Research Article Bioassay-Guided Isolation and …downloads.hindawi.com/journals/bmri/2014/549836.pdfResearch Article Bioassay-Guided Isolation and Antioxidant Evaluation of Flavonoid

Research ArticleBioassay-Guided Isolation and Antioxidant Evaluation ofFlavonoid Compound from Aerial Parts of Lippia nodiflora L

A Sudha and P Srinivasan

Department of Bioinformatics Alagappa University Karaikudi Tamil Nadu 630004 India

Correspondence should be addressed to P Srinivasan sribioinformaticsgmailcom

Received 27 February 2014 Revised 16 April 2014 Accepted 16 April 2014 Published 25 May 2014

Academic Editor Kota V Ramana

Copyright copy 2014 A Sudha and P Srinivasan This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present study was designed to identify the DPPH (2 2-diphenyl-1-picrylhydrazyl) free-radical scavenging constituents frommethanol extract of L nodiflora using bioassay-guided fractionationThe ethyl acetate fraction (EAF) revealed a strong antioxidantactivity compared to other fractions through in vitro DPPH radical-scavenging assay The repeated fractionation of active EAF bysilica gel column chromatography yielded a compound with strong antioxidant potential The isolated bioactive compound wasdetermined as 2-(3 4-dimethoxyphenyl)-5-hydroxy-7-methoxy-4H-chromen-4-one (5-hydroxy-31015840 41015840 7-trimethoxyflavone) bycomparing spectral data (UV IR 1H NMR 13C NMR and MS) with literature reports The isolated compound demonstrated anexcellent antioxidant activity through all antioxidant assays and also significantly inhibited lipid peroxidation at a concentration of50 120583gmL The results obtained suggested that extracts from L nodiflora or its derived phytocompound can be used potentially asa bioactive source of natural antioxidants by contributing beneficial health effects

1 Introduction

Reactive oxygen species (ROS) are continuously formed as aby-product of metabolisms in aerobic organisms and are alsoproduced on exposure to tobacco smoke ozone radiationsorganic solvents and other environmental pollutants [1]ROS play an important role in various physiological pro-cesses including energy production phagocytosis cellularsignal transduction cell proliferation differentiation andapoptosis On the other hand increasing evidence highlightsthat overproduction of ROS can induce oxidative damageto all biomolecules (lipids carbohydrates proteins enzymesDNA and RNA) and acts as a mediator of numerousdisorders for example inflammation arthritis diabetesarteriosclerosis cancer genotoxicity and neurological disor-ders such as Alzheimerrsquos disease [2] Antioxidants are veryessential for averting degenerative reactions produced byfree radicals and reactive oxygen species which have beenconcerned with many diseases and in food deterioration andspoilage [3] However the safety of some of the syntheticantioxidants used in the food industry has been questioned

because recent studies recognized that they might be car-cinogenic [4] Hence there is an emerging interest in naturalantioxidants which might help to prevent oxidative damage[5]

Plants and plant products are magnificent sources ofphytochemicals and have been found to hold an arrayof biological activities including antioxidant potential [6]Plants synthesize antioxidant compounds mostly flavonoidsand polyphenols which have been reported to protect thehuman body from various diseases by neutralizing ROSRecently phenolic compounds have received increasing sig-nificance among various phytochemicals due to their widedistribution in the plant kingdom and for their biologicalactivities namely anticarcinogenic antiatherogenic anti-inflammatory antimicrobial and antioxidant activities [7ndash9]Antioxidants can be either used as dietary food supplementor as a drug by the humans [10] Several studies have revealedthat the increased dietary intake of natural antioxidants suchas flavonoids and other phenolic compounds almost presentin plants exhibits potential protective effects against manydegenerative diseases [11ndash13]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 549836 10 pageshttpdxdoiorg1011552014549836

2 BioMed Research International

Lippia nodiflora Linn (Verbenaceae) commonly calledPoduthalai in Tamil is a creeping perennial herb and locallyabundant in wet regions and several medicinal propertiesare attributed to this plant in the traditional system ofmedicine The infusion of the leaves and tender stalks isgiven to children suffering from indigestion and to womenafter delivery The chutney made from the leaves and fruitsare often taken to relieve the irritation of internal piles[14] Numerous pharmacological properties of L nodifloraincluding anti-inflammatory antipyretic antitussive antidi-abetic and antimelanogenesis effects have been reported[15ndash18] The ethnopharmacological relevance of L nodiflorafor skin diseases and in folk cosmetics such as pimplescarbuncle and skin burns has also been revealed [19] Thephytochemical constituents of L nodiflora such as flavonoids[20 21] flavone glycosides [22] alkaloids essential oils resin[23] quinol [24] and steroids [25 26] have been previouslyreported Hence these phytochemicals are considered tobe accountable for the pharmacological properties of thisplant Even though there is an evidence for the antioxidantactivity of methanolic extract of L nodiflora the majorantioxidative constituents present in the aerial parts havenot been extensively investigated Hence the present studywas undertaken to isolate active compounds responsiblefor the antioxidant property of methanol extract of Lippianodiflora L through bioassay-guided fractionation using invitro DPPH assay

2 Materials and Methods

21 General Nuclear magnetic resonance spectra wererecorded on BRUKER Avance 400MHz (Switzerland) NMRinstrument operating at 400MHz for 1H and 100MHz for13Cnuclei at room temperature and referenced to the residualsolvent signal The chemical shift and coupling constants(119869) values are reported in ppm and Hz respectively HPLCanalysis was performed using a C-18 column (250 times 46mm5 120583) in a Shimadzu LC-8A chromatographic apparatus (Shi-madzu Singapore)Themobile phase consisted of methanol-05 phosphoric acid in water (60 40 vv) and the flowrate was held constant at 1mLmin The peaks are detectedat 280 nm using variable wavelength UV detector Silica gel60 F254

plates (20 times 20 cm 02mm thick E-Merck Germany)were used for thin-layer chromatography (TLC) analysisThe ultraviolet spectra were recorded using Varian Cary500 scan spectrophotometer 120582max (log 120576) in nm whereas theFTIR spectrum was obtained using a Nicolet 380 (ThermoScientific USA) The functional group was identified usingpotassium bromide (KBr) and scanned in the range of 4000ndash400 cmminus1 Sample was dissolved in methanol and ESI massspectra were obtainedwith aThermo Scientific ExactiveMassSpectrometer (Thermo Fisher Scientific USA)

The reagents such as 2-deoxy-D-ribose butylated hy-droxyl toluene (BHT) 2 2-diphenyl-1-picrylhydrazyl (DPPH)phenazine methosulphate (PMS) nitroblue tetrazolium(NBT) sodium nitroprusside and Griess reagent wereobtained from (Sigma Chemicals St Louis MO USA) 2 46-Tripyridyl-S-triazine (TPTZ) thiobarbituric acid (TBA)

trichloroacetic acid (TCA) ethylene diaminetetraacetic acid(EDTA) ferric chloride (FeCl

3) hydrogen peroxide (H

2O2)

and nicotinamide adenine dinucleotide-reduced (NADH)were obtained from Ms (Sisco Research laboratoriesMumbai India) HPLC grade solvents and reagents usedfor extraction and silica gel (0075ndash015mm) for columnchromatography were obtained from Ms (Sisco Researchlaboratories Mumbai India) All other chemicals andreagents used in this study were of analytical grade

22 Plant Materials The plant materials were freshly col-lected between August and September 2011 from KaraikudiSivagangai district Tamil NaduThe plant was taxonomicallyidentified and authenticated by Dr G V S Murthy JointDirector Botanical Survey of India Tamil Nadu Agricul-tural University Campus Coimbatore (BSISRC5232012-13Tech-19) A voucher specimen of the plant L nodiflora(DBI-HP Specimen-1) was prepared and preserved in theMolecular Biology Lab Department of Bioinformatics Ala-gappa University Karaikudi Tamil Nadu

23 Extraction Fractionation and Isolation of AntioxidantCompound The aerial parts (stem leaves and flowers) werewashed with tap water shade-dried and reduced to finepowder The powdered aerial parts of L nodiflora (15 kg)were extracted with 90 methanol (45 L times 2) at roomtemperature The mixture was filtered through Whatmannumber l filter paper and the solvents from the combinedextract were concentrated using a vacuum rotary evaporator(Superfit India) at 60∘C to afford 647 g of crude methanolextract (431) The solvent was selected based on its yieldfrom preliminary extraction and phytochemical screeningstudies The extract was dissolved in 500mL of warm waterand the resulting aqueous portion was partitioned with ethylacetate (EtOAc) (4 times 200mL) using a separating funnel toafford ethyl acetate fraction (EAF) and aqueous portionThe aqueous phase was then successively partitioned withn-butanol (3 times 200mL) thus obtaining n-butanol solublefraction (BF) and water fraction (WF) All the fractionswere collected separately and reduced using a vacuum rotaryevaporator to remove the solvent and the resultant aqueousextract was lyophilized in vacuo The samples were thentested for its antioxidant property using 2 2-diphenyl-1-picrylhydrazyl (DPPH∙) assay

The DPPH∙ active EAF (172 g) was loaded as a driedslurry of silica gel to column chromatography (45 times 35 cm)and eluted with petroleum ether EtOAc gradient elution(100 0mdash0 100) in increasing order of polarity A total of92 fractions of 100mL each were collected and analyzed byTLC (Silica gel F

254plates 20 times 20 cm Merck Germany)

TLC analysis was carried out using ethyl acetate chloro-form formic acid (5 4 1) as the mobile phase and theseparated bands were visualized using iodine vapors andvanillin-sulphuric acid reagent These fractions were pooledto afford seven major fractions (Fr A 1ndash13 Fr B 14ndash35Fr C 36ndash48 Fr D 49ndash60 Fr E 61ndash70 Fr F 71ndash80 andFr G 81ndash92) based on TLC analysis These fractions weretested for bioactivity using DPPH spectrophotometric assay

BioMed Research International 3

For further purification the highly active Fr B (28 g) wasloaded on a silica gel column eluted with petroleum ether-EtOAc gradients and the ethyl acetate content of the mixturewas increased in a series of 5 steps The inactive and lessactive proved fractions were discarded Finally the active FrB2eluted with petroleum ether-ethyl acetate (85 15) yielded

117mg of compound The purity of isolated compound wasestablished byHPLC and its structurewas confirmed throughthe interpretation of the spectral data (UV FT-IR 1H13C NMR and MS) and further tested for its antioxidanteffects

24 Antioxidant Activities

241 DPPH Radical Scavenging Assay The DPPH radicalscavenging activities of methanol extract EAF BF and WFwere tested according to Yamaguchi et al [27] Briefly 02mLof the sample solutions of different concentrations was addedto 1mL of 01mM of freshly prepared DPPH solution Thereaction mixtures were shaken vigorously and absorbance at517 nm was determined after 20min at room temperatureControl sample was prepared containing the same volumewithout test compounds or reference antioxidants whileDMSO was used as blank The reference antioxidant BHTwas used as the positive control in all the assays Theradical scavenging activity was measured as a decrease in theabsorbance of DPPH∙ and calculated as follows

Scavenging effect () = [119860control minus 119860 sample

119860control] times 100 (1)

where 119860control is the absorbance of the control and 119860 sample isthe absorbance of the extract or fractions or standard

242 Superoxide Radical-Scavenging Assay The superoxideradical-scavenging effect was determined by the method ofNishikimi et al [28] The reaction mixture with NBT (1mM)in phosphate buffer (01M pH 74) NADH (1mM) with orwithout samples and PMS (01mM) was incubated at roomtemperature for 5min and the absorbance was recorded at560 nm The inhibition percentage was calculated againsta control without the samples The scavenging ability wascalculated using the equation as described for DPPH assay

243 Hydroxyl Radical Scavenging Assay The capacity of theextract and compound to reduce hydroxyl radical-mediatedperoxidation was carried out by the method of Hinneburget al [29] Briefly 05mL of 56mM 2-deoxy-D-ribose inKH2PO4minusNaOH buffer (50mM pH 74) 02mL of 100 120583M

FeCl3 and 104mM EDTA (1 1 vv) solution were added to

01mL of different concentrations of test samples followed by100 120583L of 10mM H

2O2and 01mL of 10mM aqueous BHT

The reaction mixtures were shaken vigorously and incubatedat 50∘C for 30min Subsequently 1mL of 28 TCA and 1mLof 10 TBA were added to each tube containing reactionmixture and samples were mixed well again and boiled in awater bath at 50∘C for 30minThe absorbance of solutionwasread at 532 nm The hydroxyl radical scavenging ability was

calculated using the formula as described forDPPHassay andthe values are presented as means of triplicate analyses

244 FRAP (Ferric Reducing Antioxidant Power) Assay TheFRAP assay was determined by the method of Benzie andStrain [30] with minor modifications It depends on theability of the sample to reduce the ferric tripyridyltriazine(Fe (III)-TPTZ) complex to ferrous tripyridyltriazine (Fe (II)-TPTZ) at low pH Fe (II)-TPTZ has an intensive blue colorwhich can be read at 593 nm The stock solutions consistof 300mM acetate buffer (pH 36) 10mM 2 4 6 tripyridylS triazine (TPTZ) in 40mM of HCl and 20mM ferricchloride solution The fresh working solution was preparedby mixing 25mL of acetate buffer 25mL of TPTZ and25mL of FeCl

3sdot6H2O and the temperature was maintained

to 37∘C before use The various concentrations of extractcompound and BHT (10ndash50120583gmL) were allowed to reactwith 2mL of the FRAP solution for 30min in the darkcondition The absorbance was recorded at 593 nm Theresults are expressed in 120583MFe (II)g and were estimatedusing aqueous FeSO

4sdot7H2O (20ndash100120583M) as standard for

calibration

245 Nitric Oxide Radical ScavengingAssay At physiologicalpH nitric oxide generated from sodium nitroprusside inaqueous solution interacts with oxygen to produce nitriteions whichweremeasured by theGriess reaction [31] Briefly3mLof the reactionmixture containing 10mMsodiumnitro-prusside and the test samples (10ndash50 120583gmL) in phosphatebuffer were incubated for 150min at 25∘C After incubation05mL of the reaction mixture was mixed with 1mL ofsulfanilic acid reagent (033 in 20 glacial acetic acid) andallowed to stand for 5min for complete diazotization Then1mL of naphthyl ethylene diamine dihydrochloride (01wv) was added and the mixture was allowed to stand for30min at 25∘C A pink colored chromophore is generatedand the absorbance was measured spectrophotometricallyat 540 nm against a blank sample The nitric oxide radicalscavenging activity of the extract and compound is reportedas inhibition and was calculated using the formula asdescribed for DPPH assay

246 Lipid Peroxidation Assay Lipid peroxidation (LPO)assay was performed according to the protocol described byDamien et al [32] tomeasure the lipid peroxide formed usingegg yolk homogenates as lipid-rich media Briefly 05mL ofegg homogenate (10 vv prepared in 115 wv KCl) wasadded to 01mL of each test samples (10ndash50120583gmL) takenin a test tube and made up to 1mL with double distilledwater Thereafter 005mL of FeSO

4(007M) was added to

induce lipid peroxidation and the mixture was incubated for30min at room temperatureThen 15mL of 35M acetic acid(pH adjusted to 35) was added followed by 15mL of TBA(006M) in sodium dodecyl sulphate (004M) The resultingmixture was vortex and heated at 95∘C for 1 hr After cooling5mL of butanol was added to each tube and centrifuged at3000 rpm for 10min The absorbance of the organic upperlayer was measured at 532 nm and the above procedure was


followed for the control by using 01mL of SDS instead ofthe test sample The percentage inhibition was calculatedaccording to the following formula

Percentage inhibition of lipid peroxidation = (1 minus 119864119862)

times 100

(2)

where 119864 is the absorbance of the test sample and 119862 is theabsorbance of the fully oxidized control

25 Statistical Analysis The experiments were carried out intriplicate and data were expressed asmeans plusmn standard devia-tion (SD) All statistical analyses were performed using graphpad prism (version 50 Graph Pad software Inc San DiegoCA USA) The IC

50value represented the concentration of

the test samples that caused 50 inhibition 119875 values lt005were considered as significant

3 Results and Discussion

31 Isolation and Structure Determination of AntioxidantCompound The methanol extract and potential antioxi-dant fractions of L nodiflora were initially screened byspectrophotometric DPPH assay and the results are shownin Table 1 The DPPH assay revealed that the methanolextract had significant scavenging effects with increasingconcentrations in the range of 10ndash50120583gmL Moreover thescavenging effect ofmethanol extractwas significantly similarto that of standard BHT At 50120583gmL methanol extractand BHT exhibited 7935 and 8642 inhibition and theIC50

values (the concentration with scavenging activity of50) were 24 and 19 120583gmL respectively The result obtainedherein was lower than the reported scavenging activity ofmethanol extract of L nodiflora (1203 120583gmL) [33] Amongthe fractions the DPPH∙ was significantly scavenged by ethylacetate fraction (EAF) in a dose-dependent manner withan IC

50value of 26120583gmL followed by WF and BF with

IC50

values of 66 and 83 120583gmL (Table 1) Hence the EAFwas subjected to repeated bioassay-guided fractionation onsilica gel column chromatography using petroleum etherEtOAc gradient elution system The further purification ofactive fractions obtained from silica gel column chromatog-raphy finally yielded a bioactive antioxidant compound fromthe petroleum ether-ethyl acetate (85 15 vv) mixture Theextraction procedure for the isolation of active compoundwas schematically shown in Figure 1 Figure 2(a) shows theHPLC profile of the chemical constituents present in the 90methanol extract of L nodiflora The HPLC chromatogramof purified compound exposed the presence of a peak with aretention time of 72min eluted isocratically with the mobilephase of methanol-05 phosphoric acid in water (60 40vv) (Figure 2(b))

The compound was obtained as pale white amorphouspowder (yield 117mg 068) The FTIR peaks determinedthe bonds relevant to alcoholic OndashH stretching (3267 cmminus1)=CndashH stretching (3010 cmminus1) ndashCndashH stretching (2975 cmminus1)ndashC=O stretching (1605 cmminus1) ndashC=C stretching (1506 cmminus1)

Table 1 DPPH radical scavenging activity of the crude extract andfractions of aerial parts of Lippia nodiflora L

Sample DPPH (IC50) (120583gmL)CME 2466 plusmn 11EAF 2606 plusmn 095BF 8362 plusmn 045WF 6639 plusmn 052BHTa 1912 plusmn 057aBHT Standard antioxidant data represented as means plusmn SD (119899 = 3)

ndashCndashH bending (1154 cmminus1) CndashO stretching (1315 cmminus1)=CndashH bending (1030 cmminus1) and OndashH bending (993 cmminus1)1HNMR (CDCl

3 400MHz) 120575 (ppm) 391 (3H 119904 41015840-OMe)

389 (3H 119904 7-OMe) 387 (3H 119904 31015840-OMe) 649 (119904 1H OH)664 (119889 119869 = 663Hz 1H H6) 6898 (119904 1H H8) 699 (119889 119869 =63Hz 1H H21015840) 752 (119889 119869 = 63Hz 1H H51015840) 8178 (s 1HH61015840) 13C NMR (CDCl3 100MHz) 120575 (ppm) 17161 (C-4)14430 (C-41015840) 12569 (C-11015840) 16287 (C-2) 16177 (C-31015840) 15682(C-5) 13087 (C-61015840) 11428 (C-51015840) 13729 (C-9) 10392 (C-21015840)15774 (C-7) 9892 (C-6) 9819 (C-8) 11147 (C-3) 11358 (C-10) 5451 (7-OMe) 5476 (31015840-OMe) 5488 (41015840-OMe) (FiguresS1and S2of the Supplementary Information available online

at httpdxdoiorg1011552014549836) The compoundexhibits a molecular weight of 329 (ESI-MS 119898119911 351084 [M+ Na]+) with an elemental formula of C

18H16O6(Figure 3)

From these spectral interpretations the isolated compoundhas been characterized as 2-(3 4-dimethoxyphenyl)-5-hydroxy-7-methoxy-4H-chromen-4-one (5-hydroxy-31015840 410158407-trimethoxyflavone) (Figure 4) which agreed with the datareported [34]

32 Antioxidant Activities of Methanol Extract and IsolatedCompound from Ethyl Acetate Fraction (EAF) Flavonoids amajor group of polyphenols are considered to be the activeprinciples in diversemedicinal plants and have been reportedto possess numerous pharmacological properties The mostessential biological activity of flavonoids ismainly due to theirantioxidant property by acting as radical scavengers hydro-gen donors reducing agents and peroxidation inhibitors[35] Previous studies reported that the pharmacologicaleffects of L nodiflora such as antioxidant diuretic anti-inflammatory and antimicrobial activities were recognizeddue to the presence of the phenol and flavonoid compounds[33 36] In the present study the isolated compound wasidentified as flavone and its antioxidant activities were exam-ined by using various in vitro antioxidant models

321 DPPH Radical Scavenging Effect The substances areconsidered to be antioxidants when they are capable ofreducing the stable DPPH radical (purple) to the nonrad-ical form DPPH-H (yellow) and thus they act as radicalscavengers due to their hydrogen donating abilities [37]The results of DPPH scavenging activity of all test samplesare presented in Figure 5(a) The scavenging activity ofmethanolic extract compound and BHT increased withan increase in sample concentration (10ndash50120583gmL) The


(145kg)

Extraction with 90 methanol (reflux 2 times)

Crude methanolic extract(647 g)

Resuspend with warm water

Extraction with ethyl acetate

Aqueous phase(367 g)

Ethyl acetate fraction(EAF 172 g)

Extraction with n-butanol

Column chromatography (CC)

Pet ether-EtOAc (100 0 sim 0 100)n-Butanol fraction

(BF 153 g)Water fraction(WF 186 g)

Fr A Fr B Fr C Fr D Fr E Fr F Fr G

CC on silica gel

Fr B1 Fr B2 Fr B3 Fr B4 Fr B5(25 75)

Compound(117mg)

(14ndash35) (36ndash48) (49ndash60) (61ndash70) (71ndash80) (81ndash92)

on silica gel

(95 05) (85 15) (70 40) (0 100)

(1ndash13)

Petether-EtOAc

Powdered aerial parts of L nodiflora

(100 0 sim 0 100)

Figure 1 Extraction scheme for the isolation of antioxidant compound from aerial parts of Lippia nodiflora L

10000

5000

0

0 5 10 15 20 25 30 35 40

Retention time (min)

35480

27889

17308

10056

6562

7234

5903

4912

2995

Volts

uV

(a)

0 5 10 15 20 25 30 4035 45


2000

1000

0

7258

Volts

uV

(b)

Figure 2 (a) HPLC chromatogram of whole methanol extract of L nodiflora (b) HPLC chromatogram of isolated compound (5-hydroxy-3101584041015840 7-trimethoxyflavone) at 280 nmThe HPLC profile shows the purity of the isolated compound from aerial parts of L nodiflora


10095908580757065605550454035302520151050100 200 300 400 500 600 700 800 900 1000

Rela

tive a

bund

ance

6791790239211072

35108417

32910233

mz

Figure 3 Molecular mass of isolated compoundfrom aerial parts ofL nodiflora

highestDPPHscavenging activity for extract compound andBHT was found to be 7935 7266 and 8609 respec-tively at 50120583gmL It should be noted that the scavengingactivity of compoundwas found to be close to the extractThe IC

50values of scavenging activity on DPPH radical of

extract and BHT are given in Table 1 whereas for compoundit is found to be 27 120583gmL From these obtained datathe extract and compound were considered as an effectivefree-radical inhibitor as well as the primary antioxidantswhich may limit free-radical damage that takes place in thebody

322 Superoxide Radical Scavenging Effect The formation ofreactive oxygen species such as hydroxyl radical hydrogenperoxide and singlet oxygen in living system was mainlydue to the participation of superoxide anion radicals eitherdirectly or widely through enzyme or metal catalyzed pro-gression [38] It was therefore anticipated to evaluate therelative interceptive capacity of the extract and compoundto scavenge the superoxide radical From the data presentedin Figure 5(b) it was noted that the extract compoundand BHT showed the highest radical scavenging activities(7107ndash8464) at 50 120583gmL and the scavenging activityincreased with increasing concentration of the samples Thescavenging ability of compound on superoxide radicals wasfound to be moderate compared to methanolic extractHowever the scavenging activities of extract (8309) werefound to be very closer to that of BHT (8464) which isconsidered to be a strong superoxide radical scavenger TheIC50

value of plant extract compound and BHT was foundto be 32 38 and 26 120583gmL respectively

323 Hydroxyl Radical Scavenging Effect Hydroxyl radicalan extremely known reactive oxygen species was competentto attack and spoil almost every molecule in the living cells[11] They were also capable of stimulating lipid peroxidationprocess rapidly by attacking the fatty acid side chains of themembrane phospholipids [38] The scavenging activities ofmethanolic extract compound and BHT on hydroxyl radical

O

O

OO

O

OH

H3CCH3

CH3

Figure 4 Structure of isolated compound (5-hydroxy-31015840 41015840 7-trimethoxyflavone) from ethyl acetate (EAF) fraction of aerial partsof L nodiflora

inhibition are shown in Figure 6 All the examined samplesshowed significant hydroxyl radical scavenging activity at50 120583gmL concentration and the scavenging activity forextract compound and BHT was 6838 6090 and7416 respectively The methanolic extract of L nodiflora(IC50

= 36 120583gmL) was more powerful than the compound(IC50= 43 120583gmL) The positive control BHT was extremely

effectual on hydroxyl radical scavenging with an IC50

valueof 32 120583gmL This observed capacity of the extract and com-pounds to scavenge ∙OH radical pointed out that the testedsamples could considerably inhibit lipid peroxidation since∙OH radicals are extremely distressed during peroxidation

324 FRAP (Ferric Reducing Antioxidant Power) Assay Theferric reducingantioxidant power (FRAP assay) is widelyused in the assessment of the antioxidant component indietary polyphenols [39]The reducing properties are usuallyrelated to the presence of compoundswhich exert their actionby breaking the free-radical chain by donating a hydrogenatom [40] The results of reductive potential of plant extractand compound relative to BHT a well-known antioxidantdata are shown in Table 2 The reducing ability of the extractwas in the range of 2346ndash7114120583MFe (II)gThe FRAP valuesfor the methanol extract were significantly higher than thatof compound and BHT while the compound revealed thelowest FRAP values (1118ndash5170 120583MFe (II)g) At 50 120583gmLthe FRAP value of L nodiflora extract was found to be7114 compared to compound and BHT with FRAP valueof 5170 and 6318 respectively (Table 2) This result impliesthat the isolated compound did not show reliable reducingpower when compared to its DPPH and superoxide radicalscavenging abilities

325 Nitric Oxide Radical Scavenging Assay Nitric oxideradicals (NO∙) play a vital role in inducing inflammatoryresponse and their toxic effects increase only when theyreact with superoxide radicals that damage biomolecules likeproteins lipids and nucleic acids [41] The suppression ofNO radicals release may be attributed to a direct (NO∙)scavenging effect as both extract and compound decreasedthe amount of nitrite generated from the decomposition ofsodium nitroprusside through in vitro studies as shown in


10 20 30 40 50

100

80

60

40

20

0

Concentration (120583gmL)

Methanolic extractCompoundBHT

Inhi

bitio

n (

)

(a)

10 20 30 40 50

100

80

60

40

20

0

Concentration (120583gmL)Methanolic extractCompoundBHT

Inhi

bitio

n (

)

(b)

Figure 5 (a) DPPH radical scavenging and (b) superoxide radical scavenging activities of extract compound and BHT at differentconcentrations Values are mean plusmn SD (119899 = 3)

10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

Figure 6 Hydroxyl radical scavenging effect of extract compoundand BHT at different concentrations Values are mean plusmn SD (119899 = 3)

Figure 7(a)The extract and compound at 50120583gmL exhibited486 and 40 inhibition which was comparable to thestandard BHTwhich exhibited 498 inhibition at 40120583gmLHowever both extract (IC

50= 54 120583gmL) and compound

(IC50

= 67 120583gmL) can inhibit NO radicals but lesser whencompared to standard BHT (IC

50= 41 120583gmL) The NO

radical-scavenging activities of the extract and compoundalso followed a concentration-dependent pattern The resultssuggest that the isolated flavone compound of L nodifloramight be an effective and novel therapeutic agent for scav-enging and regulating the pathological conditions causedby excessive generation of NO and its oxidation productperoxynitrite

Table 2 Antioxidant potentials of extract compound and BHT atdifferent concentrations examined by FRAP assay

Sample Test concentration(120583gmL)

FRAP value(120583M Fe(II)g)a

Methanol extract

10 234 plusmn 05420 275 plusmn 12430 525 plusmn 06940 639 plusmn 07050 711 plusmn 169

Compound


BHT


aThe values are the average of three independent experiments

326 Inhibitory Activity towards Lipid Peroxidation Thedisturbances in the membrane assembly lead to changesin fluidity and permeability modifications of ion trans-port and inhibition of metabolic processes altogetherrevealed to be the collective consequence of reactive oxygenspecies formed during lipid peroxidation [42] The extractand compound were capable of preventing MDA forma-tion in a concentration-dependent manner (Figure 7(b))At 50 120583gmL methanolic extract and compound possessed6244 and 5495 inhibition while BHT exhibited 6864


10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(a)

10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(b)

Figure 7 (a) Nitric oxide radical scavenging and (b) lipid peroxidation inhibition activities of extract compound and BHT at differentconcentrations Values are mean plusmn SD (119899 = 3)

inhibition at the same concentration The IC50

values forextract and compound were found to be 39 and 46 120583gmLThere is no significant difference in the IC

50values of extract

and compound (119875 lt 005) However IC50

values of extractwere in good agreement with BHT (IC

50= 34 120583gmL) The

presence of hydroxyl and electron-donating methoxy groupin the compound (5-hydroxy-31015840 41015840 7-trimethoxyflavone)may be accountable for the antioxidant activity in all theexperimental assays

Based on in vitro antioxidant results of the present workthe methanol extract and isolated compound from ethylacetate fraction of L nodiflorawere believed to be an electrondonor capable of counteracting free radicals This is the firststudy to give an account on the antioxidant and free-radicalscavenging activity of 5-hydroxy-31015840 41015840 7-trimethoxyflavonefrom L nodifloraThe results of the present work also proposethat the numerous pharmacological properties exerted by Lnodiflora may be partly due to the presence of antioxidantflavone compound

4 Conclusion

The present study was projected to assess the antioxidantand free-radical scavenging activities of extract and fractionsfrom aerial parts of L nodiflora by using in vitro antiox-idant models The ethyl acetate fraction (EAF) exhibitedhighest free-radical scavenging activity among the fractionsA bioassay-guided fractionation and purification of EAFresulted in the identification of the flavone compoundnamely 5-hydroxy-31015840 41015840 7-trimethoxyflavone The mea-surement of antioxidant activity of the flavone compoundby using various in vitro antioxidant models proved it tobe a potent antioxidant compound These results signifythat methanol extract ethyl acetate fractions and isolatedcompound exhibited interesting antioxidant properties and

afford an essential basis for the use of L nodiflora in thetreatment of oxidative damages Furthermore these findingshold great perception in the development of alternativeantioxidant agents and still further work is warranted tosort out and characterize the active principles from otherfractions in order to establish their therapeutic efficacy andmechanism of action

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was financially supported by Department of Sci-ence and Technology (DST WOS-A) New Delhi (Grant noSRWOS-ALS-2372010) The authors thank Dr K Kulan-giappar Electro Organic Division Central ElectrochemicalResearch Institute Karaikudi for helping in NMR interpre-tation of the isolated compound

References

[1] B N Singh B R Singh R L Singh et al ldquoOxidative DNAdam-age protective activity antioxidant and anti-quorum sensingpotentials of Moringa oleiferardquo Food and Chemical Toxicologyvol 47 no 6 pp 1109ndash1116 2009

[2] S Shukla A Mehta J John S Singh P Mehta and S P VyasldquoAntioxidant activity and total phenolic content of ethanolicextract of Caesalpinia bonducella seedsrdquo Food and ChemicalToxicology vol 47 no 8 pp 1848ndash1851 2009

[3] I I Koleva H A G Niederlander and T A Van BeekldquoAn on-line HPLC method for detection of radical scavengingcompounds in complexmixturesrdquoAnalytical Chemistry vol 72no 10 pp 2323ndash2328 2000


[4] J Whysner C X Wang E Zang M J Iatropoulos and G MWilliams ldquoDose response of promotion by butylated hydrox-yanisole in chemically initiated tumours of the rat forestomachrdquoFood and Chemical Toxicology vol 32 no 3 pp 215ndash222 1994

[5] B A Silva F Ferreres J O Malva and A C P Dias ldquoPhyto-chemical and antioxidant characterization of Hypericum per-foratum alcoholic extractsrdquo Food Chemistry vol 90 no 1-2 pp157ndash167 2005

[6] A R Verma M Vijayakumar C S Mathela and C V Rao ldquoInVitro and in vivo antioxidant properties of different fractions ofMoringa oleifera leavesrdquo Food and Chemical Toxicology vol 47no 9 pp 2196ndash2201 2009

[7] K Saito M Kohno F Yoshizaki and Y Niwano ldquoExtensivescreening for edible herbal extracts with potent scavengingactivity against superoxide anionsrdquo Plant Foods for HumanNutrition vol 63 no 2 pp 65ndash70 2008

[8] J Delgado Adamez E Gamero Samino E Valdes Sanchez andD Gonzalez-Gomez ldquoIn Vitro estimation of the antibacterialactivity and antioxidant capacity of aqueous extracts fromgrape-seeds (Vitis vinifera L)rdquo Food Control vol 24 no 1-2 pp136ndash141 2012

[9] S Sreelatha and P R Padma ldquoAntioxidant activity and totalphenolic content of Moringa oleifera leaves in two stages ofmaturityrdquo Plant Foods for Human Nutrition vol 64 no 4 pp303ndash311 2009

[10] E S Abdel-Hameed ldquoTotal phenolic contents and free radicalscavenging activity of certain Egyptian Ficus species leaf sam-plesrdquo Food Chemistry vol 114 no 4 pp 1271ndash1277 2009

[11] B Halliwell ldquoDietary polyphenols good bad or indifferent foryour healthrdquo Cardiovascular Research vol 73 no 2 pp 341ndash347 2007

[12] V Lopez S Akerreta E Casanova J M Garcıa-Mina R YCavero andM I Calvo ldquoIn Vitro antioxidant and anti-rhizopusactivities of lamiaceae herbal extractsrdquo Plant Foods for HumanNutrition vol 62 no 4 pp 151ndash155 2007

[13] D O Rios L M G Antunes and M D L P Bianchi ldquoBixinand lycopene modulation of free radical generation induced bycisplatin-DNA interactionrdquo Food Chemistry vol 113 no 4 pp1113ndash1118 2009

[14] M Yusuf J Begum M N Hoque and J U ChowdhuryMedic-inal Plants of Bangladesh BCSIR Laboratories ChittagongBangladesh 2009 Revised and Enlarged

[15] A M Forestieri M T Monforte S Ragusa A Trovato andL Iauk ldquoAntiinflammatory analgesic and antipyretic activity inrodents of plant extracts used in African medicinerdquo Phytothea-phyr Research vol 10 no 2 pp 100ndash106 1996

[16] G Balakrishnan L Janakarajan A Balakrishnan and B SLakshmi ldquoMolecular basis of the anti-inflammatory propertyexhibited by cyclo-pentano phenanthrenol isolated from Lippianodiflorardquo Immunological Investigations vol 39 no 7 pp 713ndash739 2010

[17] R Balamurugan and S Ignacimuthu ldquoAntidiabetic and hypol-ipidemic effect of methanol extract of Lippia nodiflora L instreptozotocin induced diabetic ratsrdquo Asian Pacific Journal ofTropical Biomedicine vol 1 supplement 1 pp S30ndashS36 2011

[18] L Yen M C Wang C J Liang H H Ko and C WLee ldquoMelanogenesis inhibitor(s) from Phyla nodiflora extractrdquoEvidence Based Complementary and Alternative Medicine vol2012 Article ID 867494 9 pages 2012

[19] A M Abbasi M A Khan M Ahmad M Zafar S Jahanand S Sultana ldquoEthnopharmacological application ofmedicinal

plants to cure skin diseases and in folk cosmetics among thetribal communities of North-West Frontier Province PakistanrdquoJournal of Ethnopharmacology vol 128 no 2 pp 322ndash335 2010

[20] K Basu P Chakraborti and P K Sanyal ldquoNodifloretin- anew flavone from Lippia nodiflorardquo Journal of Indian ChemicalSociety vol 46 no 4 pp 271ndash272 1969

[21] F A Tomas-Barberan J B Harborne and R Self ldquoTwelve6-oxygenated flavone sulphates from Lippia nodiflora and Lcanescensrdquo Phytochemistry vol 26 no 8 pp 2281ndash2284 1987

[22] G R Nair P Ramesh and S Nagarajan ldquoNew flavones glyco-sides from Lippia nodiflorardquo Indian Journal of Chemistry vol 2pp 1316ndash1317 1973

[23] S D Elakovich and K L Stevens ldquoVolatile constituents ofLippia nodiflorardquo Journal of Natural Products vol 48 no 3 pp504ndash506 1985

[24] V Ravikanth P Ramesh P V Diwan and Y VenkateswarluldquoHalleridone and Hallerone from Phyla nodiflora as taxonomicmarkersrdquo Biochemical Systematics and Ecology vol 28 no 9 pp905ndash906 2000

[25] B S Siddiqui F Ahmed S K Ali S Perwaiz and S BegumldquoSteroidal constituents from the aerial parts of Lippia nodifloraLinnrdquo Natural Product Research vol 23 no 5 pp 436ndash4412009

[26] R Balamurugan V Duraipandiyan and S IgnacimuthuldquoAntidiabetic activity of 120574-sitosterol isolated from Lippia nod-iflora L in streptozotocin induced diabetic ratsrdquo EuropeanJournal of Pharmacology vol 667 no 1ndash3 pp 410ndash418 2011

[27] T Yamaguchi H Takamura T Matoba and J Terao ldquoHPLCmethod for evaluation of the free radical-scavenging activityof foods by using 11-diphenyl-2-picrylhydrazylrdquo BioscienceBiotechnology and Biochemistry vol 62 no 6 pp 1201ndash12041998

[28] M Nishikimi N Appaji Rao and K Yagi ldquoThe occurrence ofsuperoxide anion in the reaction of reduced phenazine metho-sulfate and molecular oxygenrdquo Biochemical and BiophysicalResearch Communications vol 46 no 2 pp 849ndash854 1972

[29] I Hinneburg H J Damien Dorman and R Hiltunen ldquoAntiox-idant activities of extracts from selected culinary herbs andspicesrdquo Food Chemistry vol 97 no 1 pp 122ndash129 2006

[30] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of antioxidant power the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[31] L C Green D A Wagner and J Glogowski ldquoAnalysis ofnitrate nitrite and [15N]nitrate in biological fluidsrdquo AnalyticalBiochemistry vol 126 no 1 pp 131ndash138 1982

[32] H J Damien Dorman S G Deans R C Noble and PSurai ldquoEvaluation in vitro of plant essential oils as naturalantioxidantsrdquo Journal of Essential Oil Research vol 7 no 6 pp645ndash651 1995

[33] D Ashokkumar V Thamilselvan G P Senthilkumar U KMazumder and M Gupta ldquoAntioxidant and free radical scav-enging effects of Lippia nodiflorardquo Pharmaceutical Biology vol46 no 10-11 pp 762ndash771 2008

[34] T Ersoz S I Harput I Saracoglu I Calis andYOgihara ldquoPhe-nolic compounds from Scutellaria ponticardquo Turkish Journal ofChemistry vol 26 no 4 pp 581ndash588 2002

[35] T T Mai N Fumie and N Van Chuyen ldquoAntioxidant activitiesand hypolipidemic effects of an aqueous extract from flowerbuds of Cleistocalyx operculatus (Roxb) Merr and PerryrdquoJournal of Food Biochemistry vol 33 no 6 pp 790ndash807 2009


[36] S Shukla R Patel and R Kukkar ldquoStudy of phytochemicaland diuretic potential of methanol and aqueous extracts ofaerial parts of Phyla nodiflora Linnrdquo International Journal ofPharmacy and Pharmaceutical Sciences vol 1 no 1 pp 85ndash912009

[37] R P Singh K N Chidambara Murthy and G K JayaprakashaldquoStudies on the antioxidant activity of pomegranate (Punicagranatum) peel and seed extracts using in vitromodelsrdquo Journalof Agricultural and Food Chemistry vol 50 no 1 pp 81ndash862002

[38] M Valko D Leibfritz J Moncol M T D Cronin M Mazurand J Telser ldquoFree radicals and antioxidants in normal physi-ological functions and human diseaserdquo International Journal ofBiochemistry and Cell Biology vol 39 no 1 pp 44ndash84 2007

[39] A Luximon-Ramma T Bahorun M A Soobrattee and O IAruoma ldquoAntioxidant activities of phenolic proanthocyanidinand flavonoid components in extracts of Cassia fistulardquo Journalof Agricultural and Food Chemistry vol 50 no 18 pp 5042ndash5047 2002

[40] P D Duh P C Du andG C Yen ldquoAction ofmethanolic extractof mung bean hulls as inhibitors of lipid peroxidation and non-lipid oxidative damagerdquo Food and Chemical Toxicology vol 37no 11 pp 1055ndash1061 1999

[41] S Moncada R M J Palmer and E A Higgs ldquoNitric oxidephysiology pathophysiology and pharmacologyrdquo Pharmaco-logical Reviews vol 43 no 2 pp 109ndash142 1991

[42] S Nigam and T Schewe ldquoPhospholipase A2s and lipid per-oxidationrdquo Biochimica et Biophysica Acta-Molecular and CellBiology of Lipids vol 1488 no 1-2 pp 167ndash181 2000

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


Lippia nodiflora Linn (Verbenaceae) commonly calledPoduthalai in Tamil is a creeping perennial herb and locallyabundant in wet regions and several medicinal propertiesare attributed to this plant in the traditional system ofmedicine The infusion of the leaves and tender stalks isgiven to children suffering from indigestion and to womenafter delivery The chutney made from the leaves and fruitsare often taken to relieve the irritation of internal piles[14] Numerous pharmacological properties of L nodifloraincluding anti-inflammatory antipyretic antitussive antidi-abetic and antimelanogenesis effects have been reported[15ndash18] The ethnopharmacological relevance of L nodiflorafor skin diseases and in folk cosmetics such as pimplescarbuncle and skin burns has also been revealed [19] Thephytochemical constituents of L nodiflora such as flavonoids[20 21] flavone glycosides [22] alkaloids essential oils resin[23] quinol [24] and steroids [25 26] have been previouslyreported Hence these phytochemicals are considered tobe accountable for the pharmacological properties of thisplant Even though there is an evidence for the antioxidantactivity of methanolic extract of L nodiflora the majorantioxidative constituents present in the aerial parts havenot been extensively investigated Hence the present studywas undertaken to isolate active compounds responsiblefor the antioxidant property of methanol extract of Lippianodiflora L through bioassay-guided fractionation using invitro DPPH assay

2 Materials and Methods

21 General Nuclear magnetic resonance spectra wererecorded on BRUKER Avance 400MHz (Switzerland) NMRinstrument operating at 400MHz for 1H and 100MHz for13Cnuclei at room temperature and referenced to the residualsolvent signal The chemical shift and coupling constants(119869) values are reported in ppm and Hz respectively HPLCanalysis was performed using a C-18 column (250 times 46mm5 120583) in a Shimadzu LC-8A chromatographic apparatus (Shi-madzu Singapore)Themobile phase consisted of methanol-05 phosphoric acid in water (60 40 vv) and the flowrate was held constant at 1mLmin The peaks are detectedat 280 nm using variable wavelength UV detector Silica gel60 F254

plates (20 times 20 cm 02mm thick E-Merck Germany)were used for thin-layer chromatography (TLC) analysisThe ultraviolet spectra were recorded using Varian Cary500 scan spectrophotometer 120582max (log 120576) in nm whereas theFTIR spectrum was obtained using a Nicolet 380 (ThermoScientific USA) The functional group was identified usingpotassium bromide (KBr) and scanned in the range of 4000ndash400 cmminus1 Sample was dissolved in methanol and ESI massspectra were obtainedwith aThermo Scientific ExactiveMassSpectrometer (Thermo Fisher Scientific USA)

The reagents such as 2-deoxy-D-ribose butylated hy-droxyl toluene (BHT) 2 2-diphenyl-1-picrylhydrazyl (DPPH)phenazine methosulphate (PMS) nitroblue tetrazolium(NBT) sodium nitroprusside and Griess reagent wereobtained from (Sigma Chemicals St Louis MO USA) 2 46-Tripyridyl-S-triazine (TPTZ) thiobarbituric acid (TBA)

trichloroacetic acid (TCA) ethylene diaminetetraacetic acid(EDTA) ferric chloride (FeCl

3) hydrogen peroxide (H

2O2)

and nicotinamide adenine dinucleotide-reduced (NADH)were obtained from Ms (Sisco Research laboratoriesMumbai India) HPLC grade solvents and reagents usedfor extraction and silica gel (0075ndash015mm) for columnchromatography were obtained from Ms (Sisco Researchlaboratories Mumbai India) All other chemicals andreagents used in this study were of analytical grade

22 Plant Materials The plant materials were freshly col-lected between August and September 2011 from KaraikudiSivagangai district Tamil NaduThe plant was taxonomicallyidentified and authenticated by Dr G V S Murthy JointDirector Botanical Survey of India Tamil Nadu Agricul-tural University Campus Coimbatore (BSISRC5232012-13Tech-19) A voucher specimen of the plant L nodiflora(DBI-HP Specimen-1) was prepared and preserved in theMolecular Biology Lab Department of Bioinformatics Ala-gappa University Karaikudi Tamil Nadu

23 Extraction Fractionation and Isolation of AntioxidantCompound The aerial parts (stem leaves and flowers) werewashed with tap water shade-dried and reduced to finepowder The powdered aerial parts of L nodiflora (15 kg)were extracted with 90 methanol (45 L times 2) at roomtemperature The mixture was filtered through Whatmannumber l filter paper and the solvents from the combinedextract were concentrated using a vacuum rotary evaporator(Superfit India) at 60∘C to afford 647 g of crude methanolextract (431) The solvent was selected based on its yieldfrom preliminary extraction and phytochemical screeningstudies The extract was dissolved in 500mL of warm waterand the resulting aqueous portion was partitioned with ethylacetate (EtOAc) (4 times 200mL) using a separating funnel toafford ethyl acetate fraction (EAF) and aqueous portionThe aqueous phase was then successively partitioned withn-butanol (3 times 200mL) thus obtaining n-butanol solublefraction (BF) and water fraction (WF) All the fractionswere collected separately and reduced using a vacuum rotaryevaporator to remove the solvent and the resultant aqueousextract was lyophilized in vacuo The samples were thentested for its antioxidant property using 2 2-diphenyl-1-picrylhydrazyl (DPPH∙) assay

The DPPH∙ active EAF (172 g) was loaded as a driedslurry of silica gel to column chromatography (45 times 35 cm)and eluted with petroleum ether EtOAc gradient elution(100 0mdash0 100) in increasing order of polarity A total of92 fractions of 100mL each were collected and analyzed byTLC (Silica gel F

254plates 20 times 20 cm Merck Germany)

TLC analysis was carried out using ethyl acetate chloro-form formic acid (5 4 1) as the mobile phase and theseparated bands were visualized using iodine vapors andvanillin-sulphuric acid reagent These fractions were pooledto afford seven major fractions (Fr A 1ndash13 Fr B 14ndash35Fr C 36ndash48 Fr D 49ndash60 Fr E 61ndash70 Fr F 71ndash80 andFr G 81ndash92) based on TLC analysis These fractions weretested for bioactivity using DPPH spectrophotometric assay




















calibration








times 100

(2)









IC50






3 400MHz) 120575 (ppm) 391 (3H 119904 41015840-OMe)



18H16O6(Figure 3)





(145kg)












CC on silica gel


Compound(117mg)


on silica gel

(95 05) (85 15) (70 40) (0 100)

(1ndash13)

Petether-EtOAc


(100 0 sim 0 100)


10000

5000

0

0 5 10 15 20 25 30 35 40


35480

27889

17308

10056

6562

7234

5903

4912

2995

Volts

uV

(a)

0 5 10 15 20 25 30 4035 45


2000

1000

0

7258

Volts

uV

(b)



10095908580757065605550454035302520151050100 200 300 400 500 600 700 800 900 1000

Rela

tive a

bund

ance

6791790239211072

35108417

32910233

mz








O

O

OO

O

OH

H3CCH3

CH3









10 20 30 40 50

100

80

60

40

20

0



Inhi

bitio

n (

)

(a)

10 20 30 40 50

100

80

60

40

20

0


Inhi

bitio

n (

)

(b)


10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)




(IC50


50= 41 120583gmL) The NO





Methanol extract


Compound


BHT





10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(a)

10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(b)




50values of extract



50= 34 120583gmL) The



4 Conclusion





Acknowledgments


References


















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




















calibration








times 100

(2)









IC50






3 400MHz) 120575 (ppm) 391 (3H 119904 41015840-OMe)



18H16O6(Figure 3)





(145kg)












CC on silica gel


Compound(117mg)


on silica gel

(95 05) (85 15) (70 40) (0 100)

(1ndash13)

Petether-EtOAc


(100 0 sim 0 100)


10000

5000

0

0 5 10 15 20 25 30 35 40


35480

27889

17308

10056

6562

7234

5903

4912

2995

Volts

uV

(a)

0 5 10 15 20 25 30 4035 45


2000

1000

0

7258

Volts

uV

(b)



10095908580757065605550454035302520151050100 200 300 400 500 600 700 800 900 1000

Rela

tive a

bund

ance

6791790239211072

35108417

32910233

mz








O

O

OO

O

OH

H3CCH3

CH3









10 20 30 40 50

100

80

60

40

20

0



Inhi

bitio

n (

)

(a)

10 20 30 40 50

100

80

60

40

20

0


Inhi

bitio

n (

)

(b)


10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)




(IC50


50= 41 120583gmL) The NO





Methanol extract


Compound


BHT





10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(a)

10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(b)




50values of extract



50= 34 120583gmL) The



4 Conclusion





Acknowledgments


References


















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




times 100

(2)









IC50






3 400MHz) 120575 (ppm) 391 (3H 119904 41015840-OMe)



18H16O6(Figure 3)





(145kg)












CC on silica gel


Compound(117mg)


on silica gel

(95 05) (85 15) (70 40) (0 100)

(1ndash13)

Petether-EtOAc


(100 0 sim 0 100)


10000

5000

0

0 5 10 15 20 25 30 35 40


35480

27889

17308

10056

6562

7234

5903

4912

2995

Volts

uV

(a)

0 5 10 15 20 25 30 4035 45


2000

1000

0

7258

Volts

uV

(b)



10095908580757065605550454035302520151050100 200 300 400 500 600 700 800 900 1000

Rela

tive a

bund

ance

6791790239211072

35108417

32910233

mz








O

O

OO

O

OH

H3CCH3

CH3









10 20 30 40 50

100

80

60

40

20

0



Inhi

bitio

n (

)

(a)

10 20 30 40 50

100

80

60

40

20

0


Inhi

bitio

n (

)

(b)


10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)




(IC50


50= 41 120583gmL) The NO





Methanol extract


Compound


BHT





10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(a)

10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(b)




50values of extract



50= 34 120583gmL) The



4 Conclusion





Acknowledgments


References


















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(145kg)












CC on silica gel


Compound(117mg)


on silica gel

(95 05) (85 15) (70 40) (0 100)

(1ndash13)

Petether-EtOAc


(100 0 sim 0 100)


10000

5000

0

0 5 10 15 20 25 30 35 40


35480

27889

17308

10056

6562

7234

5903

4912

2995

Volts

uV

(a)

0 5 10 15 20 25 30 4035 45


2000

1000

0

7258

Volts

uV

(b)



10095908580757065605550454035302520151050100 200 300 400 500 600 700 800 900 1000

Rela

tive a

bund

ance

6791790239211072

35108417

32910233

mz








O

O

OO

O

OH

H3CCH3

CH3









10 20 30 40 50

100

80

60

40

20

0



Inhi

bitio

n (

)

(a)

10 20 30 40 50

100

80

60

40

20

0


Inhi

bitio

n (

)

(b)


10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)




(IC50


50= 41 120583gmL) The NO





Methanol extract


Compound


BHT





10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(a)

10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(b)




50values of extract



50= 34 120583gmL) The



4 Conclusion





Acknowledgments


References


















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


10095908580757065605550454035302520151050100 200 300 400 500 600 700 800 900 1000

Rela

tive a

bund

ance

6791790239211072

35108417

32910233

mz








O

O

OO

O

OH

H3CCH3

CH3









10 20 30 40 50

100

80

60

40

20

0



Inhi

bitio

n (

)

(a)

10 20 30 40 50

100

80

60

40

20

0


Inhi

bitio

n (

)

(b)


10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)




(IC50


50= 41 120583gmL) The NO





Methanol extract


Compound


BHT





10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(a)

10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(b)




50values of extract



50= 34 120583gmL) The



4 Conclusion





Acknowledgments


References


















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


10 20 30 40 50

100

80

60

40

20

0



Inhi

bitio

n (

)

(a)

10 20 30 40 50

100

80

60

40

20

0


Inhi

bitio

n (

)

(b)


10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)




(IC50


50= 41 120583gmL) The NO





Methanol extract


Compound


BHT





10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(a)

10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(b)




50values of extract



50= 34 120583gmL) The



4 Conclusion





Acknowledgments


References


















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(a)

10 20 30 40 50

80

60

40

20

0


Inhi

bitio

n (

)

(b)




50values of extract



50= 34 120583gmL) The



4 Conclusion





Acknowledgments


References


















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

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AddictionJournal of






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Journal of


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Documents

Research Article Bioassay-Guided Isolation and …downloads.hindawi.com/journals/bmri/2014/549836.pdfResearch Article Bioassay-Guided Isolation and Antioxidant Evaluation of Flavonoid