research article phytochemical analysis and anti ...research article phytochemical analysis and...

Research ArticlePhytochemical Analysis and Anti-Inflammatory Activity ofthe Extracts of the African Medicinal Plant Ximenia caffra

Jing Zhen12 Yue Guo3 Tom Villani12 Steve Carr4 Thomas Brendler5

Davis R Mumbengegwi6 Ah-Ng Tony Kong3 James E Simon12 and Qingli Wu12

1New Use Agriculture and Natural Plant Products Program Department of Plant Biology and Pathology Rutgers University59 Dudley Road New Brunswick NJ 08901 USA2Department of Medicinal Chemistry Ernest Mario School of Pharmacy Rutgers University 160 Frelinghuysen Road PiscatawayNJ 08854 USA3Department of Pharmaceutics Ernest Mario School of Pharmacy Rutgers University 160 Frelinghuysen Road PiscatawayNJ 08854 USA4National Botanical Research Institute Windhoek Namibia5PlantaPhile 705B Park Avenue Collingswood NJ 08108 USA6Drug Discovery and Development Program Science Technology and Innovation Division Multidisciplinary Research CenterUniversity of Namibia 340 Mandume Ndemufayo Avenue Private Bag Box 13301 Windhoek Namibia

Correspondence should be addressed to James E Simon jimsimonrcirutgersedu and Qingli Wu qlwuaesoprutgersedu

Received 11 December 2014 Accepted 1 February 2015

Academic Editor Mengxia Xie

Copyright copy 2015 Jing Zhen et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A method was developed for identification and quantification of polyphenols in the leaves of Ximenia caffra using HPLCUVMSBased on analyzing the MS and UV data and in comparison to the authentic standards a total of 10 polyphenols were identifiedand quantified including gallic acid catechin quercetin kaempferol and their derivatives The total content of these compoundswas found to be approximately 1945mgg in the leaf and the most abundant is quercetin-rutinoside (908mgg)The total phenoliccontent as measured by Folin-Ciocalteu assay was 26187 plusmn 711mgGAEg and the total antioxidant capacity as measured in vitrowas 146 plusmn 001mmol TroloxgThe antiproliferative effect of the leaf extract was measured by MTS assay with IC

50value of 2390 plusmn

445 120583gmL Cell-based assays show that the leaf extract inhibits the mRNA expression of proinflammatory genes (IL-6 iNOS andTNF-120572) by using RT-qPCR implying its anti-inflammatory effects It was further demonstrated that the underlying therapeuticmechanism involves the suppression of NF-120581B a shared pathway between cell death and inflammation

1 Introduction

Ximenia caffra also known as ldquolarge sourplumrdquo is a memberof a genus of flowering plants in the Olacaceae family whichgrows in the southern African region [1 2] X caffra has beenused in foods and traditional medicine Its fruit consideredto be rich in vitamin C potassium and protein has beencommonly added into porridges and made into jam [3 4]The dried seed of X caffra contains a substantial quantity ofunsaturated fatty acids themost abundant is oleic acid [5] Assuch the extracted seed oil would be used as a feed ingredient

and domestic biofuel [6] As a traditional medicine localherbalists have been using the leaves and root of X caffrafor treatment of wounds infections fever infertility anddiarrhea [7ndash9] Recent research in laboratory confirmed thatthe leaf and root extracts of X caffra have antigonococcalantibacterial and antifungal activities which corroboratewith its traditional use very well [7 8 10 11] Comparedwith a number of researches on its pharmacological activitiesonly one paper has been published on the phytochemicalcomposition analysis of X caffra leaf in which the totalflavonoid content was only roughly estimated using vanillin

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2015 Article ID 948262 9 pageshttpdxdoiorg1011552015948262

2 Journal of Analytical Methods in Chemistry

assay [8] Bioactive phytochemicals contained in the leaf ofrelated species X americana from western and eastern sub-Sahara Africa were investigated and led to the identificationof sambunigrin gallic acid and quercetin and the glycosidesby spectrometric methods [12]

Most of the studies with X caffra focus on the fruit andseed as these represent the products of current commerceand traditional use rather than the leaf In this studywe purposefully investigate the phytochemical profile ofX caffra leaf which has not been previously examinedusing LCUVMS Interestingly though not unexpectedly anumber of polyphenol compounds were discovered in theleaf Polyphenols are a class of compounds characterized bythe presence of large multiples of phenol structural unitsand abundantly exist in food products including crops fruitvegetables tea and wine [13ndash15] These compounds haveshown a wide range of biological activities including anti-inflammation and the prevention of cancer and cardiovascu-lar diseases [16 17]

Based upon the chemical profile determined and to fur-ther explore new applications for the leaves while extendingthe traditional uses we investigated the leaves for theirantioxidant activity total phenolic content and antipro-liferation activity using the Folin-Ciocalteu assay Troloxequivalent antioxidant capacity radical scavenging assay andthe MTS cell proliferation assay respectively Additionallythe effects of extracts on the mRNA expression of proin-flammatory genes were assessed by RT-qPCR through thequantification of inducible nitric oxide synthetase (iNOS)interleukin 6 (IL-6) and tumor necrosis factor 120572 (TNF-120572) induced by lipopolysaccharide (LPS) in murine RAW2647 macrophage cells Interleukin 6 (IL-6) a cytokinesecreted by T cells and macrophages is produced at theinflammation sites during infection and tissue damage IL-6 has stimulatory effects on immune cells and can inducechronic inflammatory responses Thus targeting IL-6 wouldhelp prevent or treat rheumatoid arthritis and other chronicinflammatory diseases [18] iNOS is one of the key enzymesgenerating nitric oxide (NO) which regulates a number ofpathophysiological conditions including infection inflam-mation and neoplastic diseases Reagents that can inhibitiNOS would reduce the generation of NO and have potentialanti-inflammatory effects [16] Anther biomarker TNF-120572 anadipokine is involved in series of inflammation reactionsduring immune response Its antagonists have also showneffects in the treatment of a range of inflammatory diseasesincluding rheumatoid arthritis psoriasis and ankylosingspondylitis [19]

To further explore the mechanism of antiproliferationand anti-inflammatory activities extracts of X caffra wereevaluated in cells for suppression of NF-120581B (nuclear factorkappa-light-chain-enhancer of activated B cells) a proteincomplex that regulates the transcription of DNA and isinvolved in cellular responses to stress The activation ofNF-120581B is generally considered to upregulate a range ofgenes encoding growth factors cytokines apoptosis andimmunomodulatory molecules As a shared key signal path-way of proliferation and inflammation in cells the prolongedactivation of NF-120581B has been reported to be involved in

inflammatory conditions autoimmune disease and cancer[20ndash24] The results demonstrate that the underlying ther-apeutic mechanism involves the suppression of NF-120581B ashared pathway between cell death and inflammation

2 Experimental

21 Materials Standard compounds gallic acid rutin cat-echin quercetin kaempferol and ABTS (221015840-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) reagents were pur-chased from Sigma Aldrich (St Louis MO) HPLC-grademethanol (MeOH) acetonitrile (ACN) and formic acidwere purchased from Fisher Scientific Co (Fair Lawn NJ)Folin-Ciocalteursquos phenol reagent was purchased from MPBiomedicals (Solon OH) The plant samples of X caffraleaves were field-collected from Northeastern Namibia usinga research permit from the Ministry of Environment andTourism Namibia Voucher specimens were prepared andsent to the National herbarium at the National BotanicalResearch Institute (NBRI) where the collected plants wereauthenticated The voucher specimens were also depositedin both the National Botanical Research Institute Wind-hoek Namibia and the Chrysler Herbarium amp MycologicalCollection Biological Sciences Building Rutgers UniversityNew Brunswick NJ The leaves were manually harvestedusing NBRI guidelines and were air-dried under ambienttemperature for two weeks and then packaged for laterexperimental analysis The dried leaf materials were air-shipped to Rutgers University where each was ground intoa fine powder with a laboratory mill (Perten 3100) and storedat room temperature in the dark

22 Analytical Equipment Separation on HPLC was per-formed using column Prodigy ODS3 5120583m 150 times 32mm5 micro (Phenomenex Inc Torrance CA) The separa-tion identification and quantification were performed onHewlett-Packard Agilent 1100 series HPLC-MS (AgilentTechnologies Waldbronn Germany) equipped with a qua-ternary pump system a degasser an autosampler a DADdetector an MSD trap with an electrospray ion source (ESI)and software HP ChemStation Data Analysis 42

23 LCMS Conditions for Qualification and Quantificationof the Polyphenols HPLC separation was performed withmobile phase of solvents A and B in gradient where A was01 formic acid (vv) in water and B was 01 formic acid(vv) in acetonitrileThe gradient starts from5B to 30B in50minThe detection wavelength was set at 280 nm for gallicacid and catechin and 370 nm for quercetin and kaempferolderivativesThe flow rate is 10mLminThe electrospray wasconducted in negative and positive modes in two separatedrunsThe optimized collision energy is of 80 scanning frommz 100 to 700 ESI was conducted at voltage of 35 kV forpositive model and minus35 kV for negative model High-purityliquid nitrogen 99999 was used as dry gas and nebulizerat a flow rate of 12 Lmin and the capillary temperature was350∘C Nitrogen was used as nebulizer at 60 psi and heliumas collision gas The ESI interface and mass spectrometerparameters were optimized to obtain maximum sensitivity

Journal of Analytical Methods in Chemistry 3

24 Sample Preparation For quantitative and qualitativestudy sim500mg of the finely grinded plant material wasextracted using 10mL 70 methanol in water with 01acetic acid and sonicated for 10min Then the extract wasconditioned at room temperature for overnight and filteredthrough a 045 120583m filter 10 120583L of the extract was injectedfor both qualitative and quantitative analyses The extrac-tion procedure was adopted from our prior studies [25]For total phenolic assay in vitro antioxidant assay around20mg of each plant sample was extracted using 10mL 70methanolwater (01 acetic acid) followed by sonicationfor 10min and then conditioned at room temperature forovernight The extract was filtered through a 045 120583m nylonfilter For cell-based bioactivity assays the leaf extract abovewas dried on rotavapor and lyophilizer to powder andsubsequently dissolved in DMSO to different concentrations

Individual stock solution (sim10mgmL) of the standardcompound was prepared by dissolving sim100mg of eachcompound in 10mLmethanolThe stock solutionwas dilutedwith methanol to obtain working solutions ranging from391 to 500 120583gmL Calibration curve was built upon eachgroup of the working solutions In each standard curve sixor more concentration levels were used for calibration onHPLCThe concentration of quercetin (119910 = 0052119909 minus 082571198772= 09999) and kaempferol (119910 = 00383119909 minus 17818

1198772= 09999) ranges from 391 to 500 120583gmL For gallic acid

(119910 = 00373119909 minus 12437 1198772 = 09999) and catechin (119910 =01516119909 minus 35055 1198772 = 09999) the concentration rangesfrom 3125 to 500120583gmL All calibrations curves have goodlinearity

25 Cell Culture and Treatment The murine RAW 2647macrophage cell a widely usedmodel for inflammatory stud-ies was obtained from the American Type Culture Collection(ATCC) PC-3N cell a stable clone transfected with anNF-120581B luciferase construct was established and obtainedfrom Dr Xi Zhengrsquos laboratory as previously described [26]The RAW cell and PC-3N cell were routinely cultured inDulbeccorsquos Modified Eaglersquos Medium (DMEM InvitrogenCarlsbad CA USA) supplemented with 10 fetal bovineserum (FBS Invitrogen Carlsbad CA USA) 100UmLpenicillin and streptomycin in a humidified incubator with5 CO

2at 37∘C

26 Folin-Ciocalteu Assay Total phenolic content was mea-sured on UV-Vis spectrophotometer according to the Folin-Ciocalteursquos method [27] 40 120583L of the extract was mixed with900120583L Folin-Ciocalteursquos reagent followed by incubation atroom temperature for 5min After that 400 120583L of 15 sodiumcarbonate was added and the mixture stayed at ambientroom temperature for 45min Then the UV absorptionat wavelength of 752 nm was measured against a blanksolution The standard curve was measured based on theprepared gallic acid standard solution (10 05 025 0125 and00625mgmL) and the result was transformed as milligramsof gallic acid per gram of sample plusmn SD

27 ABTS Scavenging Assay The total antioxidant capacityof leaf extract was measured according to the decolorization

of the ABTS radical cation as percentage of inhibition sim384mg of ABTS and 66mg potassium persulfate (K

2S2O8)

were codissolved in 10mL of water and reacted for 16 hoursin dark environment to form stable radicalsTheABTSwork-ing solution was prepared by dissolving ABTS radicalizedsolution in ethanol to an absorbance of 0700 plusmn 020 at 120582 =734 nm The wavelength selected is corresponding to thehighest extinction coefficient (120576 = 17 times 104molminus1L cmminus1in ethanol) 990 120583L of the diluted ABTS radical solution wasmixed with 10 120583L of the sample extract followed by reactionfor 20 minutes at room temperature The decoloration of thesolution indicates that ABTS radical cations were reduced bythe antioxidants in the sample and was determined by themeasurement of the decrease of absorbance at 734 nm againsta blank solution of ethanol at 25∘C The percentage decreaseof absorbance at 734 nm has a linear relationship with theamount of antioxidant compounds in the extract and can becalculated using the following equation

percentage inhibition ()

= [1 minus

Absorbancesample

Absorbancecontrol] times 100

(1)

Standard solution of Trolox was prepared in ethanolThe lin-ear relationship was built up between decreased absorbancepercentage and Trolox concentration (1199032 = 099) All samplesare prepared in triplicate and the result was expressed asmmol of Trolox per gram of sample plusmn SD

28 Antiproliferation Assay The antiproliferative effectof X caffra leaf extract was tested in RAW cells usingthe MTS assay MTS assay usually called ldquoone steprdquoMTT assay is used for assessing cell viability NAD(P)H-dependent cellular oxidoreductase enzymes from live cellscan reduce MTS reagent and form a formazan productwith maximum absorption at 490ndash500 nm The RAWcells were seeded in a 96-well tissue culture plate at aninitial density of 10000 cellsmL After 24 hr the cells weretreated with various concentrations of X caffra leaf extractin DMSO for another 24 hr The MTS assay was thenperformed using the CellTiter 96 aqueous nonradioactivecell proliferation assay kit [MTS 3-(45-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet-razolium inner salt] (Promega Madison WI USA)as previously described [24 28] The absorbance of theformazan product was measured at 490 nm using a 120583QuantBiomolecular Spectrophotometer (Bio-Tek Instruments IncWinooski VT USA) The cell viability () was expressedas follows (optical density of sample)(optical density ofDMSO) times 100

29 Quantitative Real-Time Polymerase Chain Reaction(qPCR) The RAW cells were seeded in 6-well tissue cultureplates at the density of 10000 cellsmL After 24 hoursthe cells were treated with LPS (1 120583gmL Sigma St LouisMO USA) alone or cotreated with the leaf extract (7813ndash3125 120583gmL) dissolved in DMSOThen RAW cells were har-vested 24 hours after each treatment Total RNA was isolated


and purified using RNeasy Mini Kit (Qiagen Valencia CAUSA) according to the manufacturerrsquos manual The cDNAwas synthesized from 1120583g of total RNA by using the TaqManReverse Transcription Reagents (Applied Biosystems FosterCity CA USA) The gene expression of IL-6 iNOS andTNF-120572 was quantitated by qPCR performed on an AppliedBiosystems 7900HT Fast Real-Time PCR System (AppliedBiosystems Foster City CA USA) with SYBR Green PCRMasterMixThe primer sequences were described previously[29]

210 Luciferase Assay of NF-120581B-Dependent Reporter GeneActivity The PC-3N cells were seeded in 12-well tissueculture plates at an initial density of 10000 cellsmL After24 hours the cells were treated with the leaf extract (7813ndash3125 120583gmL) dissolved in DMSO Then RAW cells wereharvested 24 hours after each treatment The luciferaseactivity was measured using a luciferase reporter assaysystem (Promega Madison WI USA) according to themanufacturerrsquos manual Briefly the treated PC-3N cells werewashed twice with ice-cold phosphate-buffered saline (1XPBS pH 74) and harvested in 1X reporter lysis bufferAfter centrifugation at 12000 rpm for 5min at 4∘C a 10 120583Laliquot of the supernatants wasmixedwith 50120583L of luciferaseassay substrate and the mixture was assayed for luciferaseactivity using a SIRIUS luminometer (Berthold DetectionSystemGmbH Pforzheim Germany) After normalized withprotein concentration detected by BCA protein assay (PierceRockford IL USA) NF-120581B luciferase activity was expressedas percent of the control

3 Results and Discussion

31 Identification of Phytochemical Constituents Identifica-tion of each compound was mainly based on the massspectrometric data UV-Vis spectrum and comparison withauthentic standards Each compound was analyzed on massspectrometry under both negative and positive modes TheUV and MS chromatograms are illustrated in Figure 1 Theidentity retention time molecular ions fragment ions andmaximum UV absorption wavelength for each identity aresummarized in Table 1

The major compounds (3ndash10) detected in X caffra leafare flavonol glycosides Compounds 3ndash10 were identified asglycosylated derivatives of quercetin and kaempferol basedon their characteristic aglycone fragment ions (mz = 303mz = 287 under positive ionization) The main glycosidesattached are galactosideglucoside xyloside and rutinosideThe UV-Vis absorption of these identified compounds has amaximum absorption at sim356 nm which is consistent withprevious reports [30] The structure of each derivative wasidentified based on the molecular and fragment ions ForexampleMS spectrumof compound6 (119905

119877243min) indicates

that it has molecular ions at mz 611 ([M + H]+) and mz609 ([M minus H]minus) and it is fragmented to mz 465 [M minusrhamnosyl +H]+ andmz 303 ([M minus rhamnosyl minus glucosyl +H]+) which corresponds to quercetin-rutinoside and theidentity was further confirmed with commercially available

1

2

00 50 100 150 200 300250Time (min)

(a)

3 4 5

6

87

910

00 50 100 150 200 300250Time (min)

(b)

3 4 5

6

87

9102

00 50 100 150 200 300250Time (min)

(c)

Figure 1 Chromatograms of Ximenia caffra leaf extract (a) UVchromatogram at 280 nm (b) UV chromatogram at 370 nm (c)processed MS chromatogram

authentic standard The same identification was appliedto the derivatives of galloylglucosidegalloylgalactoside Forexample compound 34 (119905

119877221min or 119905

119877231min) has

a molecular ion at mz 617 ([M + H]+) and it is furtherfragmented tomz 465 ([Mminusgalloyl+H]+) andmz 303 ([Mminusgalloylglucoside+H]+ or [Mminus galloylgalactoside+H]+)TheMS spectra of the flavonol glycosides under positive ionmodeare illustrated in Figure 2 and the structures of representativecompounds are shown in Figure 3

Some components give the same MS UV-Vis spectrumsuch as compound 34 and 56 Compounds 3 and 4 mayhave different glycosides (galactoside or glucoside) attachedor same glycosides but at different positions Compound 6can be differentiated from 5 based on a comparison withcommercial available standard and can be determined asquercetin-3-O-rutinoside

Other polyphenol compounds including gallic acid andcatechin were also determined Their detection under nega-tive mode yields better sensitivity Gallic acid has a molecularion at mz 169 ([M minus H]minus) and a fragment ion at mz 125([M minus CO

2minus H]minus) Catechin has a molecular ion at mz 289

([M minus H]minus) Both have characteristic maximum absorptionat sim280 nm To further confirm the identities of gallic acidand catechin each sample was coinjected with respectiveauthentic standard

32 Quantification of Each Phytochemical The leaves of Xcaffra were found to contain copious amounts of differentflavonol glycosides and other polyphenol compounds Thecontents of flavonol glycoside products were quantified atUV wavelength of 370 nm To approximate the content of


Table 1 Phytochemicals identified from the leaf extract of Ximenia caffra

Peak Retention time (min) UV-Vis120582max (nm)

Molecular and fragment ions Compound name Content (mgg)Negative ions Positive ions

1 24 278 125 169 mdash Gallic acidlowast 0962 100 280 289 291 Catechinlowast 1773 221 356 615 303 465 617 Quercetin-G-Gall 1704 231 358 615 303 465 617 Quercetin-G-Gall 1595 236 357 609 303 465 611 Quercetin-G-Rha 1126 243 357 463 609 303 465 611 Quercetin-Glc-Rha (Rutin)lowast 9087 251 356 463 303 465 Quercetin-G 2038 272 356 433 303 435 Quercetin-Xyl 0119 279 356 593 287 449 595 Kaempferol-G-Rha 08210 289 356 447 287 449 Kaempferol-G 026G glucosylgalactosyl Glc glucosyl Gla galactosyl Rha rhamnosyl Gall galloyl Xyl xylosyl lowast indicates that compounds were unambiguously confirmedthrough the comparison with authentic standards

3

5

6

7

8

9

10

4

303

303

303

303

303

303

287

287

465

465

465

465

465

435

449

449

617

617

611

611

595

200 300 400 500 600 700mz

Peak

Peak

Peak

Peak

Peak

Peak

Peak

Peak

Figure 2 The mass spectra of flavonol glycosides (peak 3 to peak10) under positive ion mode

compounds without commercially available standards theresponse factor was used and adjusted by the ratio of themolecular weight to that of the aglycone [31] Gallic acidand catechin were quantified at 280 nm The content ofeach individual polyphenol is included in Table 1 The totalamount of polyphenols measured by HPLC is 1946mgg81 of which is contributed by quercetin and its derivatives(1564mgg) while kaempferol derivatives represent only 5of the total amount The most abundant single compound isrutin (908mgg) accounting for 47 of the total composi-tion Gallic acid a phenolic acid is 095mgg and represents5 of the total amountThe only flavanol compound catechin(177mgg) contributes 9 of the total composition

33 Total Phenolics and In Vitro Antioxidant Capacities Thecontent of phenolics in the sample is expressed as mg of gallicacid equivalents (GAE) per gram of ground powderThe totalcontent of phenolics determined by Folin-Ciocalteu assay inthe leaf is 26187 plusmn 711mgGAEg The antioxidant capacityof the samples is indicated using mmol of Trolox equivalentsper gram of ground plant material The total antioxidantcapacity of the leaf extract determined byABTS radical cationis 146plusmn001mmol Troloxg It has been widely accepted thatpolyphenol compounds can reduce radicals in vitro and invivo so it is highly possible that this activity is due to itscontent of flavonoids described above

34 Antiproliferation Analysis TheMTS assay was applied toinvestigate the effect of X caffra leaf extract on the growthof cultured RAW 2647 cells As illustrated in Figure 4 after24 hours of treatment with leaf extract the cell viabilityof RAW cells decreased the dose dependently When theconcentration of raw leaf extract is 3125 120583gmL the viabilityof the RAW cell is 4683 plusmn 346 The IC

50value of the raw

leaf extract was determined to be 2390 plusmn 445 120583gmL Thisdata implies that the leaf extract inhibits the growth of RAWcell potentially possessing a cytotoxic effect

35 Anti-Inflammatory Analysis To evaluate the effect of Xcaffra leaf extract on the LPS-stimulated expression of inflam-matory enzymes and proinflammatory cytokines the mRNAexpressions of inflammatorymarkers IL-6 iNOS and TNF-120572were measured by qPCR Treatment of cells with leaf extractat a variety of concentrations demonstrated a dose-dependentresponse on the expression of IL-6 iNOS and TNF-120572 asshown in Figure 5 The strongest response was seen for IL-6 (Figure 5(a)) wherein treatment at 3125 120583gmL induceda nearly 10-fold decrease in expression versus backgroundexpression and nearly 100-fold decrease in expression versusLPS induced cells which were not treated by X caffra leafextract This demonstrates clearly the impact of X caffra


O

O

O

O

OH

OH

OH OH

OH

OH

OH

OH

OH

HO

HO HO HOR

1 2 3

Figure 3 Representative structures of identified polyphenols (1) Gallic acid (2) Catechin (3) The aglycone of compound 3ndash10 quercetin(R=OH) and kaempferol (R=H) Sugar moieties are usually attached at position 3

Cell

viab

ility

()

Concentration (120583gmL)0 100 200 300

100

80

60

40

20

0

lowast

lowastlowast

Figure 4 Cell viability versus the concentrations of leaf extractmeasured by MTS assay

leaf extract on the expression of IL-6 an indication ofpotentially significant anti-inflammatory activity The effectof X caffra extract on TNF-120572 and iNOS was measurablebut less significant than the results for IL-6 The expressionof iNOS in cells treated with 3125120583gmL leaf extract wascomparable to background expression a twofold decrease inexpression compared to LPS induced cells (Figure 5(b)) Atwofold decrease in the expression of TNF-120572wasmeasured incells treated with 3125120583gmLwhen compared to backgroundexpression which is a threefold decrease in the expressioncompared to LPS induced cells that were not treated withX caffra extract (Figure 5(c)) These results corroborate wellwith IL-6 results further supporting the anti-inflammatoryactivity of X caffra leaf extracts in a cell system

36 NF-120581B Transcription Activity As described above theleaf extract of X caffra has shown both antiproliferation andanti-inflammatory activities in RAW cells NF-120581B proteincomplex is involved in a number of cellular responses tostimuli such as free radical stress and cytokine [32 33] Toinvestigate the effects of X caffra leaf extract on the NF-120581B transcription activity the luciferase activity of PC-3N

cells with a stably transfected NF-120581B luciferase constructswas measured In this assay protein NF-120581B was coexpressedwith protein luciferase and directly quantified by lumi-nometer The data demonstrated that X caffra leaf extractsignificantly attenuated the NF-120581B transcription activity in adose-dependent manner after 24 h treatment (Figure 6) Atthe concentration of 3125 120583gmL the expression of NF-120581Bwas decreased to approximately 60 of the vehicle controlThe data obtained is consistent with the results from MTSand qPCR assays indicating that the inhibition of NF-120581Bactivation might be one of the mechanisms underlying theantiproliferation and anti-inflammatory effects of the leafextract of X caffra

4 Conclusion

This is the first study examining the leaves rather than thefruit or seeds of X caffra which can be sustainably harvestedbut underutilized by local indigenous peoples The chemicalprofile of X caffra leaf was comprehensively analyzed and ledto the identification of 10 polyphenol compounds includingphenolic acid and flavonoids The individual polyphenolswere successfully quantitated using UV detection Furtherbioactivity investigations showed that the extracts of Xcaffra leaf exhibit antioxidant antiproliferation and anti-inflammatory activities The underlying molecular mecha-nism may partially be contributed by the inhibition of NF-120581B activation a shared signal pathway between proliferationand inflammation Further investigations are needed toexplore whether these polyphenol compounds could worksynergistically to achieve much improved activities thaneach single component The chemical profile and bioactiv-ities determined support its traditional use and may helpfor its further pharmacological studies and nutraceuticalapplications Considering that some botanical supplementson market have similar polyphenol profile the compoundsfound inX caffra leaf would also be of interest as a new sourceof these natural products The development of a sustainableproduction and collection system for X caffra leaves couldcompliment interest in this plantrsquos fruit and seeds


IL-6

mRN

A e

xpre

ssio

n (fo

ld)

00 7813 15625 3124

201510

5

10

08

06

04

02

00

lowastlowast

lowastlowast

lowast

LPS (1120583gmL)

Concentration (1120583gmL)

(a)

iNO

Sm

RNA

exp

ress

ion

(fold

)

00 7813 15625 3124

20

15

10

05

00

25 LPS (1120583gmL)


(b)

00 7813 15625 3124

20

15

10

05

00

TNF-120572

mRN

A ex

pres

sion

(fold

) LPS (1120583gmL)


(c)

Figure 5 Anti-inflammatory activity of Ximenia caffra leaf extract measured by RT-qPCR in RAW 2647 macrophage cells (a) IL-6 mRNAexpressions after treatment (b) iNOS mRNA expression after treatment (c) TNF-120572mRNA expression after treatment

0 7813 15625 3124

120

100

80

60

40

20

0

lowastlowast


Luci

fera

se ac

tivity

( o

f con

trol)

Figure 6 NF-120581B transcription activity versus the concentrations of Ximenia caffra leaf extract after treatment


Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was commissioned by the Millennium ChallengeAccount Namibia with funding from the Millennium Chal-lenge Corporation in support of the Commissioned ProjectInventory and Rapid Throughput Screening of Indigenous Uti-lized Plant Species for Development of New Natural Products(MCANLMEF201001) The authors thank Dave Cole andLoide Uahengo of MCA-Namibia for their support and guid-ance the National Botanical Research Institute for providingthe botanical authentication services and their collaborativepartnership Danielle Mortensen for her assistance in fieldcollection the Namibian Indigenous Plant Task Team forproviding oversight to our work the Namibian Ministry ofEnvironment and Tourism for their assistance with permitsand other compliance requirements and both the Universityof Namibia and the Rutgers New Jersey Agricultural Stationwho also provided financial and logistical support

References

[1] B-E van Wyk and N Gericke Peoplersquos Plants Briza Publica-tions 2000

[2] A Cheikhyoussef M Shapi K Matengu and H M AshekeleldquoEthnobotanical study of indigenous knowledge on medicinalplant use by traditional healers in Oshikoto region NamibiardquoJournal of Ethnobiology and Ethnomedicine vol 7 article 102011

[3] B-E Van Wyk ldquoThe potential of South African plants in thedevelopment of new food and beverage productsrdquo SouthAfricanJournal of Botany vol 77 no 4 pp 857ndash868 2011

[4] A R Ndhlala M Muchuweti C Mupure et al ldquoPhenoliccontent and profiles of selected wild fruits of Zimbabwe Xime-nia caffra Artobotrys brachypetalus and Syzygium cordatumrdquoInternational Journal of Food Science and Technology vol 43 no8 pp 1333ndash1337 2008

[5] L W Khumalo L Majoko J S Read and I Ncube ldquoCharacter-isation of some underutilised vegetable oils and their evaluationas starting materials for lipase-catalysed production of cocoabutter equivalentsrdquo Industrial Crops and Products vol 16 no3 pp 237ndash244 2002

[6] E Chivandi B C Davidson and K H Erlwanger ldquoRedsour plum (Ximenia caffra) seed a potential non-conventionalenergy and protein source for livestock feedsrdquo InternationalJournal of Agriculture and Biology vol 14 no 4 pp 540ndash5442012

[7] J J Nair R B Mulaudzi J C Chukwujekwu F R van Heerdenand J van Staden ldquoAntigonococcal activity of Ximenia caffraSond (Olacaceae) and identification of the active principlerdquoSouth African Journal of Botany vol 86 pp 111ndash115 2013

[8] R B Mulaudzi A R Ndhlala M G Kulkarni J F Finnie andJ van Staden ldquoAntimicrobial properties and phenolic contentsof medicinal plants used by the Venda people for conditionsrelated to venereal diseasesrdquo Journal of Ethnopharmacology vol135 no 2 pp 330ndash337 2011

[9] H deWet V N Nzama and S F vanVuuren ldquoMedicinal plantsused for the treatment of sexually transmitted infections bylay people in northern Maputaland KwaZulu-Natal ProvinceSouth Africardquo South African Journal of Botany vol 78 pp 12ndash20 2012

[10] W Fabry P O Okemo and R Ansorg ldquoAntibacterial activity ofEast African medicinal plantsrdquo Journal of Ethnopharmacologyvol 60 no 1 pp 79ndash84 1998

[11] TMunodawafa L S Chagonda and S RMoyo ldquoAntimicrobialand phytochemical screening of some Zimbabwean medicinalplantsrdquo Journal of Biologically Active Products from Nature vol3 no 5-6 pp 323ndash330 2013

[12] N H T Le K E Malterud D Diallo B S Paulsen CS Nergard and H Wangensteen ldquoBioactive polyphenols inXimenia americana and the traditional use among Malianhealersrdquo Journal of Ethnopharmacology vol 139 no 3 pp 858ndash862 2012

[13] I Erlund ldquoReview of the flavonoids quercetin hesperetin andnaringenin Dietary sources bioactivities bioavailability andepidemiologyrdquo Nutrition Research vol 24 no 10 pp 851ndash8742004

[14] D Vauzour A Rodriguez-Mateos G Corona M J Oruna-Concha and J P E Spencer ldquoPolyphenols and human healthprevention of disease andmechanisms of actionrdquoNutrients vol2 no 11 pp 1106ndash1131 2010

[15] E-Q Xia G-F Deng Y-J Guo and H-B Li ldquoBiologicalactivities of polyphenols from grapesrdquo International Journal ofMolecular Sciences vol 11 no 2 pp 622ndash646 2010

[16] M Hamalainen R Nieminen P Vuorela M Heinonen and EMoilanen ldquoAnti-inflammatory effects of flavonoids Genisteinkaempferol quercetin and daidzein inhibit STAT-1 and NF-kappaB activations whereas flavone isorhamnetin naringeninand pelargonidin inhibit only NF-kappaB activation along withtheir inhibitory effect on iNOS expression and NO productionin activatedmacrophagesrdquoMediators of Inflammation vol 2007Article ID 45673 10 pages 2007

[17] A Scalbert I T Johnson and M Saltmarsh ldquoPolyphenolsantioxidants and beyondrdquo The American Journal of ClinicalNutrition vol 81 no 1 pp 215Sndash217S 2005

[18] C Gabay ldquoInterleukin-6 and chronic inflammationrdquo ArthritisResearch ampTherapy vol 8 supplement 2 p S3 2006

[19] J R Bradley ldquoTNF-mediated inflammatory diseaserdquo The Jour-nal of Pathology vol 214 no 2 pp 149ndash160 2008

[20] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[21] J Dabek A Kułach and Z Gasior ldquoNuclear factor kappa-light-chain-enhancer of activated B cells (NF-120581B) a new potentialtherapeutic target in atherosclerosisrdquo Pharmacological Reportsvol 62 no 5 pp 778ndash783 2010

[22] P P Tak and G S Firestein ldquoNF-120581B a key role in inflammatorydiseasesrdquoThe Journal of Clinical Investigation vol 107 no 1 pp7ndash11 2001

[23] J Dutta Y Fan N Gupta G Fan and C Gelinas ldquoCurrentinsights into the regulation of programmed cell death by NF-120581Brdquo Oncogene vol 25 no 51 pp 6800ndash6816 2006

[24] L Wang C Zhang Y Guo et al ldquoBlocking of JB6 celltransformation by tanshinone IIA epigenetic reactivation ofNrf2 antioxidative stress pathwayrdquo The AAPS Journal vol 16no 6 pp 1214ndash1225 2014

[25] QWuMWang and J E Simon ldquoDetermination of isoflavonesin red clover and related species by high-performance liquid


chromatography combined with ultraviolet and mass spectro-metric detectionrdquo Journal of Chromatography A vol 1016 no 2pp 195ndash209 2003

[26] D-Y Zhou N Ding J van Doren et al ldquoEffects of curcuminanalogues for inhibiting human prostate cancer cells and thegrowth of human PC-3 prostate xenografts in immunodeficientmicerdquo Biological amp Pharmaceutical Bulletin vol 37 no 6 pp1029ndash1034 2014

[27] V L Singleton ldquoCitation classicmdashcolorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquo Cur-rent ContentsAgriculture Biology amp Environmental Sciencesvol 48 p 18 1985

[28] C L Saw Y Guo A Y Yang et al ldquoThe berry constituentsquercetin kaempferol and pterostilbene synergistically atten-uate reactive oxygen species Involvement of the Nrf2-AREsignaling pathwayrdquo Food and Chemical Toxicology vol 72 pp303ndash311 2014

[29] J H Lee K-R Lee Z-Y Su et al ldquoIn vitro and in vivoanti-inflammatory effects of a novel 46-bis ((119864)-4-hydroxy-3-methoxystyryl)-1-phenethylpyrimidine-2(1119867)-thionerdquo Chemi-cal Research in Toxicology vol 27 no 1 pp 34ndash41 2014

[30] B Ducrey J L Wolfender A Marston and K HostettmannldquoAnalysis of flavonol glycosides of thirteen Epilobium species(Onagraceae) by LC-UV and thermospray LC-MSrdquo Phytochem-istry vol 38 no 1 pp 129ndash137 1995

[31] M-J Dubber and I Kanfer ldquoHigh-performance liquid chro-matographic determination of selected flavonols in Ginkgobiloba solid oral dosage formsrdquo Journal of Pharmacy amp Phar-maceutical Sciences vol 7 no 3 pp 303ndash309 2004

[32] C L L Saw A Y Yang Y Guo and A N T Kong ldquoAstaxanthinand omega-3 fatty acids individually and in combination protectagainst oxidative stress via the Nrf2-ARE pathwayrdquo Food andChemical Toxicology vol 62 pp 869ndash875 2013

[33] C L-L SawM Cintron T-YWu et al ldquoPharmacodynamics ofdietary phytochemical indoles I3C andDIM induction ofNrf2-mediated phase II drug metabolizing and antioxidant genesand synergism with isothiocyanatesrdquo Biopharmaceutics amp DrugDisposition vol 32 no 5 pp 289ndash300 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


assay [8] Bioactive phytochemicals contained in the leaf ofrelated species X americana from western and eastern sub-Sahara Africa were investigated and led to the identificationof sambunigrin gallic acid and quercetin and the glycosidesby spectrometric methods [12]

Most of the studies with X caffra focus on the fruit andseed as these represent the products of current commerceand traditional use rather than the leaf In this studywe purposefully investigate the phytochemical profile ofX caffra leaf which has not been previously examinedusing LCUVMS Interestingly though not unexpectedly anumber of polyphenol compounds were discovered in theleaf Polyphenols are a class of compounds characterized bythe presence of large multiples of phenol structural unitsand abundantly exist in food products including crops fruitvegetables tea and wine [13ndash15] These compounds haveshown a wide range of biological activities including anti-inflammation and the prevention of cancer and cardiovascu-lar diseases [16 17]

Based upon the chemical profile determined and to fur-ther explore new applications for the leaves while extendingthe traditional uses we investigated the leaves for theirantioxidant activity total phenolic content and antipro-liferation activity using the Folin-Ciocalteu assay Troloxequivalent antioxidant capacity radical scavenging assay andthe MTS cell proliferation assay respectively Additionallythe effects of extracts on the mRNA expression of proin-flammatory genes were assessed by RT-qPCR through thequantification of inducible nitric oxide synthetase (iNOS)interleukin 6 (IL-6) and tumor necrosis factor 120572 (TNF-120572) induced by lipopolysaccharide (LPS) in murine RAW2647 macrophage cells Interleukin 6 (IL-6) a cytokinesecreted by T cells and macrophages is produced at theinflammation sites during infection and tissue damage IL-6 has stimulatory effects on immune cells and can inducechronic inflammatory responses Thus targeting IL-6 wouldhelp prevent or treat rheumatoid arthritis and other chronicinflammatory diseases [18] iNOS is one of the key enzymesgenerating nitric oxide (NO) which regulates a number ofpathophysiological conditions including infection inflam-mation and neoplastic diseases Reagents that can inhibitiNOS would reduce the generation of NO and have potentialanti-inflammatory effects [16] Anther biomarker TNF-120572 anadipokine is involved in series of inflammation reactionsduring immune response Its antagonists have also showneffects in the treatment of a range of inflammatory diseasesincluding rheumatoid arthritis psoriasis and ankylosingspondylitis [19]

To further explore the mechanism of antiproliferationand anti-inflammatory activities extracts of X caffra wereevaluated in cells for suppression of NF-120581B (nuclear factorkappa-light-chain-enhancer of activated B cells) a proteincomplex that regulates the transcription of DNA and isinvolved in cellular responses to stress The activation ofNF-120581B is generally considered to upregulate a range ofgenes encoding growth factors cytokines apoptosis andimmunomodulatory molecules As a shared key signal path-way of proliferation and inflammation in cells the prolongedactivation of NF-120581B has been reported to be involved in

inflammatory conditions autoimmune disease and cancer[20ndash24] The results demonstrate that the underlying ther-apeutic mechanism involves the suppression of NF-120581B ashared pathway between cell death and inflammation

2 Experimental

21 Materials Standard compounds gallic acid rutin cat-echin quercetin kaempferol and ABTS (221015840-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) reagents were pur-chased from Sigma Aldrich (St Louis MO) HPLC-grademethanol (MeOH) acetonitrile (ACN) and formic acidwere purchased from Fisher Scientific Co (Fair Lawn NJ)Folin-Ciocalteursquos phenol reagent was purchased from MPBiomedicals (Solon OH) The plant samples of X caffraleaves were field-collected from Northeastern Namibia usinga research permit from the Ministry of Environment andTourism Namibia Voucher specimens were prepared andsent to the National herbarium at the National BotanicalResearch Institute (NBRI) where the collected plants wereauthenticated The voucher specimens were also depositedin both the National Botanical Research Institute Wind-hoek Namibia and the Chrysler Herbarium amp MycologicalCollection Biological Sciences Building Rutgers UniversityNew Brunswick NJ The leaves were manually harvestedusing NBRI guidelines and were air-dried under ambienttemperature for two weeks and then packaged for laterexperimental analysis The dried leaf materials were air-shipped to Rutgers University where each was ground intoa fine powder with a laboratory mill (Perten 3100) and storedat room temperature in the dark

22 Analytical Equipment Separation on HPLC was per-formed using column Prodigy ODS3 5120583m 150 times 32mm5 micro (Phenomenex Inc Torrance CA) The separa-tion identification and quantification were performed onHewlett-Packard Agilent 1100 series HPLC-MS (AgilentTechnologies Waldbronn Germany) equipped with a qua-ternary pump system a degasser an autosampler a DADdetector an MSD trap with an electrospray ion source (ESI)and software HP ChemStation Data Analysis 42

23 LCMS Conditions for Qualification and Quantificationof the Polyphenols HPLC separation was performed withmobile phase of solvents A and B in gradient where A was01 formic acid (vv) in water and B was 01 formic acid(vv) in acetonitrileThe gradient starts from5B to 30B in50minThe detection wavelength was set at 280 nm for gallicacid and catechin and 370 nm for quercetin and kaempferolderivativesThe flow rate is 10mLminThe electrospray wasconducted in negative and positive modes in two separatedrunsThe optimized collision energy is of 80 scanning frommz 100 to 700 ESI was conducted at voltage of 35 kV forpositive model and minus35 kV for negative model High-purityliquid nitrogen 99999 was used as dry gas and nebulizerat a flow rate of 12 Lmin and the capillary temperature was350∘C Nitrogen was used as nebulizer at 60 psi and heliumas collision gas The ESI interface and mass spectrometerparameters were optimized to obtain maximum sensitivity







2at 37∘C




2S2O8)



= [1 minus

Absorbancesample


(1)












1

2

00 50 100 150 200 300250Time (min)

(a)

3 4 5

6

87

910

00 50 100 150 200 300250Time (min)

(b)

3 4 5

6

87

9102

00 50 100 150 200 300250Time (min)

(c)



119877221min or 119905

119877231min) has












3

5

6

7

8

9

10

4

303

303

303

303

303

303

287

287

465

465

465

465

465

435

449

449

617

617

611

611

595

200 300 400 500 600 700mz

Peak

Peak

Peak

Peak

Peak

Peak

Peak

Peak





50value of the raw




O

O

O

O

OH

OH

OH OH

OH

OH

OH

OH

OH

HO

HO HO HOR

1 2 3


Cell

viab

ility

()


100

80

60

40

20

0

lowast

lowastlowast





4 Conclusion



IL-6

mRN

A e

xpre

ssio

n (fo

ld)

00 7813 15625 3124

201510

5

10

08

06

04

02

00

lowastlowast

lowastlowast

lowast

LPS (1120583gmL)


(a)

iNO

Sm

RNA

exp

ress

ion

(fold

)

00 7813 15625 3124

20

15

10

05

00

25 LPS (1120583gmL)


(b)

00 7813 15625 3124

20

15

10

05

00

TNF-120572

mRN

A ex

pres

sion

(fold

) LPS (1120583gmL)


(c)


0 7813 15625 3124

120

100

80

60

40

20

0

lowastlowast


Luci

fera

se ac

tivity

( o

f con

trol)





Acknowledgments


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of







2at 37∘C




2S2O8)



= [1 minus

Absorbancesample


(1)












1

2

00 50 100 150 200 300250Time (min)

(a)

3 4 5

6

87

910

00 50 100 150 200 300250Time (min)

(b)

3 4 5

6

87

9102

00 50 100 150 200 300250Time (min)

(c)



119877221min or 119905

119877231min) has












3

5

6

7

8

9

10

4

303

303

303

303

303

303

287

287

465

465

465

465

465

435

449

449

617

617

611

611

595

200 300 400 500 600 700mz

Peak

Peak

Peak

Peak

Peak

Peak

Peak

Peak





50value of the raw




O

O

O

O

OH

OH

OH OH

OH

OH

OH

OH

OH

HO

HO HO HOR

1 2 3


Cell

viab

ility

()


100

80

60

40

20

0

lowast

lowastlowast





4 Conclusion



IL-6

mRN

A e

xpre

ssio

n (fo

ld)

00 7813 15625 3124

201510

5

10

08

06

04

02

00

lowastlowast

lowastlowast

lowast

LPS (1120583gmL)


(a)

iNO

Sm

RNA

exp

ress

ion

(fold

)

00 7813 15625 3124

20

15

10

05

00

25 LPS (1120583gmL)


(b)

00 7813 15625 3124

20

15

10

05

00

TNF-120572

mRN

A ex

pres

sion

(fold

) LPS (1120583gmL)


(c)


0 7813 15625 3124

120

100

80

60

40

20

0

lowastlowast


Luci

fera

se ac

tivity

( o

f con

trol)





Acknowledgments


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of









1

2

00 50 100 150 200 300250Time (min)

(a)

3 4 5

6

87

910

00 50 100 150 200 300250Time (min)

(b)

3 4 5

6

87

9102

00 50 100 150 200 300250Time (min)

(c)



119877221min or 119905

119877231min) has












3

5

6

7

8

9

10

4

303

303

303

303

303

303

287

287

465

465

465

465

465

435

449

449

617

617

611

611

595

200 300 400 500 600 700mz

Peak

Peak

Peak

Peak

Peak

Peak

Peak

Peak





50value of the raw




O

O

O

O

OH

OH

OH OH

OH

OH

OH

OH

OH

HO

HO HO HOR

1 2 3


Cell

viab

ility

()


100

80

60

40

20

0

lowast

lowastlowast





4 Conclusion



IL-6

mRN

A e

xpre

ssio

n (fo

ld)

00 7813 15625 3124

201510

5

10

08

06

04

02

00

lowastlowast

lowastlowast

lowast

LPS (1120583gmL)


(a)

iNO

Sm

RNA

exp

ress

ion

(fold

)

00 7813 15625 3124

20

15

10

05

00

25 LPS (1120583gmL)


(b)

00 7813 15625 3124

20

15

10

05

00

TNF-120572

mRN

A ex

pres

sion

(fold

) LPS (1120583gmL)


(c)


0 7813 15625 3124

120

100

80

60

40

20

0

lowastlowast


Luci

fera

se ac

tivity

( o

f con

trol)





Acknowledgments


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






3

5

6

7

8

9

10

4

303

303

303

303

303

303

287

287

465

465

465

465

465

435

449

449

617

617

611

611

595

200 300 400 500 600 700mz

Peak

Peak

Peak

Peak

Peak

Peak

Peak

Peak





50value of the raw




O

O

O

O

OH

OH

OH OH

OH

OH

OH

OH

OH

HO

HO HO HOR

1 2 3


Cell

viab

ility

()


100

80

60

40

20

0

lowast

lowastlowast





4 Conclusion



IL-6

mRN

A e

xpre

ssio

n (fo

ld)

00 7813 15625 3124

201510

5

10

08

06

04

02

00

lowastlowast

lowastlowast

lowast

LPS (1120583gmL)


(a)

iNO

Sm

RNA

exp

ress

ion

(fold

)

00 7813 15625 3124

20

15

10

05

00

25 LPS (1120583gmL)


(b)

00 7813 15625 3124

20

15

10

05

00

TNF-120572

mRN

A ex

pres

sion

(fold

) LPS (1120583gmL)


(c)


0 7813 15625 3124

120

100

80

60

40

20

0

lowastlowast


Luci

fera

se ac

tivity

( o

f con

trol)





Acknowledgments


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


O

O

O

O

OH

OH

OH OH

OH

OH

OH

OH

OH

HO

HO HO HOR

1 2 3


Cell

viab

ility

()


100

80

60

40

20

0

lowast

lowastlowast





4 Conclusion



IL-6

mRN

A e

xpre

ssio

n (fo

ld)

00 7813 15625 3124

201510

5

10

08

06

04

02

00

lowastlowast

lowastlowast

lowast

LPS (1120583gmL)


(a)

iNO

Sm

RNA

exp

ress

ion

(fold

)

00 7813 15625 3124

20

15

10

05

00

25 LPS (1120583gmL)


(b)

00 7813 15625 3124

20

15

10

05

00

TNF-120572

mRN

A ex

pres

sion

(fold

) LPS (1120583gmL)


(c)


0 7813 15625 3124

120

100

80

60

40

20

0

lowastlowast


Luci

fera

se ac

tivity

( o

f con

trol)





Acknowledgments


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


IL-6

mRN

A e

xpre

ssio

n (fo

ld)

00 7813 15625 3124

201510

5

10

08

06

04

02

00

lowastlowast

lowastlowast

lowast

LPS (1120583gmL)


(a)

iNO

Sm

RNA

exp

ress

ion

(fold

)

00 7813 15625 3124

20

15

10

05

00

25 LPS (1120583gmL)


(b)

00 7813 15625 3124

20

15

10

05

00

TNF-120572

mRN

A ex

pres

sion

(fold

) LPS (1120583gmL)


(c)


0 7813 15625 3124

120

100

80

60

40

20

0

lowastlowast


Luci

fera

se ac

tivity

( o

f con

trol)





Acknowledgments


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




Acknowledgments


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article phytochemical analysis and anti ...research article phytochemical analysis and...

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