoxicity as the range of concentration used in the studieshad no effect on RAW2647 cell viability (Figure 2)
Although the current study shows that an ethanol extractfrom PP leaves has in vivo anti-inflammatory activities[13] its mechanism of anti-inflammation is still unrevealedInflammatory disorders are characterized among otherevents by the production of significant amounts of freeradicals nitrogen reactive species and pro-inflammatorycytokines [10] High NO concentration combines with super-oxides to form peroxynitrite ions (OONOminus) which areresponsible for cell and tissue damage from inflammation[33] Therefore we investigated inflammatory effects of EEPand WEP on the suppression of NO production in LPS plusIFN-120574-activated RAW2647 cells At the concentration rangeof 50ndash250120583gmL both EEP and WEP dose-dependentlysuppressed NO production and the suppression was morepronounced in EEP than WEP (Figure 4) These resultsagreed with the observation that EEP was also a betterscavenger of intracellular ROS than WEP (Figures 3(b) and3(c))
As enhanced NO production by LPS and IFN-120574-stimulated RAW2647 cells mainly occurs via increasedintracellular content of iNOS [3 4] the effect of EEP andWEP on iNOS expression was investigatedThe present studyclearly indicates that the suppressive effect of EEP and WEPon NO production was mediated through the inhibition of
iNOS expression (Figures 5(a) and 5(b)) In agreement withthe study of NO suppression the suppressive effect of EEP(50ndash200120583gmL) on iNOS was more remarkable than that ofWEP
In addition to iNOS induction LPS and IFN-120574 alsoefficiently enhance COX-2 expression in RAW2647 cells [35] An increased level of COX-2 expression is also knownto account for the excessive production of PGE
2in most
if not all inflammatory cells and tissues [34] This studyshows that both EEP andWEP can exhibit anti-inflammatoryactivity by reducing high COX-2 protein levels in a dose-related manner (Figures 5(c) and 5(d)) Thus EEP and WEPmight play important roles in attenuating inflammationand cellular damage through their extra- and intracellularROS scavenging activity and downregulation of NO iNOSand COX-2 Concordantly Khumpook et al [13] recentlyreported the in vivo anti-inflammatory activity of PP leavesas evidenced by decreased lipid peroxidation and NO levelin concomitance with increased superoxide dismutase in thecotton-induced chronic inflammation in Albino rats uponexposure to an ethanol extract of PP leaves for 17 days
In fact several medicinal plant extracts with naturalantioxidant properties together with suppressive effects onNO iNOS andorCOX-2 expression inRAW2647 have beenreported to display a wide spectrum of bioactivities Theseactivities include anti-inflammation such as curcumin fromCurcuma longa resveratrol from grape skins red wines andother plants and a mixture of 120573-sitosterol and stigmasterolfrom Andrographis paniculata [8 35 36] Previous investi-gators demonstrated that pretreatment of RAW2647 withflavonoids such as apigenin genistein and kaempferol sup-pressed LPS-stimulated expression of NO iNOS and COX-2
Evidence-Based Complementary and Alternative Medicine 9
FoldiNOS
Tubulin
003 086 058 030 061 038 033 024 015
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
EEP (120583gmL)
LPSIFN-120574
(a)
Fold
Tubulin
iNOS
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
WEP (120583gmL)
LPSIFN-120574
008 111 083 052 073 063 048 036 008
(b)
COX-2
Fold
Tubulin
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
006 093 076 074 087 079 065 044 030
EEP (120583gmL)
LPSIFN-120574
(c)
Fold
Tubulin
COX-2
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
001 113 066 063 091 078 067 064 050
WEP (120583gmL)
LPSIFN-120574
(d)
Figure 5 Effect of EEP on LPS plus IFN-120574-induced iNOS (a) and COX-2 (c) andWEP on LPS plus IFN-120574-induced iNOS (b) and COX-2 (d)protein levels in RAW2647 cells The relative expression of proteins was quantified densitometrically using ImageJ software and normalizedto tubulin reference bands Data are representative of at least two independent experiments
protein production [37] Major chemical constituents of PPleaves consist of 120573-sitosterol stigmasterol kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucoside[11] All aforementioned compounds have been shown topossess anti-inflammatory properties Both kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucosidemay be metabolized into kaempferol and apigenin whichalso have antioxidant and anti-inflammatory activities Thusit is possible that phenolic and flavonoid compounds inboth EEP andWEP provide substantial antioxidant and anti-inflammatory activities
In summary the cytoprotective effects of EEP and WEPis due to their abilities to decrease ROS generation and NOradical production in cells In addition both EEP and WEPexert anti-inflammatory effects through the suppression ofNO release and decrease the protein expression of iNOSand COX-2 Thus PP leaves possess high potential forfurther exploration in the research development of anti-inflammatory medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was financially supported by a Royal GoldenJubilee Scholarship Grant (PhD02082548) and by the
National Research Council of Thailand (NRCT) throughSuranaree University of Technology (SUT-104-53-36-09)
References
[1] B Halliwell and J M C Gutteridge Free Radicals in BiologyandMedicine OxfordUniversity Press NewYork NYUSA 3rdedition 1999
[2] N Fujiwara and K Kobayashi ldquoMacrophages in inflammationrdquoCurrent Drug TargetsmdashInflammation and Allergy vol 4 no 3pp 281ndash286 2005
[3] S I Jang Y-J Kim W-Y Lee et al ldquoScoparone from Artemisiacapillaris inhibits the release of inflammatory mediators inRAW 2647 cells upon stimulation cells by interferon-120574 plusLPSrdquo Archives of Pharmacal Research vol 28 no 2 pp 203ndash208 2005
[4] R Korhonen A Lahti H Kankaanranta and E MoilanenldquoNitric oxide production and signaling in inflammationrdquo Cur-rent Drug TargetsmdashInflammation and Allergy vol 4 no 4 pp471ndash479 2005
[5] E Karpuzoglu and S A Ahmed ldquoEstrogen regulation of nitricoxide and inducible nitric oxide synthase (iNOS) in immunecells implications for immunity autoimmune diseases andapoptosisrdquo Nitric Oxide vol 15 no 3 pp 177ndash186 2006
[6] S G Harris J Padilla L Koumas D Ray and R PPhipps ldquoProstaglandins as modulators of immunityrdquo Trends inImmunology vol 23 no 3 pp 144ndash150 2002
[7] I-N Hsieh A S-Y Chang C-M Teng C-C Chen andC-R Yang ldquoAciculatin inhibits lipopolysaccharide-mediated
10 Evidence-Based Complementary and Alternative Medicine
inducible nitric oxide synthase and cyclooxygenase-2 expres-sion via suppressing NF-120581B and JNKp38 MAPK activationpathwaysrdquo Journal of Biomedical Science vol 18 no 1 article28 2011
[8] J B Calixto M F Otuki and A R S Santos ldquoAnti-inflammatory compounds of plant originmdashpart I action onarachidonic acid pathway nitric oxide and nuclear factor 120581 B(NF-120581B)rdquo Planta Medica vol 69 no 11 pp 973ndash983 2003
[9] K B Pandey and S I Rizvi ldquoCurrent understanding of dietarypolyphenols and their role in health and diseaserdquo CurrentNutrition and Food Science vol 5 no 4 pp 249ndash263 2009
[10] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer How are theylinkedrdquo Free Radical Biology and Medicine vol 49 no 11 pp1603ndash1616 2010
[11] P M Giang H V Bao and P T Son ldquoPhytochemical studyon Pseuderanthemum palatiferum (Nees) Radlk AcanthaceaerdquoJournal of Chemistry vol 41 no 2 pp 115ndash118 2003
[12] H K Dieu C B Loc S Yamasaki and Y Hirata ldquoThe ethnob-otanical and botanical study on Pseuderanthemum palatiferumas a new medicinal plant in the Mekong Delta of VietnamrdquoJapan Agricultural Research Quarterly vol 39 no 3 pp 191ndash1962005
[13] T Khumpook S Chomdej S Saenphet D Amornlerdpi-son and K Saenphet ldquoAnti-inflammatory activity of ethanolextract from the leaves of Pseuderanthemum palatiferum (Nees)RadlkrdquoChiangMai Journal of Science vol 40 no 3 pp 321ndash3312013
[14] Q-V Nguyen and J-B Eun ldquoAntioxidant activity of solventextracts from Vietnamese medicinal plantsrdquo Journal of Medic-inal Plant Research vol 5 no 13 pp 2798ndash2811 2011
[15] P M Giang H V Bao and P T Son ldquoStudy on anti-oxidativeactivities and preliminary investigation on antibacterial anti-fungal of extracted fraction rich in flavonoids from leaves ofPseuderanthemum palatiferum (Nees) Radlkrdquo TC Duoc Hocvol 9 no 9 pp 9ndash12 2005 (In Vietnamese with Englishsummary)
[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998
[17] M Liu X Q Li C Weber C Y Lee J Brown and R HLiu ldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002
[18] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[19] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoFreeradical scavenging capacity and inhibition of lipid oxidation ofwines grape juices and related polyphenolic constituentsrdquo FoodResearch International vol 32 no 6 pp 407ndash412 1999
[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958
[21] S-C Chun S Y Jee S G Lee S J Park J R Lee and SC Kim ldquoAnti-inflammatory activity of the methanol extractof Moutan Cortex in LPS-activated Raw2647 cellsrdquo Evidence-Based Complementary and Alternative Medicine vol 4 no 3pp 327ndash333 2007
[22] G-N Kim Y-I Kwon and H-D Jang ldquoProtective mech-anism of quercetin and rutin on 22rsquo-azobis(2-amidino-propane)dihydrochloride or Cu2+-induced oxidative stress inHepG2 cellsrdquoToxicology in Vitro vol 25 no 1 pp 138ndash144 2011
[23] M G B de Oliveira R B Marques M F de Santana et al ldquo120572-Terpineol reducesmechanical hypernociception and inflamma-tory responserdquoBasic ampClinical PharmacologyampToxicology vol111 no 2 pp 120ndash125 2012
[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[25] A K L Nascimento R FMelo-Silveira N Dantas-Santos et alldquoAntioxidant and antiproliferative activities of leaf extracts fromPlukenetia volubilis Linneo (Euphorbiaceae)rdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID950272 10 pages 2013
[26] A Muthuraman N Singh and A S Jaggi ldquoProtective effectof Acorus calamus L in rat model of vincristine inducedpainful neuropathy an evidence of anti-inflammatory and anti-oxidative activityrdquo Food and Chemical Toxicology vol 49 no 10pp 2557ndash2563 2011
[27] A Bunea DO Rugina AM Pintea Z Sconta C I Bunea andC Socaciu ldquoComparative polyphenolic content and antioxidantactivities of some wild and cultivated blueberries from Roma-niardquoNotulae Botanicae Horti Agrobotanici Cluj-Napoca vol 39no 2 pp 70ndash76 2011
[28] K E Heim A R Tagliaferro and D J Bobilya ldquoFlavonoidantioxidants chemistry metabolism and structure-activityrelationshipsrdquo Journal of Nutritional Biochemistry vol 13 no10 pp 572ndash584 2002
[29] J Perez-Jimenez S Arranz M Tabernero et al ldquoUpdatedmethodology to determine antioxidant capacity in plant foodsoils and beverages extraction measurement and expression ofresultsrdquo Food Research International vol 41 no 3 pp 274ndash2852008
[30] K L Wolfe and R H Lui ldquoCellular antioxidant activity(CAA) assay for assessing antioxidants foods and dietarysupplementsrdquo Journal of Agricultural and Food Chemistry vol55 no 22 pp 8896ndash8907 2007
[31] M Okawara H Katsuki E Kurimoto H Shibata T Kumeand A Akaike ldquoResveratrol protects dopaminergic neuronsin midbrain slice culture from multiple insultsrdquo BiochemicalPharmacology vol 73 no 4 pp 550ndash560 2007
[32] H Nishikawa KWakano and S Kitani ldquoInhibition of NADPHoxidase subunits translocation by tea catechin EGCG in mastcellrdquo Biochemical and Biophysical Research Communicationsvol 362 no 2 pp 504ndash509 2007
[33] J Fang T Seki and H Maeda ldquoTherapeutic strategies by mod-ulating oxygen stress in cancer and inflammationrdquo AdvancedDrug Delivery Reviews vol 61 no 4 pp 290ndash302 2009
[34] K Subbaramaiah and A J Dannenberg ldquoCyclooxygenase 2 amolecular target for cancer prevention and treatmentrdquo Trendsin Pharmacological Sciences vol 24 no 2 pp 96ndash102 2003
[35] Y-J Surh K-S ChunH-H Cha et al ldquoMolecularmechanismsunderlying chemopreventive activities of anti-inflammatoryphytochemicals down-regulation of COX-2 and iNOS throughsuppression of NF-120581B activationrdquoMutation Research vol 480-481 pp 243ndash268 2001
[36] W-W Chao Y-H Kuo and B-F Lin ldquoAnti-inflammatoryactivity of new compounds from Andrographis paniculata byNF-120581B transactivation inhibitionrdquo Journal of Agricultural andFood Chemistry vol 58 no 4 pp 2505ndash2512 2010
Evidence-Based Complementary and Alternative Medicine 11
[37] Y-C Liang Y-T Huang S-H Tsai S-Y Lin-Shiau C-FChen and J-K Lin ldquoSuppression of inducible cyclooxygenaseand inducible nitric oxide synthase by apigenin and relatedflavonoids in mouse macrophagesrdquo Carcinogenesis vol 20 no10 pp 1945ndash1952 1999
6 Evidence-Based Complementary and Alternative Medicine
80
60
40
20
0
b b b b
aab
Cel
l via
bilit
y (
)120
100
Control 005 025 050 150100
EEP (mgmL)
(a)
80
60
40
20
0
b
a
ccd
d
Cel
l via
bilit
y (
)
120
100
Control 010 050 150 450
WEP (mgmL)
(b)
Figure 2 Effect of EEP andWEP on cell viability of RAW2647 cellsThe effect of EEP (a) andWEP (b) on cell viability was assessed byMTTValues are expressed as means plusmn SEM (119899 = 3) and are representative of three independent experiments with similar results Bars marked withdifferent letters are significantly different at 119875 lt 005 as determined by one-way ANOVA
EEP is a stronger suppressant of NO induction than WEPConcentrations of 50 120583gmL of EEP and 150 and 200 120583gmLofWEPwere required to exhibit the NO suppression with thesame efficiency as 500 120583M (8806120583gmL) vitamin C
38 Suppression of iNOS and COX-2 Protein Expression byEEP and WEP in LPS Plus IFN-120574-Activated RAW2647 CellsTo determine if suppression of NO production by EEP orWEPwas related to changes in iNOS aswell as COX-2 proteinlevels Western blotting analysis was performed RAW2647cells were pretreated with antioxidants Trolox (50120583gmL)vitamin C (500120583M) or PP extracts (EEP or WEP) at 50ndash250120583gmL for 1 h prior activation with LPS (1120583gmL) plusIFN-120574 (25UmL) for 18 h Total proteins were extracted andanalyzed for the expression of iNOS and COX-2 by Westernblotting LPS plus IFN-120574 induced increases in iNOS (Figures5(a) and 5(b)) and COX-2 (Figures 5(c) and 5(d)) expressioncompared to the unstimulated cultures Antioxidant controls(Trolox and vitamin C) decreased LPS plus IFN-120574-inducediNOS and COX-2 protein levels The data also suggestedthat the suppression by 500120583M (8806120583gmL) vitamin Cis more pronounced than 50 120583gmL Trolox Compared tothe corresponding controls both EEP and WEP produced adose-dependent suppression of iNOS level in LPS plus IFN-120574-activated RAW2647 cells (Figures 5(a) and 5(b)) suggestingthat the suppression of NO production by EEP and WEP ismediated by decreasing the expression of iNOS In agreementwith the result of NO suppression 50ndash200 120583gmL EEP wasprobably more efficient than WEP in iNOS suppressionThe iNOS expression was almost completely eliminated at200120583gmL EEP and was barely observed at 250 120583gmLWEPThe inflammatory modulation of EEP and WEP was alsofurther supported by the dose-dependent suppression of theCOX-2 level by both EEP and WEP (Figures 5(c) and 5(d))in the activated RAW2647 cells Notably EEP and WEPexhibited higher suppression of iNOS than COX-2
4 Discussion
It is well known that major phytochemicals of plant leafextracts possessing antioxidant activity are flavonoids andother phenolic compounds Researchers have found thatflavonoids from PP leaves display antioxidant activity andall ethyl acetate chloroform and 119899-butanol-soluble fractionsof PP contain flavonoids [11 15] In addition Nguyen andEun [14] found phenolics and flavonoids in extracts of PPleaves when assessed with Folin-Ciocalteu and aluminumtrichloride PP leaf extracts also have antioxidant activitieswhen evaluated with DPPH and FRAP assays Similarlythe present study also showed that both EEP and WEPcontain high levels of flavonoids and phenolics and exhibitantioxidant activity The most frequently used antioxidantstandards for food samples (vitamin C Trolox catechinresveratrol and quercetin) were used as positive antioxidantcontrols in the present study
This study revealed that DPPH radical scavenging capac-ity of WEP is greater than that of EEP (Figure 1) In contrastEEP has higher ferric reducing power than WEP (Table 2)Such contradictory results between DPPH and FRAP assaysare not unusual Though both assays are based on a singleelectron transfer reaction [27] their characteristics sensitiv-ities mechanisms of the reaction and endpoints are totallydifferent For instance the DPPH method is based on thefree radical scavenging activity while FRAP measures thecapability of reducing Fe3+ to Fe2+ Depending on whatspecific phytochemical constituents present in the extractare providing the antioxidant activity their discrete chemicalstructures positions numbers and types of substitutions caninfluence their redox properties and hence their antioxidantpotentials [28]
Though both DPPH and FRAP assays are frequently usedfor assessing antioxidant capacity they have some drawbacksIn the DPPH assay interfering compounds may have sig-nificant absorption at the same measured wavelength In
Evidence-Based Complementary and Alternative Medicine 7
Figure 3 Cellular radical scavenging activity in tBuOOH-activated RAW2647 cells Intracellular ROS level generated in cells was measuredby the DCFH-DA RAW2647 cells were pretreated with indicated concentrations of antioxidants (a) EEP (b) or WEP (c) for 24 h priorto take-up of 20 120583M DCFH-DA for 30min Results are mean plusmn SEM (119899 = 4) and are representative of three independent experiments withsimilar results Pointsmarked with different letters are significantly different at119875 lt 005when compared at the same time point as determinedby two-way ANOVA
addition theDPPH radical is not present in living organismsFor the FRAPmethod compounds with low redox potentialwhich probably do not serve as antioxidants in vivo still canreduce the Fe3+ Interfering compounds may also absorb atthe same wavelength and the assay is also performed at anonphysiological pH [29] Therefore antioxidant activitiesof EEP and WEP were also evaluated by the cell-basedassay using an intracellular fluorescent probe DCFH-DAWhen the nonfluorescent DCFH-DA is taken up into cellsits diacetate moiety will be hydrolyzed by cellular esterasesto generate the more polar DCFH which is trapped insidethe cells In the presence of ROS intracellular DCFH isfurther oxidized to form the fluorescent DCF product [30]
The macrophage cell line RAW2647 is usually the cell ofchoice in studying ROS-mediated cellular events since itcan generate high amounts of ROS following an oxidantchallenge Catechin resveratrol and quercetin at the levelof concentration used in this study have been shown andoptimized to exhibit a strong suppression of intracellular ROSgeneration [22 31 32] Therefore the present study selectedthese compounds as antioxidant positive controls for theDCFH-DA assay The present study demonstrated that allantioxidant standards 250120583M catechin 20120583M resveratroland 10 120583M quercetin exerted a strong inhibition of ROSgeneration induced by tBuOOH over a period of 30 to240min In addition to extracellular antioxidant capacity
8 Evidence-Based Complementary and Alternative Medicine
45
40
35
30
25
20
15
10
5
0
NA
VH 50
150
250
e
d
c
bb
a
e
d
bcNitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
EEP (120583gmL)
(a)
b
e
cc
bc
d
f
a
45
40
35
30
25
20
15
10
5
0
NA 50
150
250
Nitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
WEP (120583gmL)
(b)
Figure 4 EEP (a) and WEP (b) suppressed LPS plus IFN-120574-induced nitrite production in RAW2647 cells RAW2647 cells were incubatedfor 24 hwith LPS (1120583gmL) plus IFN-120574 (25UmL) in the presence or absence of indicated concentrations of vitaminC (500120583M) EEP orWEPAccumulated nitrite in the culturemediumwas determined by the Griess reactionThe values aremeansplusmn SEM (119899 = 3) and are representativeof three independent experiments with similar results Bars marked with different letters are significantly different at 119875 lt 005 as determinedby one-way ANOVA
EEP and WEP also served as intracellular ROS scavengersand subsequently decreased the oxidation of DCFH (Figures3(b) and 3(c)) Both EEP and WEP were as efficient as theantioxidant standards in scavenging ROS Notably EEP was abetter reducer of DCF fluorescence thanWEPThe reductionof DCF fluorescence by EEP and WEP is not due to directcytotoxicity as the range of concentration used in the studieshad no effect on RAW2647 cell viability (Figure 2)
Although the current study shows that an ethanol extractfrom PP leaves has in vivo anti-inflammatory activities[13] its mechanism of anti-inflammation is still unrevealedInflammatory disorders are characterized among otherevents by the production of significant amounts of freeradicals nitrogen reactive species and pro-inflammatorycytokines [10] High NO concentration combines with super-oxides to form peroxynitrite ions (OONOminus) which areresponsible for cell and tissue damage from inflammation[33] Therefore we investigated inflammatory effects of EEPand WEP on the suppression of NO production in LPS plusIFN-120574-activated RAW2647 cells At the concentration rangeof 50ndash250120583gmL both EEP and WEP dose-dependentlysuppressed NO production and the suppression was morepronounced in EEP than WEP (Figure 4) These resultsagreed with the observation that EEP was also a betterscavenger of intracellular ROS than WEP (Figures 3(b) and3(c))
As enhanced NO production by LPS and IFN-120574-stimulated RAW2647 cells mainly occurs via increasedintracellular content of iNOS [3 4] the effect of EEP andWEP on iNOS expression was investigatedThe present studyclearly indicates that the suppressive effect of EEP and WEPon NO production was mediated through the inhibition of
iNOS expression (Figures 5(a) and 5(b)) In agreement withthe study of NO suppression the suppressive effect of EEP(50ndash200120583gmL) on iNOS was more remarkable than that ofWEP
In addition to iNOS induction LPS and IFN-120574 alsoefficiently enhance COX-2 expression in RAW2647 cells [35] An increased level of COX-2 expression is also knownto account for the excessive production of PGE
2in most
if not all inflammatory cells and tissues [34] This studyshows that both EEP andWEP can exhibit anti-inflammatoryactivity by reducing high COX-2 protein levels in a dose-related manner (Figures 5(c) and 5(d)) Thus EEP and WEPmight play important roles in attenuating inflammationand cellular damage through their extra- and intracellularROS scavenging activity and downregulation of NO iNOSand COX-2 Concordantly Khumpook et al [13] recentlyreported the in vivo anti-inflammatory activity of PP leavesas evidenced by decreased lipid peroxidation and NO levelin concomitance with increased superoxide dismutase in thecotton-induced chronic inflammation in Albino rats uponexposure to an ethanol extract of PP leaves for 17 days
In fact several medicinal plant extracts with naturalantioxidant properties together with suppressive effects onNO iNOS andorCOX-2 expression inRAW2647 have beenreported to display a wide spectrum of bioactivities Theseactivities include anti-inflammation such as curcumin fromCurcuma longa resveratrol from grape skins red wines andother plants and a mixture of 120573-sitosterol and stigmasterolfrom Andrographis paniculata [8 35 36] Previous investi-gators demonstrated that pretreatment of RAW2647 withflavonoids such as apigenin genistein and kaempferol sup-pressed LPS-stimulated expression of NO iNOS and COX-2
Evidence-Based Complementary and Alternative Medicine 9
FoldiNOS
Tubulin
003 086 058 030 061 038 033 024 015
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
EEP (120583gmL)
LPSIFN-120574
(a)
Fold
Tubulin
iNOS
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
WEP (120583gmL)
LPSIFN-120574
008 111 083 052 073 063 048 036 008
(b)
COX-2
Fold
Tubulin
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
006 093 076 074 087 079 065 044 030
EEP (120583gmL)
LPSIFN-120574
(c)
Fold
Tubulin
COX-2
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
001 113 066 063 091 078 067 064 050
WEP (120583gmL)
LPSIFN-120574
(d)
Figure 5 Effect of EEP on LPS plus IFN-120574-induced iNOS (a) and COX-2 (c) andWEP on LPS plus IFN-120574-induced iNOS (b) and COX-2 (d)protein levels in RAW2647 cells The relative expression of proteins was quantified densitometrically using ImageJ software and normalizedto tubulin reference bands Data are representative of at least two independent experiments
protein production [37] Major chemical constituents of PPleaves consist of 120573-sitosterol stigmasterol kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucoside[11] All aforementioned compounds have been shown topossess anti-inflammatory properties Both kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucosidemay be metabolized into kaempferol and apigenin whichalso have antioxidant and anti-inflammatory activities Thusit is possible that phenolic and flavonoid compounds inboth EEP andWEP provide substantial antioxidant and anti-inflammatory activities
In summary the cytoprotective effects of EEP and WEPis due to their abilities to decrease ROS generation and NOradical production in cells In addition both EEP and WEPexert anti-inflammatory effects through the suppression ofNO release and decrease the protein expression of iNOSand COX-2 Thus PP leaves possess high potential forfurther exploration in the research development of anti-inflammatory medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was financially supported by a Royal GoldenJubilee Scholarship Grant (PhD02082548) and by the
National Research Council of Thailand (NRCT) throughSuranaree University of Technology (SUT-104-53-36-09)
References
[1] B Halliwell and J M C Gutteridge Free Radicals in BiologyandMedicine OxfordUniversity Press NewYork NYUSA 3rdedition 1999
[2] N Fujiwara and K Kobayashi ldquoMacrophages in inflammationrdquoCurrent Drug TargetsmdashInflammation and Allergy vol 4 no 3pp 281ndash286 2005
[3] S I Jang Y-J Kim W-Y Lee et al ldquoScoparone from Artemisiacapillaris inhibits the release of inflammatory mediators inRAW 2647 cells upon stimulation cells by interferon-120574 plusLPSrdquo Archives of Pharmacal Research vol 28 no 2 pp 203ndash208 2005
[4] R Korhonen A Lahti H Kankaanranta and E MoilanenldquoNitric oxide production and signaling in inflammationrdquo Cur-rent Drug TargetsmdashInflammation and Allergy vol 4 no 4 pp471ndash479 2005
[5] E Karpuzoglu and S A Ahmed ldquoEstrogen regulation of nitricoxide and inducible nitric oxide synthase (iNOS) in immunecells implications for immunity autoimmune diseases andapoptosisrdquo Nitric Oxide vol 15 no 3 pp 177ndash186 2006
[6] S G Harris J Padilla L Koumas D Ray and R PPhipps ldquoProstaglandins as modulators of immunityrdquo Trends inImmunology vol 23 no 3 pp 144ndash150 2002
[7] I-N Hsieh A S-Y Chang C-M Teng C-C Chen andC-R Yang ldquoAciculatin inhibits lipopolysaccharide-mediated
10 Evidence-Based Complementary and Alternative Medicine
inducible nitric oxide synthase and cyclooxygenase-2 expres-sion via suppressing NF-120581B and JNKp38 MAPK activationpathwaysrdquo Journal of Biomedical Science vol 18 no 1 article28 2011
[8] J B Calixto M F Otuki and A R S Santos ldquoAnti-inflammatory compounds of plant originmdashpart I action onarachidonic acid pathway nitric oxide and nuclear factor 120581 B(NF-120581B)rdquo Planta Medica vol 69 no 11 pp 973ndash983 2003
[9] K B Pandey and S I Rizvi ldquoCurrent understanding of dietarypolyphenols and their role in health and diseaserdquo CurrentNutrition and Food Science vol 5 no 4 pp 249ndash263 2009
[10] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer How are theylinkedrdquo Free Radical Biology and Medicine vol 49 no 11 pp1603ndash1616 2010
[11] P M Giang H V Bao and P T Son ldquoPhytochemical studyon Pseuderanthemum palatiferum (Nees) Radlk AcanthaceaerdquoJournal of Chemistry vol 41 no 2 pp 115ndash118 2003
[12] H K Dieu C B Loc S Yamasaki and Y Hirata ldquoThe ethnob-otanical and botanical study on Pseuderanthemum palatiferumas a new medicinal plant in the Mekong Delta of VietnamrdquoJapan Agricultural Research Quarterly vol 39 no 3 pp 191ndash1962005
[13] T Khumpook S Chomdej S Saenphet D Amornlerdpi-son and K Saenphet ldquoAnti-inflammatory activity of ethanolextract from the leaves of Pseuderanthemum palatiferum (Nees)RadlkrdquoChiangMai Journal of Science vol 40 no 3 pp 321ndash3312013
[14] Q-V Nguyen and J-B Eun ldquoAntioxidant activity of solventextracts from Vietnamese medicinal plantsrdquo Journal of Medic-inal Plant Research vol 5 no 13 pp 2798ndash2811 2011
[15] P M Giang H V Bao and P T Son ldquoStudy on anti-oxidativeactivities and preliminary investigation on antibacterial anti-fungal of extracted fraction rich in flavonoids from leaves ofPseuderanthemum palatiferum (Nees) Radlkrdquo TC Duoc Hocvol 9 no 9 pp 9ndash12 2005 (In Vietnamese with Englishsummary)
[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998
[17] M Liu X Q Li C Weber C Y Lee J Brown and R HLiu ldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002
[18] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[19] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoFreeradical scavenging capacity and inhibition of lipid oxidation ofwines grape juices and related polyphenolic constituentsrdquo FoodResearch International vol 32 no 6 pp 407ndash412 1999
[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958
[21] S-C Chun S Y Jee S G Lee S J Park J R Lee and SC Kim ldquoAnti-inflammatory activity of the methanol extractof Moutan Cortex in LPS-activated Raw2647 cellsrdquo Evidence-Based Complementary and Alternative Medicine vol 4 no 3pp 327ndash333 2007
[22] G-N Kim Y-I Kwon and H-D Jang ldquoProtective mech-anism of quercetin and rutin on 22rsquo-azobis(2-amidino-propane)dihydrochloride or Cu2+-induced oxidative stress inHepG2 cellsrdquoToxicology in Vitro vol 25 no 1 pp 138ndash144 2011
[23] M G B de Oliveira R B Marques M F de Santana et al ldquo120572-Terpineol reducesmechanical hypernociception and inflamma-tory responserdquoBasic ampClinical PharmacologyampToxicology vol111 no 2 pp 120ndash125 2012
[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[25] A K L Nascimento R FMelo-Silveira N Dantas-Santos et alldquoAntioxidant and antiproliferative activities of leaf extracts fromPlukenetia volubilis Linneo (Euphorbiaceae)rdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID950272 10 pages 2013
[26] A Muthuraman N Singh and A S Jaggi ldquoProtective effectof Acorus calamus L in rat model of vincristine inducedpainful neuropathy an evidence of anti-inflammatory and anti-oxidative activityrdquo Food and Chemical Toxicology vol 49 no 10pp 2557ndash2563 2011
[27] A Bunea DO Rugina AM Pintea Z Sconta C I Bunea andC Socaciu ldquoComparative polyphenolic content and antioxidantactivities of some wild and cultivated blueberries from Roma-niardquoNotulae Botanicae Horti Agrobotanici Cluj-Napoca vol 39no 2 pp 70ndash76 2011
[28] K E Heim A R Tagliaferro and D J Bobilya ldquoFlavonoidantioxidants chemistry metabolism and structure-activityrelationshipsrdquo Journal of Nutritional Biochemistry vol 13 no10 pp 572ndash584 2002
[29] J Perez-Jimenez S Arranz M Tabernero et al ldquoUpdatedmethodology to determine antioxidant capacity in plant foodsoils and beverages extraction measurement and expression ofresultsrdquo Food Research International vol 41 no 3 pp 274ndash2852008
[30] K L Wolfe and R H Lui ldquoCellular antioxidant activity(CAA) assay for assessing antioxidants foods and dietarysupplementsrdquo Journal of Agricultural and Food Chemistry vol55 no 22 pp 8896ndash8907 2007
[31] M Okawara H Katsuki E Kurimoto H Shibata T Kumeand A Akaike ldquoResveratrol protects dopaminergic neuronsin midbrain slice culture from multiple insultsrdquo BiochemicalPharmacology vol 73 no 4 pp 550ndash560 2007
[32] H Nishikawa KWakano and S Kitani ldquoInhibition of NADPHoxidase subunits translocation by tea catechin EGCG in mastcellrdquo Biochemical and Biophysical Research Communicationsvol 362 no 2 pp 504ndash509 2007
[33] J Fang T Seki and H Maeda ldquoTherapeutic strategies by mod-ulating oxygen stress in cancer and inflammationrdquo AdvancedDrug Delivery Reviews vol 61 no 4 pp 290ndash302 2009
[34] K Subbaramaiah and A J Dannenberg ldquoCyclooxygenase 2 amolecular target for cancer prevention and treatmentrdquo Trendsin Pharmacological Sciences vol 24 no 2 pp 96ndash102 2003
[35] Y-J Surh K-S ChunH-H Cha et al ldquoMolecularmechanismsunderlying chemopreventive activities of anti-inflammatoryphytochemicals down-regulation of COX-2 and iNOS throughsuppression of NF-120581B activationrdquoMutation Research vol 480-481 pp 243ndash268 2001
[36] W-W Chao Y-H Kuo and B-F Lin ldquoAnti-inflammatoryactivity of new compounds from Andrographis paniculata byNF-120581B transactivation inhibitionrdquo Journal of Agricultural andFood Chemistry vol 58 no 4 pp 2505ndash2512 2010
Evidence-Based Complementary and Alternative Medicine 11
[37] Y-C Liang Y-T Huang S-H Tsai S-Y Lin-Shiau C-FChen and J-K Lin ldquoSuppression of inducible cyclooxygenaseand inducible nitric oxide synthase by apigenin and relatedflavonoids in mouse macrophagesrdquo Carcinogenesis vol 20 no10 pp 1945ndash1952 1999
Figure 3 Cellular radical scavenging activity in tBuOOH-activated RAW2647 cells Intracellular ROS level generated in cells was measuredby the DCFH-DA RAW2647 cells were pretreated with indicated concentrations of antioxidants (a) EEP (b) or WEP (c) for 24 h priorto take-up of 20 120583M DCFH-DA for 30min Results are mean plusmn SEM (119899 = 4) and are representative of three independent experiments withsimilar results Pointsmarked with different letters are significantly different at119875 lt 005when compared at the same time point as determinedby two-way ANOVA
addition theDPPH radical is not present in living organismsFor the FRAPmethod compounds with low redox potentialwhich probably do not serve as antioxidants in vivo still canreduce the Fe3+ Interfering compounds may also absorb atthe same wavelength and the assay is also performed at anonphysiological pH [29] Therefore antioxidant activitiesof EEP and WEP were also evaluated by the cell-basedassay using an intracellular fluorescent probe DCFH-DAWhen the nonfluorescent DCFH-DA is taken up into cellsits diacetate moiety will be hydrolyzed by cellular esterasesto generate the more polar DCFH which is trapped insidethe cells In the presence of ROS intracellular DCFH isfurther oxidized to form the fluorescent DCF product [30]
The macrophage cell line RAW2647 is usually the cell ofchoice in studying ROS-mediated cellular events since itcan generate high amounts of ROS following an oxidantchallenge Catechin resveratrol and quercetin at the levelof concentration used in this study have been shown andoptimized to exhibit a strong suppression of intracellular ROSgeneration [22 31 32] Therefore the present study selectedthese compounds as antioxidant positive controls for theDCFH-DA assay The present study demonstrated that allantioxidant standards 250120583M catechin 20120583M resveratroland 10 120583M quercetin exerted a strong inhibition of ROSgeneration induced by tBuOOH over a period of 30 to240min In addition to extracellular antioxidant capacity
8 Evidence-Based Complementary and Alternative Medicine
45
40
35
30
25
20
15
10
5
0
NA
VH 50
150
250
e
d
c
bb
a
e
d
bcNitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
EEP (120583gmL)
(a)
b
e
cc
bc
d
f
a
45
40
35
30
25
20
15
10
5
0
NA 50
150
250
Nitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
WEP (120583gmL)
(b)
Figure 4 EEP (a) and WEP (b) suppressed LPS plus IFN-120574-induced nitrite production in RAW2647 cells RAW2647 cells were incubatedfor 24 hwith LPS (1120583gmL) plus IFN-120574 (25UmL) in the presence or absence of indicated concentrations of vitaminC (500120583M) EEP orWEPAccumulated nitrite in the culturemediumwas determined by the Griess reactionThe values aremeansplusmn SEM (119899 = 3) and are representativeof three independent experiments with similar results Bars marked with different letters are significantly different at 119875 lt 005 as determinedby one-way ANOVA
EEP and WEP also served as intracellular ROS scavengersand subsequently decreased the oxidation of DCFH (Figures3(b) and 3(c)) Both EEP and WEP were as efficient as theantioxidant standards in scavenging ROS Notably EEP was abetter reducer of DCF fluorescence thanWEPThe reductionof DCF fluorescence by EEP and WEP is not due to directcytotoxicity as the range of concentration used in the studieshad no effect on RAW2647 cell viability (Figure 2)
Although the current study shows that an ethanol extractfrom PP leaves has in vivo anti-inflammatory activities[13] its mechanism of anti-inflammation is still unrevealedInflammatory disorders are characterized among otherevents by the production of significant amounts of freeradicals nitrogen reactive species and pro-inflammatorycytokines [10] High NO concentration combines with super-oxides to form peroxynitrite ions (OONOminus) which areresponsible for cell and tissue damage from inflammation[33] Therefore we investigated inflammatory effects of EEPand WEP on the suppression of NO production in LPS plusIFN-120574-activated RAW2647 cells At the concentration rangeof 50ndash250120583gmL both EEP and WEP dose-dependentlysuppressed NO production and the suppression was morepronounced in EEP than WEP (Figure 4) These resultsagreed with the observation that EEP was also a betterscavenger of intracellular ROS than WEP (Figures 3(b) and3(c))
As enhanced NO production by LPS and IFN-120574-stimulated RAW2647 cells mainly occurs via increasedintracellular content of iNOS [3 4] the effect of EEP andWEP on iNOS expression was investigatedThe present studyclearly indicates that the suppressive effect of EEP and WEPon NO production was mediated through the inhibition of
iNOS expression (Figures 5(a) and 5(b)) In agreement withthe study of NO suppression the suppressive effect of EEP(50ndash200120583gmL) on iNOS was more remarkable than that ofWEP
In addition to iNOS induction LPS and IFN-120574 alsoefficiently enhance COX-2 expression in RAW2647 cells [35] An increased level of COX-2 expression is also knownto account for the excessive production of PGE
2in most
if not all inflammatory cells and tissues [34] This studyshows that both EEP andWEP can exhibit anti-inflammatoryactivity by reducing high COX-2 protein levels in a dose-related manner (Figures 5(c) and 5(d)) Thus EEP and WEPmight play important roles in attenuating inflammationand cellular damage through their extra- and intracellularROS scavenging activity and downregulation of NO iNOSand COX-2 Concordantly Khumpook et al [13] recentlyreported the in vivo anti-inflammatory activity of PP leavesas evidenced by decreased lipid peroxidation and NO levelin concomitance with increased superoxide dismutase in thecotton-induced chronic inflammation in Albino rats uponexposure to an ethanol extract of PP leaves for 17 days
In fact several medicinal plant extracts with naturalantioxidant properties together with suppressive effects onNO iNOS andorCOX-2 expression inRAW2647 have beenreported to display a wide spectrum of bioactivities Theseactivities include anti-inflammation such as curcumin fromCurcuma longa resveratrol from grape skins red wines andother plants and a mixture of 120573-sitosterol and stigmasterolfrom Andrographis paniculata [8 35 36] Previous investi-gators demonstrated that pretreatment of RAW2647 withflavonoids such as apigenin genistein and kaempferol sup-pressed LPS-stimulated expression of NO iNOS and COX-2
Evidence-Based Complementary and Alternative Medicine 9
FoldiNOS
Tubulin
003 086 058 030 061 038 033 024 015
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
EEP (120583gmL)
LPSIFN-120574
(a)
Fold
Tubulin
iNOS
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
WEP (120583gmL)
LPSIFN-120574
008 111 083 052 073 063 048 036 008
(b)
COX-2
Fold
Tubulin
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
006 093 076 074 087 079 065 044 030
EEP (120583gmL)
LPSIFN-120574
(c)
Fold
Tubulin
COX-2
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
001 113 066 063 091 078 067 064 050
WEP (120583gmL)
LPSIFN-120574
(d)
Figure 5 Effect of EEP on LPS plus IFN-120574-induced iNOS (a) and COX-2 (c) andWEP on LPS plus IFN-120574-induced iNOS (b) and COX-2 (d)protein levels in RAW2647 cells The relative expression of proteins was quantified densitometrically using ImageJ software and normalizedto tubulin reference bands Data are representative of at least two independent experiments
protein production [37] Major chemical constituents of PPleaves consist of 120573-sitosterol stigmasterol kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucoside[11] All aforementioned compounds have been shown topossess anti-inflammatory properties Both kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucosidemay be metabolized into kaempferol and apigenin whichalso have antioxidant and anti-inflammatory activities Thusit is possible that phenolic and flavonoid compounds inboth EEP andWEP provide substantial antioxidant and anti-inflammatory activities
In summary the cytoprotective effects of EEP and WEPis due to their abilities to decrease ROS generation and NOradical production in cells In addition both EEP and WEPexert anti-inflammatory effects through the suppression ofNO release and decrease the protein expression of iNOSand COX-2 Thus PP leaves possess high potential forfurther exploration in the research development of anti-inflammatory medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was financially supported by a Royal GoldenJubilee Scholarship Grant (PhD02082548) and by the
National Research Council of Thailand (NRCT) throughSuranaree University of Technology (SUT-104-53-36-09)
References
[1] B Halliwell and J M C Gutteridge Free Radicals in BiologyandMedicine OxfordUniversity Press NewYork NYUSA 3rdedition 1999
[2] N Fujiwara and K Kobayashi ldquoMacrophages in inflammationrdquoCurrent Drug TargetsmdashInflammation and Allergy vol 4 no 3pp 281ndash286 2005
[3] S I Jang Y-J Kim W-Y Lee et al ldquoScoparone from Artemisiacapillaris inhibits the release of inflammatory mediators inRAW 2647 cells upon stimulation cells by interferon-120574 plusLPSrdquo Archives of Pharmacal Research vol 28 no 2 pp 203ndash208 2005
[4] R Korhonen A Lahti H Kankaanranta and E MoilanenldquoNitric oxide production and signaling in inflammationrdquo Cur-rent Drug TargetsmdashInflammation and Allergy vol 4 no 4 pp471ndash479 2005
[5] E Karpuzoglu and S A Ahmed ldquoEstrogen regulation of nitricoxide and inducible nitric oxide synthase (iNOS) in immunecells implications for immunity autoimmune diseases andapoptosisrdquo Nitric Oxide vol 15 no 3 pp 177ndash186 2006
[6] S G Harris J Padilla L Koumas D Ray and R PPhipps ldquoProstaglandins as modulators of immunityrdquo Trends inImmunology vol 23 no 3 pp 144ndash150 2002
[7] I-N Hsieh A S-Y Chang C-M Teng C-C Chen andC-R Yang ldquoAciculatin inhibits lipopolysaccharide-mediated
10 Evidence-Based Complementary and Alternative Medicine
inducible nitric oxide synthase and cyclooxygenase-2 expres-sion via suppressing NF-120581B and JNKp38 MAPK activationpathwaysrdquo Journal of Biomedical Science vol 18 no 1 article28 2011
[8] J B Calixto M F Otuki and A R S Santos ldquoAnti-inflammatory compounds of plant originmdashpart I action onarachidonic acid pathway nitric oxide and nuclear factor 120581 B(NF-120581B)rdquo Planta Medica vol 69 no 11 pp 973ndash983 2003
[9] K B Pandey and S I Rizvi ldquoCurrent understanding of dietarypolyphenols and their role in health and diseaserdquo CurrentNutrition and Food Science vol 5 no 4 pp 249ndash263 2009
[10] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer How are theylinkedrdquo Free Radical Biology and Medicine vol 49 no 11 pp1603ndash1616 2010
[11] P M Giang H V Bao and P T Son ldquoPhytochemical studyon Pseuderanthemum palatiferum (Nees) Radlk AcanthaceaerdquoJournal of Chemistry vol 41 no 2 pp 115ndash118 2003
[12] H K Dieu C B Loc S Yamasaki and Y Hirata ldquoThe ethnob-otanical and botanical study on Pseuderanthemum palatiferumas a new medicinal plant in the Mekong Delta of VietnamrdquoJapan Agricultural Research Quarterly vol 39 no 3 pp 191ndash1962005
[13] T Khumpook S Chomdej S Saenphet D Amornlerdpi-son and K Saenphet ldquoAnti-inflammatory activity of ethanolextract from the leaves of Pseuderanthemum palatiferum (Nees)RadlkrdquoChiangMai Journal of Science vol 40 no 3 pp 321ndash3312013
[14] Q-V Nguyen and J-B Eun ldquoAntioxidant activity of solventextracts from Vietnamese medicinal plantsrdquo Journal of Medic-inal Plant Research vol 5 no 13 pp 2798ndash2811 2011
[15] P M Giang H V Bao and P T Son ldquoStudy on anti-oxidativeactivities and preliminary investigation on antibacterial anti-fungal of extracted fraction rich in flavonoids from leaves ofPseuderanthemum palatiferum (Nees) Radlkrdquo TC Duoc Hocvol 9 no 9 pp 9ndash12 2005 (In Vietnamese with Englishsummary)
[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998
[17] M Liu X Q Li C Weber C Y Lee J Brown and R HLiu ldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002
[18] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[19] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoFreeradical scavenging capacity and inhibition of lipid oxidation ofwines grape juices and related polyphenolic constituentsrdquo FoodResearch International vol 32 no 6 pp 407ndash412 1999
[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958
[21] S-C Chun S Y Jee S G Lee S J Park J R Lee and SC Kim ldquoAnti-inflammatory activity of the methanol extractof Moutan Cortex in LPS-activated Raw2647 cellsrdquo Evidence-Based Complementary and Alternative Medicine vol 4 no 3pp 327ndash333 2007
[22] G-N Kim Y-I Kwon and H-D Jang ldquoProtective mech-anism of quercetin and rutin on 22rsquo-azobis(2-amidino-propane)dihydrochloride or Cu2+-induced oxidative stress inHepG2 cellsrdquoToxicology in Vitro vol 25 no 1 pp 138ndash144 2011
[23] M G B de Oliveira R B Marques M F de Santana et al ldquo120572-Terpineol reducesmechanical hypernociception and inflamma-tory responserdquoBasic ampClinical PharmacologyampToxicology vol111 no 2 pp 120ndash125 2012
[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[25] A K L Nascimento R FMelo-Silveira N Dantas-Santos et alldquoAntioxidant and antiproliferative activities of leaf extracts fromPlukenetia volubilis Linneo (Euphorbiaceae)rdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID950272 10 pages 2013
[26] A Muthuraman N Singh and A S Jaggi ldquoProtective effectof Acorus calamus L in rat model of vincristine inducedpainful neuropathy an evidence of anti-inflammatory and anti-oxidative activityrdquo Food and Chemical Toxicology vol 49 no 10pp 2557ndash2563 2011
[27] A Bunea DO Rugina AM Pintea Z Sconta C I Bunea andC Socaciu ldquoComparative polyphenolic content and antioxidantactivities of some wild and cultivated blueberries from Roma-niardquoNotulae Botanicae Horti Agrobotanici Cluj-Napoca vol 39no 2 pp 70ndash76 2011
[28] K E Heim A R Tagliaferro and D J Bobilya ldquoFlavonoidantioxidants chemistry metabolism and structure-activityrelationshipsrdquo Journal of Nutritional Biochemistry vol 13 no10 pp 572ndash584 2002
[29] J Perez-Jimenez S Arranz M Tabernero et al ldquoUpdatedmethodology to determine antioxidant capacity in plant foodsoils and beverages extraction measurement and expression ofresultsrdquo Food Research International vol 41 no 3 pp 274ndash2852008
[30] K L Wolfe and R H Lui ldquoCellular antioxidant activity(CAA) assay for assessing antioxidants foods and dietarysupplementsrdquo Journal of Agricultural and Food Chemistry vol55 no 22 pp 8896ndash8907 2007
[31] M Okawara H Katsuki E Kurimoto H Shibata T Kumeand A Akaike ldquoResveratrol protects dopaminergic neuronsin midbrain slice culture from multiple insultsrdquo BiochemicalPharmacology vol 73 no 4 pp 550ndash560 2007
[32] H Nishikawa KWakano and S Kitani ldquoInhibition of NADPHoxidase subunits translocation by tea catechin EGCG in mastcellrdquo Biochemical and Biophysical Research Communicationsvol 362 no 2 pp 504ndash509 2007
[33] J Fang T Seki and H Maeda ldquoTherapeutic strategies by mod-ulating oxygen stress in cancer and inflammationrdquo AdvancedDrug Delivery Reviews vol 61 no 4 pp 290ndash302 2009
[34] K Subbaramaiah and A J Dannenberg ldquoCyclooxygenase 2 amolecular target for cancer prevention and treatmentrdquo Trendsin Pharmacological Sciences vol 24 no 2 pp 96ndash102 2003
[35] Y-J Surh K-S ChunH-H Cha et al ldquoMolecularmechanismsunderlying chemopreventive activities of anti-inflammatoryphytochemicals down-regulation of COX-2 and iNOS throughsuppression of NF-120581B activationrdquoMutation Research vol 480-481 pp 243ndash268 2001
[36] W-W Chao Y-H Kuo and B-F Lin ldquoAnti-inflammatoryactivity of new compounds from Andrographis paniculata byNF-120581B transactivation inhibitionrdquo Journal of Agricultural andFood Chemistry vol 58 no 4 pp 2505ndash2512 2010
Evidence-Based Complementary and Alternative Medicine 11
[37] Y-C Liang Y-T Huang S-H Tsai S-Y Lin-Shiau C-FChen and J-K Lin ldquoSuppression of inducible cyclooxygenaseand inducible nitric oxide synthase by apigenin and relatedflavonoids in mouse macrophagesrdquo Carcinogenesis vol 20 no10 pp 1945ndash1952 1999
8 Evidence-Based Complementary and Alternative Medicine
45
40
35
30
25
20
15
10
5
0
NA
VH 50
150
250
e
d
c
bb
a
e
d
bcNitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
EEP (120583gmL)
(a)
b
e
cc
bc
d
f
a
45
40
35
30
25
20
15
10
5
0
NA 50
150
250
Nitr
ite (120583
M)
LPS
IFN
-120574
Vita
min
C
100
200
WEP (120583gmL)
(b)
Figure 4 EEP (a) and WEP (b) suppressed LPS plus IFN-120574-induced nitrite production in RAW2647 cells RAW2647 cells were incubatedfor 24 hwith LPS (1120583gmL) plus IFN-120574 (25UmL) in the presence or absence of indicated concentrations of vitaminC (500120583M) EEP orWEPAccumulated nitrite in the culturemediumwas determined by the Griess reactionThe values aremeansplusmn SEM (119899 = 3) and are representativeof three independent experiments with similar results Bars marked with different letters are significantly different at 119875 lt 005 as determinedby one-way ANOVA
EEP and WEP also served as intracellular ROS scavengersand subsequently decreased the oxidation of DCFH (Figures3(b) and 3(c)) Both EEP and WEP were as efficient as theantioxidant standards in scavenging ROS Notably EEP was abetter reducer of DCF fluorescence thanWEPThe reductionof DCF fluorescence by EEP and WEP is not due to directcytotoxicity as the range of concentration used in the studieshad no effect on RAW2647 cell viability (Figure 2)
Although the current study shows that an ethanol extractfrom PP leaves has in vivo anti-inflammatory activities[13] its mechanism of anti-inflammation is still unrevealedInflammatory disorders are characterized among otherevents by the production of significant amounts of freeradicals nitrogen reactive species and pro-inflammatorycytokines [10] High NO concentration combines with super-oxides to form peroxynitrite ions (OONOminus) which areresponsible for cell and tissue damage from inflammation[33] Therefore we investigated inflammatory effects of EEPand WEP on the suppression of NO production in LPS plusIFN-120574-activated RAW2647 cells At the concentration rangeof 50ndash250120583gmL both EEP and WEP dose-dependentlysuppressed NO production and the suppression was morepronounced in EEP than WEP (Figure 4) These resultsagreed with the observation that EEP was also a betterscavenger of intracellular ROS than WEP (Figures 3(b) and3(c))
As enhanced NO production by LPS and IFN-120574-stimulated RAW2647 cells mainly occurs via increasedintracellular content of iNOS [3 4] the effect of EEP andWEP on iNOS expression was investigatedThe present studyclearly indicates that the suppressive effect of EEP and WEPon NO production was mediated through the inhibition of
iNOS expression (Figures 5(a) and 5(b)) In agreement withthe study of NO suppression the suppressive effect of EEP(50ndash200120583gmL) on iNOS was more remarkable than that ofWEP
In addition to iNOS induction LPS and IFN-120574 alsoefficiently enhance COX-2 expression in RAW2647 cells [35] An increased level of COX-2 expression is also knownto account for the excessive production of PGE
2in most
if not all inflammatory cells and tissues [34] This studyshows that both EEP andWEP can exhibit anti-inflammatoryactivity by reducing high COX-2 protein levels in a dose-related manner (Figures 5(c) and 5(d)) Thus EEP and WEPmight play important roles in attenuating inflammationand cellular damage through their extra- and intracellularROS scavenging activity and downregulation of NO iNOSand COX-2 Concordantly Khumpook et al [13] recentlyreported the in vivo anti-inflammatory activity of PP leavesas evidenced by decreased lipid peroxidation and NO levelin concomitance with increased superoxide dismutase in thecotton-induced chronic inflammation in Albino rats uponexposure to an ethanol extract of PP leaves for 17 days
In fact several medicinal plant extracts with naturalantioxidant properties together with suppressive effects onNO iNOS andorCOX-2 expression inRAW2647 have beenreported to display a wide spectrum of bioactivities Theseactivities include anti-inflammation such as curcumin fromCurcuma longa resveratrol from grape skins red wines andother plants and a mixture of 120573-sitosterol and stigmasterolfrom Andrographis paniculata [8 35 36] Previous investi-gators demonstrated that pretreatment of RAW2647 withflavonoids such as apigenin genistein and kaempferol sup-pressed LPS-stimulated expression of NO iNOS and COX-2
Evidence-Based Complementary and Alternative Medicine 9
FoldiNOS
Tubulin
003 086 058 030 061 038 033 024 015
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
EEP (120583gmL)
LPSIFN-120574
(a)
Fold
Tubulin
iNOS
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
WEP (120583gmL)
LPSIFN-120574
008 111 083 052 073 063 048 036 008
(b)
COX-2
Fold
Tubulin
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
006 093 076 074 087 079 065 044 030
EEP (120583gmL)
LPSIFN-120574
(c)
Fold
Tubulin
COX-2
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
001 113 066 063 091 078 067 064 050
WEP (120583gmL)
LPSIFN-120574
(d)
Figure 5 Effect of EEP on LPS plus IFN-120574-induced iNOS (a) and COX-2 (c) andWEP on LPS plus IFN-120574-induced iNOS (b) and COX-2 (d)protein levels in RAW2647 cells The relative expression of proteins was quantified densitometrically using ImageJ software and normalizedto tubulin reference bands Data are representative of at least two independent experiments
protein production [37] Major chemical constituents of PPleaves consist of 120573-sitosterol stigmasterol kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucoside[11] All aforementioned compounds have been shown topossess anti-inflammatory properties Both kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucosidemay be metabolized into kaempferol and apigenin whichalso have antioxidant and anti-inflammatory activities Thusit is possible that phenolic and flavonoid compounds inboth EEP andWEP provide substantial antioxidant and anti-inflammatory activities
In summary the cytoprotective effects of EEP and WEPis due to their abilities to decrease ROS generation and NOradical production in cells In addition both EEP and WEPexert anti-inflammatory effects through the suppression ofNO release and decrease the protein expression of iNOSand COX-2 Thus PP leaves possess high potential forfurther exploration in the research development of anti-inflammatory medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was financially supported by a Royal GoldenJubilee Scholarship Grant (PhD02082548) and by the
National Research Council of Thailand (NRCT) throughSuranaree University of Technology (SUT-104-53-36-09)
References
[1] B Halliwell and J M C Gutteridge Free Radicals in BiologyandMedicine OxfordUniversity Press NewYork NYUSA 3rdedition 1999
[2] N Fujiwara and K Kobayashi ldquoMacrophages in inflammationrdquoCurrent Drug TargetsmdashInflammation and Allergy vol 4 no 3pp 281ndash286 2005
[3] S I Jang Y-J Kim W-Y Lee et al ldquoScoparone from Artemisiacapillaris inhibits the release of inflammatory mediators inRAW 2647 cells upon stimulation cells by interferon-120574 plusLPSrdquo Archives of Pharmacal Research vol 28 no 2 pp 203ndash208 2005
[4] R Korhonen A Lahti H Kankaanranta and E MoilanenldquoNitric oxide production and signaling in inflammationrdquo Cur-rent Drug TargetsmdashInflammation and Allergy vol 4 no 4 pp471ndash479 2005
[5] E Karpuzoglu and S A Ahmed ldquoEstrogen regulation of nitricoxide and inducible nitric oxide synthase (iNOS) in immunecells implications for immunity autoimmune diseases andapoptosisrdquo Nitric Oxide vol 15 no 3 pp 177ndash186 2006
[6] S G Harris J Padilla L Koumas D Ray and R PPhipps ldquoProstaglandins as modulators of immunityrdquo Trends inImmunology vol 23 no 3 pp 144ndash150 2002
[7] I-N Hsieh A S-Y Chang C-M Teng C-C Chen andC-R Yang ldquoAciculatin inhibits lipopolysaccharide-mediated
10 Evidence-Based Complementary and Alternative Medicine
inducible nitric oxide synthase and cyclooxygenase-2 expres-sion via suppressing NF-120581B and JNKp38 MAPK activationpathwaysrdquo Journal of Biomedical Science vol 18 no 1 article28 2011
[8] J B Calixto M F Otuki and A R S Santos ldquoAnti-inflammatory compounds of plant originmdashpart I action onarachidonic acid pathway nitric oxide and nuclear factor 120581 B(NF-120581B)rdquo Planta Medica vol 69 no 11 pp 973ndash983 2003
[9] K B Pandey and S I Rizvi ldquoCurrent understanding of dietarypolyphenols and their role in health and diseaserdquo CurrentNutrition and Food Science vol 5 no 4 pp 249ndash263 2009
[10] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer How are theylinkedrdquo Free Radical Biology and Medicine vol 49 no 11 pp1603ndash1616 2010
[11] P M Giang H V Bao and P T Son ldquoPhytochemical studyon Pseuderanthemum palatiferum (Nees) Radlk AcanthaceaerdquoJournal of Chemistry vol 41 no 2 pp 115ndash118 2003
[12] H K Dieu C B Loc S Yamasaki and Y Hirata ldquoThe ethnob-otanical and botanical study on Pseuderanthemum palatiferumas a new medicinal plant in the Mekong Delta of VietnamrdquoJapan Agricultural Research Quarterly vol 39 no 3 pp 191ndash1962005
[13] T Khumpook S Chomdej S Saenphet D Amornlerdpi-son and K Saenphet ldquoAnti-inflammatory activity of ethanolextract from the leaves of Pseuderanthemum palatiferum (Nees)RadlkrdquoChiangMai Journal of Science vol 40 no 3 pp 321ndash3312013
[14] Q-V Nguyen and J-B Eun ldquoAntioxidant activity of solventextracts from Vietnamese medicinal plantsrdquo Journal of Medic-inal Plant Research vol 5 no 13 pp 2798ndash2811 2011
[15] P M Giang H V Bao and P T Son ldquoStudy on anti-oxidativeactivities and preliminary investigation on antibacterial anti-fungal of extracted fraction rich in flavonoids from leaves ofPseuderanthemum palatiferum (Nees) Radlkrdquo TC Duoc Hocvol 9 no 9 pp 9ndash12 2005 (In Vietnamese with Englishsummary)
[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998
[17] M Liu X Q Li C Weber C Y Lee J Brown and R HLiu ldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002
[18] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[19] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoFreeradical scavenging capacity and inhibition of lipid oxidation ofwines grape juices and related polyphenolic constituentsrdquo FoodResearch International vol 32 no 6 pp 407ndash412 1999
[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958
[21] S-C Chun S Y Jee S G Lee S J Park J R Lee and SC Kim ldquoAnti-inflammatory activity of the methanol extractof Moutan Cortex in LPS-activated Raw2647 cellsrdquo Evidence-Based Complementary and Alternative Medicine vol 4 no 3pp 327ndash333 2007
[22] G-N Kim Y-I Kwon and H-D Jang ldquoProtective mech-anism of quercetin and rutin on 22rsquo-azobis(2-amidino-propane)dihydrochloride or Cu2+-induced oxidative stress inHepG2 cellsrdquoToxicology in Vitro vol 25 no 1 pp 138ndash144 2011
[23] M G B de Oliveira R B Marques M F de Santana et al ldquo120572-Terpineol reducesmechanical hypernociception and inflamma-tory responserdquoBasic ampClinical PharmacologyampToxicology vol111 no 2 pp 120ndash125 2012
[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[25] A K L Nascimento R FMelo-Silveira N Dantas-Santos et alldquoAntioxidant and antiproliferative activities of leaf extracts fromPlukenetia volubilis Linneo (Euphorbiaceae)rdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID950272 10 pages 2013
[26] A Muthuraman N Singh and A S Jaggi ldquoProtective effectof Acorus calamus L in rat model of vincristine inducedpainful neuropathy an evidence of anti-inflammatory and anti-oxidative activityrdquo Food and Chemical Toxicology vol 49 no 10pp 2557ndash2563 2011
[27] A Bunea DO Rugina AM Pintea Z Sconta C I Bunea andC Socaciu ldquoComparative polyphenolic content and antioxidantactivities of some wild and cultivated blueberries from Roma-niardquoNotulae Botanicae Horti Agrobotanici Cluj-Napoca vol 39no 2 pp 70ndash76 2011
[28] K E Heim A R Tagliaferro and D J Bobilya ldquoFlavonoidantioxidants chemistry metabolism and structure-activityrelationshipsrdquo Journal of Nutritional Biochemistry vol 13 no10 pp 572ndash584 2002
[29] J Perez-Jimenez S Arranz M Tabernero et al ldquoUpdatedmethodology to determine antioxidant capacity in plant foodsoils and beverages extraction measurement and expression ofresultsrdquo Food Research International vol 41 no 3 pp 274ndash2852008
[30] K L Wolfe and R H Lui ldquoCellular antioxidant activity(CAA) assay for assessing antioxidants foods and dietarysupplementsrdquo Journal of Agricultural and Food Chemistry vol55 no 22 pp 8896ndash8907 2007
[31] M Okawara H Katsuki E Kurimoto H Shibata T Kumeand A Akaike ldquoResveratrol protects dopaminergic neuronsin midbrain slice culture from multiple insultsrdquo BiochemicalPharmacology vol 73 no 4 pp 550ndash560 2007
[32] H Nishikawa KWakano and S Kitani ldquoInhibition of NADPHoxidase subunits translocation by tea catechin EGCG in mastcellrdquo Biochemical and Biophysical Research Communicationsvol 362 no 2 pp 504ndash509 2007
[33] J Fang T Seki and H Maeda ldquoTherapeutic strategies by mod-ulating oxygen stress in cancer and inflammationrdquo AdvancedDrug Delivery Reviews vol 61 no 4 pp 290ndash302 2009
[34] K Subbaramaiah and A J Dannenberg ldquoCyclooxygenase 2 amolecular target for cancer prevention and treatmentrdquo Trendsin Pharmacological Sciences vol 24 no 2 pp 96ndash102 2003
[35] Y-J Surh K-S ChunH-H Cha et al ldquoMolecularmechanismsunderlying chemopreventive activities of anti-inflammatoryphytochemicals down-regulation of COX-2 and iNOS throughsuppression of NF-120581B activationrdquoMutation Research vol 480-481 pp 243ndash268 2001
[36] W-W Chao Y-H Kuo and B-F Lin ldquoAnti-inflammatoryactivity of new compounds from Andrographis paniculata byNF-120581B transactivation inhibitionrdquo Journal of Agricultural andFood Chemistry vol 58 no 4 pp 2505ndash2512 2010
Evidence-Based Complementary and Alternative Medicine 11
[37] Y-C Liang Y-T Huang S-H Tsai S-Y Lin-Shiau C-FChen and J-K Lin ldquoSuppression of inducible cyclooxygenaseand inducible nitric oxide synthase by apigenin and relatedflavonoids in mouse macrophagesrdquo Carcinogenesis vol 20 no10 pp 1945ndash1952 1999
Evidence-Based Complementary and Alternative Medicine 9
FoldiNOS
Tubulin
003 086 058 030 061 038 033 024 015
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
EEP (120583gmL)
LPSIFN-120574
(a)
Fold
Tubulin
iNOS
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
WEP (120583gmL)
LPSIFN-120574
008 111 083 052 073 063 048 036 008
(b)
COX-2
Fold
Tubulin
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
006 093 076 074 087 079 065 044 030
EEP (120583gmL)
LPSIFN-120574
(c)
Fold
Tubulin
COX-2
NA
VH
Trol
ox
Vita
min
C 50 100
150
200
250
001 113 066 063 091 078 067 064 050
WEP (120583gmL)
LPSIFN-120574
(d)
Figure 5 Effect of EEP on LPS plus IFN-120574-induced iNOS (a) and COX-2 (c) andWEP on LPS plus IFN-120574-induced iNOS (b) and COX-2 (d)protein levels in RAW2647 cells The relative expression of proteins was quantified densitometrically using ImageJ software and normalizedto tubulin reference bands Data are representative of at least two independent experiments
protein production [37] Major chemical constituents of PPleaves consist of 120573-sitosterol stigmasterol kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucoside[11] All aforementioned compounds have been shown topossess anti-inflammatory properties Both kaempferol 3-methyl ether 7-O-120573-glucoside and apigenin 7-O-120573-glucosidemay be metabolized into kaempferol and apigenin whichalso have antioxidant and anti-inflammatory activities Thusit is possible that phenolic and flavonoid compounds inboth EEP andWEP provide substantial antioxidant and anti-inflammatory activities
In summary the cytoprotective effects of EEP and WEPis due to their abilities to decrease ROS generation and NOradical production in cells In addition both EEP and WEPexert anti-inflammatory effects through the suppression ofNO release and decrease the protein expression of iNOSand COX-2 Thus PP leaves possess high potential forfurther exploration in the research development of anti-inflammatory medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was financially supported by a Royal GoldenJubilee Scholarship Grant (PhD02082548) and by the
National Research Council of Thailand (NRCT) throughSuranaree University of Technology (SUT-104-53-36-09)
References
[1] B Halliwell and J M C Gutteridge Free Radicals in BiologyandMedicine OxfordUniversity Press NewYork NYUSA 3rdedition 1999
[2] N Fujiwara and K Kobayashi ldquoMacrophages in inflammationrdquoCurrent Drug TargetsmdashInflammation and Allergy vol 4 no 3pp 281ndash286 2005
[3] S I Jang Y-J Kim W-Y Lee et al ldquoScoparone from Artemisiacapillaris inhibits the release of inflammatory mediators inRAW 2647 cells upon stimulation cells by interferon-120574 plusLPSrdquo Archives of Pharmacal Research vol 28 no 2 pp 203ndash208 2005
[4] R Korhonen A Lahti H Kankaanranta and E MoilanenldquoNitric oxide production and signaling in inflammationrdquo Cur-rent Drug TargetsmdashInflammation and Allergy vol 4 no 4 pp471ndash479 2005
[5] E Karpuzoglu and S A Ahmed ldquoEstrogen regulation of nitricoxide and inducible nitric oxide synthase (iNOS) in immunecells implications for immunity autoimmune diseases andapoptosisrdquo Nitric Oxide vol 15 no 3 pp 177ndash186 2006
[6] S G Harris J Padilla L Koumas D Ray and R PPhipps ldquoProstaglandins as modulators of immunityrdquo Trends inImmunology vol 23 no 3 pp 144ndash150 2002
[7] I-N Hsieh A S-Y Chang C-M Teng C-C Chen andC-R Yang ldquoAciculatin inhibits lipopolysaccharide-mediated
10 Evidence-Based Complementary and Alternative Medicine
inducible nitric oxide synthase and cyclooxygenase-2 expres-sion via suppressing NF-120581B and JNKp38 MAPK activationpathwaysrdquo Journal of Biomedical Science vol 18 no 1 article28 2011
[8] J B Calixto M F Otuki and A R S Santos ldquoAnti-inflammatory compounds of plant originmdashpart I action onarachidonic acid pathway nitric oxide and nuclear factor 120581 B(NF-120581B)rdquo Planta Medica vol 69 no 11 pp 973ndash983 2003
[9] K B Pandey and S I Rizvi ldquoCurrent understanding of dietarypolyphenols and their role in health and diseaserdquo CurrentNutrition and Food Science vol 5 no 4 pp 249ndash263 2009
[10] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer How are theylinkedrdquo Free Radical Biology and Medicine vol 49 no 11 pp1603ndash1616 2010
[11] P M Giang H V Bao and P T Son ldquoPhytochemical studyon Pseuderanthemum palatiferum (Nees) Radlk AcanthaceaerdquoJournal of Chemistry vol 41 no 2 pp 115ndash118 2003
[12] H K Dieu C B Loc S Yamasaki and Y Hirata ldquoThe ethnob-otanical and botanical study on Pseuderanthemum palatiferumas a new medicinal plant in the Mekong Delta of VietnamrdquoJapan Agricultural Research Quarterly vol 39 no 3 pp 191ndash1962005
[13] T Khumpook S Chomdej S Saenphet D Amornlerdpi-son and K Saenphet ldquoAnti-inflammatory activity of ethanolextract from the leaves of Pseuderanthemum palatiferum (Nees)RadlkrdquoChiangMai Journal of Science vol 40 no 3 pp 321ndash3312013
[14] Q-V Nguyen and J-B Eun ldquoAntioxidant activity of solventextracts from Vietnamese medicinal plantsrdquo Journal of Medic-inal Plant Research vol 5 no 13 pp 2798ndash2811 2011
[15] P M Giang H V Bao and P T Son ldquoStudy on anti-oxidativeactivities and preliminary investigation on antibacterial anti-fungal of extracted fraction rich in flavonoids from leaves ofPseuderanthemum palatiferum (Nees) Radlkrdquo TC Duoc Hocvol 9 no 9 pp 9ndash12 2005 (In Vietnamese with Englishsummary)
[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998
[17] M Liu X Q Li C Weber C Y Lee J Brown and R HLiu ldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002
[18] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[19] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoFreeradical scavenging capacity and inhibition of lipid oxidation ofwines grape juices and related polyphenolic constituentsrdquo FoodResearch International vol 32 no 6 pp 407ndash412 1999
[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958
[21] S-C Chun S Y Jee S G Lee S J Park J R Lee and SC Kim ldquoAnti-inflammatory activity of the methanol extractof Moutan Cortex in LPS-activated Raw2647 cellsrdquo Evidence-Based Complementary and Alternative Medicine vol 4 no 3pp 327ndash333 2007
[22] G-N Kim Y-I Kwon and H-D Jang ldquoProtective mech-anism of quercetin and rutin on 22rsquo-azobis(2-amidino-propane)dihydrochloride or Cu2+-induced oxidative stress inHepG2 cellsrdquoToxicology in Vitro vol 25 no 1 pp 138ndash144 2011
[23] M G B de Oliveira R B Marques M F de Santana et al ldquo120572-Terpineol reducesmechanical hypernociception and inflamma-tory responserdquoBasic ampClinical PharmacologyampToxicology vol111 no 2 pp 120ndash125 2012
[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[25] A K L Nascimento R FMelo-Silveira N Dantas-Santos et alldquoAntioxidant and antiproliferative activities of leaf extracts fromPlukenetia volubilis Linneo (Euphorbiaceae)rdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID950272 10 pages 2013
[26] A Muthuraman N Singh and A S Jaggi ldquoProtective effectof Acorus calamus L in rat model of vincristine inducedpainful neuropathy an evidence of anti-inflammatory and anti-oxidative activityrdquo Food and Chemical Toxicology vol 49 no 10pp 2557ndash2563 2011
[27] A Bunea DO Rugina AM Pintea Z Sconta C I Bunea andC Socaciu ldquoComparative polyphenolic content and antioxidantactivities of some wild and cultivated blueberries from Roma-niardquoNotulae Botanicae Horti Agrobotanici Cluj-Napoca vol 39no 2 pp 70ndash76 2011
[28] K E Heim A R Tagliaferro and D J Bobilya ldquoFlavonoidantioxidants chemistry metabolism and structure-activityrelationshipsrdquo Journal of Nutritional Biochemistry vol 13 no10 pp 572ndash584 2002
[29] J Perez-Jimenez S Arranz M Tabernero et al ldquoUpdatedmethodology to determine antioxidant capacity in plant foodsoils and beverages extraction measurement and expression ofresultsrdquo Food Research International vol 41 no 3 pp 274ndash2852008
[30] K L Wolfe and R H Lui ldquoCellular antioxidant activity(CAA) assay for assessing antioxidants foods and dietarysupplementsrdquo Journal of Agricultural and Food Chemistry vol55 no 22 pp 8896ndash8907 2007
[31] M Okawara H Katsuki E Kurimoto H Shibata T Kumeand A Akaike ldquoResveratrol protects dopaminergic neuronsin midbrain slice culture from multiple insultsrdquo BiochemicalPharmacology vol 73 no 4 pp 550ndash560 2007
[32] H Nishikawa KWakano and S Kitani ldquoInhibition of NADPHoxidase subunits translocation by tea catechin EGCG in mastcellrdquo Biochemical and Biophysical Research Communicationsvol 362 no 2 pp 504ndash509 2007
[33] J Fang T Seki and H Maeda ldquoTherapeutic strategies by mod-ulating oxygen stress in cancer and inflammationrdquo AdvancedDrug Delivery Reviews vol 61 no 4 pp 290ndash302 2009
[34] K Subbaramaiah and A J Dannenberg ldquoCyclooxygenase 2 amolecular target for cancer prevention and treatmentrdquo Trendsin Pharmacological Sciences vol 24 no 2 pp 96ndash102 2003
[35] Y-J Surh K-S ChunH-H Cha et al ldquoMolecularmechanismsunderlying chemopreventive activities of anti-inflammatoryphytochemicals down-regulation of COX-2 and iNOS throughsuppression of NF-120581B activationrdquoMutation Research vol 480-481 pp 243ndash268 2001
[36] W-W Chao Y-H Kuo and B-F Lin ldquoAnti-inflammatoryactivity of new compounds from Andrographis paniculata byNF-120581B transactivation inhibitionrdquo Journal of Agricultural andFood Chemistry vol 58 no 4 pp 2505ndash2512 2010
Evidence-Based Complementary and Alternative Medicine 11
[37] Y-C Liang Y-T Huang S-H Tsai S-Y Lin-Shiau C-FChen and J-K Lin ldquoSuppression of inducible cyclooxygenaseand inducible nitric oxide synthase by apigenin and relatedflavonoids in mouse macrophagesrdquo Carcinogenesis vol 20 no10 pp 1945ndash1952 1999
10 Evidence-Based Complementary and Alternative Medicine
inducible nitric oxide synthase and cyclooxygenase-2 expres-sion via suppressing NF-120581B and JNKp38 MAPK activationpathwaysrdquo Journal of Biomedical Science vol 18 no 1 article28 2011
[8] J B Calixto M F Otuki and A R S Santos ldquoAnti-inflammatory compounds of plant originmdashpart I action onarachidonic acid pathway nitric oxide and nuclear factor 120581 B(NF-120581B)rdquo Planta Medica vol 69 no 11 pp 973ndash983 2003
[9] K B Pandey and S I Rizvi ldquoCurrent understanding of dietarypolyphenols and their role in health and diseaserdquo CurrentNutrition and Food Science vol 5 no 4 pp 249ndash263 2009
[10] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer How are theylinkedrdquo Free Radical Biology and Medicine vol 49 no 11 pp1603ndash1616 2010
[11] P M Giang H V Bao and P T Son ldquoPhytochemical studyon Pseuderanthemum palatiferum (Nees) Radlk AcanthaceaerdquoJournal of Chemistry vol 41 no 2 pp 115ndash118 2003
[12] H K Dieu C B Loc S Yamasaki and Y Hirata ldquoThe ethnob-otanical and botanical study on Pseuderanthemum palatiferumas a new medicinal plant in the Mekong Delta of VietnamrdquoJapan Agricultural Research Quarterly vol 39 no 3 pp 191ndash1962005
[13] T Khumpook S Chomdej S Saenphet D Amornlerdpi-son and K Saenphet ldquoAnti-inflammatory activity of ethanolextract from the leaves of Pseuderanthemum palatiferum (Nees)RadlkrdquoChiangMai Journal of Science vol 40 no 3 pp 321ndash3312013
[14] Q-V Nguyen and J-B Eun ldquoAntioxidant activity of solventextracts from Vietnamese medicinal plantsrdquo Journal of Medic-inal Plant Research vol 5 no 13 pp 2798ndash2811 2011
[15] P M Giang H V Bao and P T Son ldquoStudy on anti-oxidativeactivities and preliminary investigation on antibacterial anti-fungal of extracted fraction rich in flavonoids from leaves ofPseuderanthemum palatiferum (Nees) Radlkrdquo TC Duoc Hocvol 9 no 9 pp 9ndash12 2005 (In Vietnamese with Englishsummary)
[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998
[17] M Liu X Q Li C Weber C Y Lee J Brown and R HLiu ldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002
[18] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[19] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoFreeradical scavenging capacity and inhibition of lipid oxidation ofwines grape juices and related polyphenolic constituentsrdquo FoodResearch International vol 32 no 6 pp 407ndash412 1999
[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958
[21] S-C Chun S Y Jee S G Lee S J Park J R Lee and SC Kim ldquoAnti-inflammatory activity of the methanol extractof Moutan Cortex in LPS-activated Raw2647 cellsrdquo Evidence-Based Complementary and Alternative Medicine vol 4 no 3pp 327ndash333 2007
[22] G-N Kim Y-I Kwon and H-D Jang ldquoProtective mech-anism of quercetin and rutin on 22rsquo-azobis(2-amidino-propane)dihydrochloride or Cu2+-induced oxidative stress inHepG2 cellsrdquoToxicology in Vitro vol 25 no 1 pp 138ndash144 2011
[23] M G B de Oliveira R B Marques M F de Santana et al ldquo120572-Terpineol reducesmechanical hypernociception and inflamma-tory responserdquoBasic ampClinical PharmacologyampToxicology vol111 no 2 pp 120ndash125 2012
[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[25] A K L Nascimento R FMelo-Silveira N Dantas-Santos et alldquoAntioxidant and antiproliferative activities of leaf extracts fromPlukenetia volubilis Linneo (Euphorbiaceae)rdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID950272 10 pages 2013
[26] A Muthuraman N Singh and A S Jaggi ldquoProtective effectof Acorus calamus L in rat model of vincristine inducedpainful neuropathy an evidence of anti-inflammatory and anti-oxidative activityrdquo Food and Chemical Toxicology vol 49 no 10pp 2557ndash2563 2011
[27] A Bunea DO Rugina AM Pintea Z Sconta C I Bunea andC Socaciu ldquoComparative polyphenolic content and antioxidantactivities of some wild and cultivated blueberries from Roma-niardquoNotulae Botanicae Horti Agrobotanici Cluj-Napoca vol 39no 2 pp 70ndash76 2011
[28] K E Heim A R Tagliaferro and D J Bobilya ldquoFlavonoidantioxidants chemistry metabolism and structure-activityrelationshipsrdquo Journal of Nutritional Biochemistry vol 13 no10 pp 572ndash584 2002
[29] J Perez-Jimenez S Arranz M Tabernero et al ldquoUpdatedmethodology to determine antioxidant capacity in plant foodsoils and beverages extraction measurement and expression ofresultsrdquo Food Research International vol 41 no 3 pp 274ndash2852008
[30] K L Wolfe and R H Lui ldquoCellular antioxidant activity(CAA) assay for assessing antioxidants foods and dietarysupplementsrdquo Journal of Agricultural and Food Chemistry vol55 no 22 pp 8896ndash8907 2007
[31] M Okawara H Katsuki E Kurimoto H Shibata T Kumeand A Akaike ldquoResveratrol protects dopaminergic neuronsin midbrain slice culture from multiple insultsrdquo BiochemicalPharmacology vol 73 no 4 pp 550ndash560 2007
[32] H Nishikawa KWakano and S Kitani ldquoInhibition of NADPHoxidase subunits translocation by tea catechin EGCG in mastcellrdquo Biochemical and Biophysical Research Communicationsvol 362 no 2 pp 504ndash509 2007
[33] J Fang T Seki and H Maeda ldquoTherapeutic strategies by mod-ulating oxygen stress in cancer and inflammationrdquo AdvancedDrug Delivery Reviews vol 61 no 4 pp 290ndash302 2009
[34] K Subbaramaiah and A J Dannenberg ldquoCyclooxygenase 2 amolecular target for cancer prevention and treatmentrdquo Trendsin Pharmacological Sciences vol 24 no 2 pp 96ndash102 2003
[35] Y-J Surh K-S ChunH-H Cha et al ldquoMolecularmechanismsunderlying chemopreventive activities of anti-inflammatoryphytochemicals down-regulation of COX-2 and iNOS throughsuppression of NF-120581B activationrdquoMutation Research vol 480-481 pp 243ndash268 2001
[36] W-W Chao Y-H Kuo and B-F Lin ldquoAnti-inflammatoryactivity of new compounds from Andrographis paniculata byNF-120581B transactivation inhibitionrdquo Journal of Agricultural andFood Chemistry vol 58 no 4 pp 2505ndash2512 2010
Evidence-Based Complementary and Alternative Medicine 11
[37] Y-C Liang Y-T Huang S-H Tsai S-Y Lin-Shiau C-FChen and J-K Lin ldquoSuppression of inducible cyclooxygenaseand inducible nitric oxide synthase by apigenin and relatedflavonoids in mouse macrophagesrdquo Carcinogenesis vol 20 no10 pp 1945ndash1952 1999
