Research Article Topical Anti-Inflammatory Effects of ...downloads.hindawi.com/journals/bmri/2015/847320.pdf · Isorhamnetin Glycosides in OFI Extract. Identi cation and quanti cation

Research ArticleTopical Anti-Inflammatory Effects of Isorhamnetin GlycosidesIsolated from Opuntia ficus-indica

Marilena Antunes-Ricardo Janet A Gutieacuterrez-UribeCarlos Martiacutenez-Vitela and Sergio O Serna-Saldiacutevar

Centro de Biotecnologıa-FEMSA Tecnologico de Monterrey Avenida Eugenio Garza Sada 2501 Sur 64849 Monterrey NL Mexico

Correspondence should be addressed to Janet A Gutierrez-Uribe jaguitesmmx

Received 9 October 2014 Revised 15 December 2014 Accepted 16 December 2014

Academic Editor Maxim E Darvin

Copyright copy 2015 Marilena Antunes-Ricardo et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Opuntia ficus-indica (OFI) has been widely used in Mexico as a food and for the treatment of different health disorders such asinflammation and skin aging Its biological properties have been attributed to different phytochemicals such as the isorhamnetinglycosides which are themost abundant flavonoidsMoreover these compounds are considered a chemotaxonomic characteristic ofOFI speciesThe aim of this studywas to evaluate the effect of OFI extract and its isorhamnetin glycosides on different inflammatorymarkers in vitro and in vivo OFI extract was obtained by alkaline hydrolysis of OFI cladodes powder and pure compounds wereobtained by preparative chromatography Nitric oxide (NO) cyclooxygenase-2 (COX-2) tumor necrosis factor- (TNF-) 120572 andinterleukin- (IL-) 6 production were measured NO production was tested in lipopolysaccharide-stimulated RAW 2647 cellswhile in vivo studies were carried on croton oil-induced ear edema model OFI extract and diglycoside isorhamnetin-glucosyl-rhamnoside (IGR) at 125 ngmL suppressed the NO production in vitro (735 plusmn 48 and 687 plusmn 50 resp) without affectingcell viability Likewise IGR inhibited the ear edema (774 plusmn 57) equating the indomethacin effects (695 plusmn 53) Both IGR andOFI extract significantly inhibited the COX-2 TNF-120572 and IL-6 production IGR seems to be a suitable natural compound fordevelopment of new anti-inflammatory ingredient

1 Introduction

The skin is the first line of defense against the externalenvironment and xenobiotic agents It interacts with bothendogenous and environmental oxidant species which areinvolved in the pathogenesis of many inflammatory skin dis-eases [1ndash4] Cutaneous injury causes keratinocyte activationproliferation and releasing of inflammatory mediators suchas nitric oxide (NO) tumor necrosis factor alpha (TNF-120572)and interleukin- (IL-) 1 and IL-6 [5 6]

Different inflammation models have been used to under-stand the anti-inflammatory mechanisms of synthetic drugsor compounds from natural sources [7] Lee et al [8]and Yoon et al [9] observed that the extracts of Chrysan-themum indicum and Scutellaria baicalensis induced anti-inflammatory responses in vivo and in vitro These effectswere evidenced by a reduction on the NO productioncytokines chemokine and growth factors along with

different histopathological parameters Additionally topicalformulationswhich combine naturally occurring compoundsand nutritional supplements have been developed and thesehave shown great potential to inhibit oxidative stress and skininflammation [10]

The ear edema inflammation model has been widelyused to evaluate the anti-inflammatory potential of naturalcompounds The topical application of croton oil as anirritant agent increases vascular permeability and induces thesynthesis of arachidonic acid metabolites and the expressionof COX-2 IL-1120573 TNF-120572 and the adhesionmolecule ICAM-1[11]

Different flavonoids-rich extracts and isolated flavonoidshave been tested in vitro and in vivo to evaluate theiranti-inflammatory effects For example a flavonoid-enrichedfraction obtained from Cayaponia tayuya roots (05mgear)showed an inhibition of 66 in acute TPA-induced edemain mouse ears When the extract was tested at 2230 120583gml

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 847320 9 pageshttpdxdoiorg1011552015847320

2 BioMed Research International

on RAW 2647 macrophages it inhibited the expressionsof iNOS and COX-2 by 97 and 65 respectively [15]Likewise flavonoids contained in a flavonoid-enriched frac-tion (50 120583gmL) extracted from the rhizomes of Sophoraflavescens also showed in vitro inhibitory effects greater than50 on the NO production [16] Isorhamnetin kaempferoland quercetin inhibited iNOS protein andmRNA expressionwhen tested in macrophages stimulated with lipopolysac-charide (LPS) [17] Also these compounds inhibited theactivation of nuclear transcription factor-120581B (NF-120581B)

In nature flavonoids are generally attached to sugarresidues that modify the mechanism of absorption and theirability to enter cells or to interact with transporters andcellular lipoproteins [18] Therefore there are differencesamong the biological effects exerted by flavonoid glycosidesaccording to the type number and position of sugar moieties[19 20] de Melo et al [21] showed that kaempferol 3-O-120573-glucopyranoside-7-O-120572-rhamnopyranoside and kaempferol7-O-120572-rhamnopyranoside inhibited the croton oil-inducedear edema by 465 and 333 respectively Likewisekaempferol 3-O-120573-D-apiofuranosyl-(1rarr 2)-120572-L-arabinofur-anosyl-7-O-120572-L-rhamnopyranoside (10 120583M) showed betterinhibitory effects on TNF-120572 and IL-12 production thankaempferol 3-O-120573-D-apiofuranosy-(1rarr 4)-120572-L-rhamnopy-ranosyl-7-O-120572-L-rhamnopyranoside (40 120583M) [22]

Opuntia ficus-indica (OFI) has been used in Mexico as afood and as a remedy against different health disorders relatedto skin such as inflammation [23 24] This plant is a naturalsource of flavonoids mainly isorhamnetin glycosides [12ndash1425] Isorhamnetin and isorhamnetin-3-O-galactoside possessin vitro anti-inflammatory activity [26 27]

Considering all the above the aim of this research workwas to evaluate the in vitro and in vivo anti-inflammatoryeffects of an OFI extract and compare the bioactivity ofdifferent isorhamnetin glycosides isolated from this sourceOFI extract and isolated isorhamnetin diglycosides andtriglycosides were tested on RAW 2647 macrophage cellsin order to evaluate their effect on NO production Laterthese compoundswere tested in a rat ear edema inflammationmodel to evaluate their effect on COX-2 TNF-120572 and IL-6levels

2 Materials and Methods

21 Reagents Dulbeccorsquos Modified Eagle Medium NutrientMixture F-12 (DMEM-F12) ampicillinstreptomycin andphosphate-buffered saline (pH 74) were purchased fromGibco Invitrogen (Carlsbad CA) Trypsin-EDTA (025)and fetal bovine serumwere obtained fromHyCloneThermoScientific (Logan UT) Triton X-100 was acquired fromResearch Organics (Cleveland OH) Lipopolysaccharides(LPS) from Salmonella enterica serotype typhimurium L7261as well as croton oil indomethacin isorhamnetin stan-dard and formic acid solution HPLC grade were purchasedfrom Sigma-Aldrich (St Louis MO) Chromatography gradewater and methanol (VWR International LLC West ChesterPA) were used for high-pressure liquid chromatographequippedwith a photodiode array detector (HPLC-PDA) and

liquid chromatographmass selective detector time-of-flight(LCMSD TOF) analysis

22 Obtaining O ficus-indica Extract The taxonomic identi-fication of Opuntia ficus-indica was done by PhD RigobertoE Vazquez-Alvarado at the School of Agronomy of Uni-versidad Autonoma de Nuevo Leon (UANL) Mexico OFIcladodes were harvested at 7 months and grown in the regionof Montemorelos Nuevo Leon Mexico and then they wereprocessed into powder according to Santos-Zea et al [14]OFI extract was obtained by alkaline hydrolysis following themethod previously reported by Antunes-Ricardo et al [12]

23 Identification Quantification and Purification ofIsorhamnetin Glycosides in OFI Extract Identification andquantification of isorhamnetin glycosides were performedaccording to the method described by Antunes-Ricardoet al [12] using HPLC-PDA (Agilent 1100 Series SantaClara CA) Purification of isorhamnetin glycosides wasdone by semipreparative chromatography according to themethod described by Antunes-Ricardo et al [12] usinga semipreparative Zorbax SB-C18 (94 times 250mm 5120583m)column at 17∘C flow rate of 20mLmin and injectionvolume of 50 120583L Water with 01 formic acid (A) andmethanol 80 (B) were used as mobile phases startingwith 35 of B and increasing to 60 and 90 after 2 and19min respectively and then decreasing to 0 B for the next3min Percentages of purity of 90 or more were obtainedfor the isorhamnetin glycosides LCMSD TOF technology(Model G1969A Agilent 1100 Santa Clara CA) was usedto identify the isorhamnetin glycosides according to themethod described by Santos-Zea et al [14]

24 RAW 2647 Cell Culture RAW 2647 a murinemacrophage cell line was obtained from the AmericanType Culture Collection (ATCC Manassas VA) Cells werecultured in petri plates with Dulbeccorsquos Modified EagleMedium (DMEM) supplemented with 10 fetal bovineserum (FBS) and 1 antibiotic and incubated at 37∘C and 5of CO

2(NuAire Plymouth MN) To evaluate the effects of

an OFI extract and isolated isorhamnetin glycosides on nitricoxide production cells were plated in 96-well plate (5 times 104cellswell) and allowed to adhere for 4 h After that differentconcentrations (25 50 or 125 ng isorhamnetin equivalents(IsoEqmL) of the compounds were added and incubated foranother 24 h Following half of wells were stimulated withLPS at 10 120583gmL (final concentration) while the other halfwere used as control for each sample

25 Measurement of Nitrite Concentration Production ofnitric oxide (NO) was measured by nitrites determinationwhich is a stable and nonvolatile breakdown product of NOAfter incubation with the different treatments 100120583L ofsupernatant was transferred to another 96-well plate to mea-sure nitrites concentration using the Griess Reagent System(Promega Madison WI) at 550 nm Data was expressed aspercentage of inhibition of NO production The remaining

BioMed Research International 3

cells and medium in original plate were used to measure cellviability

26 Measurement of RAW 2647 Cell Viability Cell viabilitywas determined by CellTiter 96 AQueous One Solution CellProliferation Assay (Promega Madison WI) Absorbancewas measured at 490 nm with a 96-well microplate reader(Synergy HT Bio-Tek Winooski VM) Cell viability wascalculated by dividing the absorbance of cells treated by theabsorbance of control cells and this ratio was expressed aspercentage

27 Animals Male Wistar rats (250ndash300 g) were purchasedfrom Bioinvert (Mexico DF Mexico) Animals were dividedinto seven groups (119899 = 8) and kept in a room with controlledtemperature (25∘C) and relative humidity (50 plusmn 5) for12 h lightdark cycles with food and water ad libitum TheInstitutional Committee on Care and Use of ExperimentalAnimals at the Tecnologico deMonterrey approved this studywith the protocol number 2014-RE-001

28 Acute Ear Edema Induced by Croton Oil Experimentswere carried out as described previously [28 29] Cutaneousinflammation was induced by the application of 5 crotonoil (20120583L) in acetone in the outer surface of the right earin rats The left ear received an equal volume of vehicle(acetone) After 30 minutes OFI extract isorhamnetin gly-cosides isorhamnetin standard at dose of 10120583moL IsoEqearor indomethacin (10120583moLear) was applied topically in theright ear in the test groups The left ear of each experi-mental subject of the test groups received an equal volumeof the vehicle The control group only received vehicle astreatment Test compounds and vehicle were applied by anautomaticmicropipette Animals were euthanized by cervicaldislocation 4 hours after croton oil application Both earswere removed with a metal punch (6mm diameter disc)and weighed (APX-100 Denver Instrument Denver CO)Ear edema was calculated by subtracting the weight of theleft ear (untreated) from the right ear (treated) The resultswere expressed as percentage of inhibition of ear edemawith respect to the control group according to the followinginhibition () [(ear edema of control group) minus (ear edema oftreated group)] times 100[ear edema of control group]

29 Measurement of COX-2 TNF-120572 and IL-6 in the EarEdema Model Induced by Croton Oil Ear biopsy sampleswere pooled by group and homogenized vigorously (TissueTearor BioSpec Products RacineWI) in 15mL of extractionbuffer (containing 10mM Tris pH 74 150mM NaCl and 1Triton X-100) per gram of sample The homogenates werecentrifuged at 13000 g for 10 minutes at 4∘C in a Microfuge22R centrifuge (Beckman Coulter Atlanta GA) and thesupernatants were stored at minus80∘C until being analyzed Theinhibition of COX-2 activity was determined by the COXColorimetric Inhibitor Screening Assay (Cayman ChemicalAnn Arbor MI) The levels of TNF-120572 and IL-6 in thesupernatants were measured using commercially availablesandwich enzyme-linked immunosorbent assay (ELISA) kits

IGRR IGRP

IGP IGR

Triglycosides

Diglycosides

Retention time (min)25 50 75 10 125 15 175 200 225

0255075

100125150175200225

(mAU

)

250

Figure 1 Chromatogram obtained at 365 nm showing themost abundant isorhamnetin glycosides found in Opuntia ficus-indica extract IGRR isorhamnetin-glucosyl-rhamnosyl-rhamno-side IGRP isorhamnetin-glucosyl-rhamnosyl-pentoside IGPisorhamnetin-glucosyl-pentoside IGR isorhamnetin-glucosyl-rhamnoside

(Invitrogen Corp Camarillo CA) according to themanufac-turerrsquos instructions The results were expressed as percentageof inhibition with respect to control

210 Statistical Analysis All measurements were performedat least in triplicate and results were expressed as mean plusmnstandard deviation Statistical analyses were performed withthe JMP 80 software (SAS Institute Inc Cary NC) Data wasanalyzed by ANOVA methodology followed by Tukeyrsquos HSDtests with a significance level of 119875 lt 005

3 Results

31 Identification and Quantification of IsorhamnetinGlycoside in OFI Extract The most abundant isorhamnetindiglycosides were isorhamnetin-glucosyl-pentoside (IGP)and isorhamnetin-glucosyl-rhamnoside (IGR) whereasisorhamnetin-glucosyl-rhamnosyl-rhamnoside (IGRR) andisorhamnetin-glucosyl-rhamnosyl-pentoside (IGRP) werethe most abundant triglycosides (Figure 1) Table 1 showsquantification of isorhamnetin glycosides in the OFI extractand their identification by comparison with previous reportsof lambda maxima (UVVIS) obtained by HPLC and mz[M+H] obtained by mass spectrometry (see SupplementaryFigures S1 and S2 in Supplementary Material availableonline at httpdxdoiorg1011552015847320) Along withthe molecular ion sodium adducts were obseved in eachmass spectra Ionization conditions allowed the detectionof fragments generated by the loss of three two and onesugar moieties observed in the triglycosides mass spectrum(Figure S1) likewise fragments generated by the loss of twoand one sugar moieties were observed in the diglycosidesmass spectrum (Figure S2) The mz [31705] correspondingto isorhamnetin aglycone appears in all mass spectrum

32 Effect of OFI Extract and Isorhamnetin Glycosides inCell Viability and NO Production in RAW 2647 MacrophageCells Stimulated by LPS Isorhamnetin triglycosides IGRRand IGRP did not affect the cell viability at any of


Table 1 Identification and quantification of most abundant isorhamnetin glycosides associated with Opuntia ficus-indica extract

IDcompound Tentative structure Concentration

(120583moLg)lowastUVVIS120582max (nm)

[M+H]mz References

IGRR

OOH

HO

OH

O-GLU-RHA-RHA

OCH3

O 2276 plusmn 75 253 353 771 [12ndash14]

IGRP

OOH

HO

OH

OCH3

O-GLU-RHA-PEN

O2202 plusmn 64 253 354 757 [12ndash14]

IGP

OOH

HO

OH

O

O-GLU-PEN

OCH3

931 plusmn 33 253 353 611 [12ndash14]

IGR

OOH

HO

OH

OCH3

O

O-GLU-RHA

951 plusmn 59 254 354 625 [12ndash14]

lowastConcentration expressed in 120583moL of IsoEqg of OFI extract IGRR isorhamnetin-glucosyl-rhamnosyl-rhamnoside IGRP isorhamnetin-glucosyl-rhamnosyl-pentoside IGP isorhamnetin-glucosyl-pentoside IGR isorhamnetin-glucosyl-rhamnoside

the concentrations tested (Table 2) In contrast IGP IGR andOFI extract showed a slight decrease in cell viability (849 plusmn40 816 plusmn 30 and 899 plusmn 19 resp) when tested at125 ng IsoEqmL

Regarding the inhibitory effects of isorhamnetin glyco-sides and OFI extract on NO production (Table 3) datashowed that OFI extract was more potent inhibitor thanisorhamnetin at all tested concentrations exhibiting 735 plusmn48 as the maximum inhibition value at 125 ng IsoEqmL

At 25 ng IsoEqmL IGRP and diglycosides IGP and IGRwere better inhibiting NO production showing 532 plusmn 47574 plusmn 91 and 600 plusmn 34 of inhibition respectivelyAt the same concentration IGR showed also better effectinhibiting NO production than OFI extract But when theconcentration was increased to 50 and 125 ng IsoEqmL IGR

andOFI extract increasedNO inhibition by 359 and 327respectively Although isorhamnetin bioactivity significantlyincreased with concentration it was still the least potentinhibitor of NO production (476 plusmn 58)

33 Effect of Isorhamnetin Glycosides and OFI Extract in AcuteEar Edema Induced by Croton Oil The diglycoside IGR andindomethacin showed the greatest potential to inhibit theear edema induced by croton oil 774 plusmn 57 and 695 plusmn53 respectively (Figure 2) Inhibition exhibited by IGPwasslightly lower (653 plusmn 59) than the observed for IGR butsimilar to the observed for isorhamnetin The isorhamnetintriglycoside IGRR showed lower inhibitory effect on earinflammation (447plusmn82) than IGR and OFI extract (571plusmn97) In this work isorhamnetin glycosides IGP and IGR but


Table 2 Effect of Opuntia ficus-indica (OFI) extract isorhamnetinand its glycosides in RAW 2647 cell viability

Treatment Cell viability ()25 ngmL 50 ngmL 125 ngmL

Isorhamnetin 993 plusmn 56alowast 97 5 plusmn 53a 977 plusmn 21a

IGP 967 plusmn 24ab 889 plusmn 53b 849 plusmn 40cd

IGR 922 plusmn 31b 852 plusmn 51b 816 plusmn 30d

IGRR 994 plusmn 29a 985 plusmn 23a 957 plusmn 45ab

IGRP 994 plusmn 39a 975 plusmn 28a 942 plusmn 40ab

OFI extract 945 plusmn 12ab 926 plusmn 48ab 899 plusmn 19bcConcentration expressed in ng IsoEqmL IGP isorhamnetin-glucosyl-pentoside IGR isorhamnetin-glucosyl-rhamnoside IGRR isorhamnetin-glucosyl-rhamnosyl-rhamnoside IGRP isorhamnetin-glucosyl-rhamnosyl-pentoside lowastDifferent letters in each column denote significant differencesamong treatments (119875 lt 005)

Table 3 Percentage of inhibition of nitric oxide production in RAW2647 cells incubated with different concentrations ofOpuntia ficus-indica (OFI) extract isorhamnetin and its glycosides

TreatmentNitric oxide inhibition ()

Concentration (ng IsoEqmL)25 50 125

Isorhamnetin 196 plusmn 22Cd 476 plusmn 58Bc 555 plusmn 31Ac

IGP 574 plusmn 91Aab 587 plusmn 42Aab 607 plusmn 43Abc

IGR 600 plusmn 34Ba 654 plusmn 42ABa 687 plusmn 50Aab

IGRR 506 plusmn 40Bb 542 plusmn 28Bbc 612 plusmn 58Abc

IGRP 532 plusmn 47Aab 544 plusmn 63Abc 599 plusmn 41Ac

OFI extract 408 plusmn 45Cc 572 plusmn 57Bab 735 plusmn 48Aa

IGP isorhamnetin-glucosyl-pentoside IGR isorhamnetin-glucosyl-rham-noside IGRR isorhamnetin-glucosyl-rhamnosyl-rhamnoside IGRP iso-rhamnetin-glucosyl-rhamnosyl-pentosideabcDifferent lower case letters denote significant differences among treat-mentsABCDifferent upper case letters denote significant differences among con-centrations (119875 lt 005)

not IGRR showed better topical anti-inflammatory potentialthan OFI extract

34 Effect of Isorhamnetin Glycosides and OFI Extract inProinflammatory COX-2 Activity and TNF-120572 and IL-6 Pro-duction Proinflammatory cytokines TNF-120572 and IL-6 weredetermined in order to clarify the topical anti-inflammatorymechanisms of isorhamnetin glycosides present and isolatedfrom the OFI extract (Figure 3) The greatest suppression ofTNF-120572 production was observed in ears treated with IGR(883 plusmn 05) while the OFI extract showed greater IL-6suppression (591 plusmn 14) The pure IGR increased TNF-120572inhibition in 309 in contrast to the inhibition caused bythe OFI crude extractThe OFI extract showed more effect asan inhibitor of COX-2 (748 plusmn 29) than indomethacin andIGRwhich exerted the same inhibitory effect (709plusmn09and683 plusmn 06 resp)

abbc

bca

dc

0

20

40

60

80

100

INDO ISO IGP IGR IGRR

Ear e

dem

a inh

ibiti

on (

) lowast lowast

OFIextract

Figure 2 Effect of indomethacin (INDO) isorhamnetin (ISO)isorhamnetin glycosides and Opuntia ficus-indica (OFI) extracton croton oil-induced rat ear edema IGP isorhamnetin-glucosyl-pentoside IGR isorhamnetin-glucosyl-rhamnoside IGRR iso-rhamnetin-glucosyl-rhamnosyl-rhamnoside abcdDifferent lettersdenote significant differences among treatments (119875 lt 005)lowastTreatment with highest potential to inhibit ear edema

c

a

b

c

ba

b ba

0

20

40

60

80

100

INDO IGR OFI extractTreatments

IL-6COX-2

Inhi

bitio

n (

of co

ntro

l)

TNF-120572

Figure 3 Effect of indomethacin (INDO) isorhamnetin-glucosyl-rhamnoside (IGR) and Opuntia ficus-indica (OFI) extract on TNF-120572 and interleukin- (IL-) 6 levels and on COX-2 activity in ratears tissue homogenate Indomethacin was used as positive controlControl group only received croton oil 5 vv as irritant agentData represent means plusmn standard deviations of at least 6 replicatesabcDifferent letters denote significant differences among treatments(119875 lt 005)

4 Discussion

The observed effects on cell viability were similar tothose reported by Benayad et al [23] who tested theanti-inflammatory activities of extracts from MoroccanOpuntia ficus-indica flowers Cell viability of macrophageRAW 2647 cells was ge90 for all samples at the concentra-tions tested [23] Therefore reduction of NO was not due tothe cytotoxicity of isorhamnetin glycosides since cell viabilitywas above 80

All isolated isorhamnetin glycosides showed inhibitoryeffects higher than 50 on NO production even at concen-trations as low as 25 ng IsoEqmL Isorhamnetin diglycosideshad a higher anti-inflammatory potential than triglycosidesOther reports had demonstrated that kaempferol glyco-sides inhibited differently the NO production Particularly


kaempferol 7-O-120572-L-rhamnopyranoside showed an IC50value of 412 120583M whereas kaempferol triglycosides anddiglycosides at the dosage of 100 120583M had an inhibitoryeffect of less than 10 [30] Fang et al [22] showedthat kaempferitrin and kaempferol-3-O-120573-D-apiofuranosyl-(1-2)-120572-L-arabinofuranosyl-7-O-120572-L-rhamnopyranoside dif-ferently inhibited the NO production exhibiting an IC50 of40 120583M and 15 120583M respectively

Additionally Rho et al [31] demonstrated that theposition and the number of sugar moieties affect thesuppression of NO production exerted by flavonol glyco-sides Kaempferol 7-O-120572-L-rhamnopyranoside showed morepotential to inhibit NO production (IC50 = 377 120583M) thankaempferol 3-O-120572-L-rhamnopyranoside (IC50 gt 100 120583M)Likewise Rho et al [31] showed that the presence of anadditional rhamnose moiety at the 3-position caused a loss inNO inhibitory activity as in the case of kaempferol-3 7-O-120572-dirhamnoside (IC50 gt 100 120583M) These results are consistentwith our findings where the diglycoside IGR showed greaterinhibitory activity on NO production compared with itscorresponding triglycoside IGRR that has an additionalrhamnose moiety

Isorhamnetin glycosides had a higher inhibition on nitricoxide production compared to aglycone tested at 25 ngIsoEqmL (Table 3) In contrast previous reports indicatedthat a quercetin glycoside had a similar effect to the aglyconeonly when tested at a concentration 30 times higher [32]But baicalein and baicalein-6-120572-glucoside when they weretested at 40 120583M showed similar inhibitory activities on theNO production (approximately 60) [33]

The effect of OFI extract at 50 ng IsoEqmL was com-parable with the NO production inhibition induced by anextract ofO ficus-indica flowers (05mg extractmL) in RAW2647 cell line (gt60) [23] Isolated isorhamnetin glycosidesshowed greater NO production inhibition than OFI extractwhen tested at 25 ng IsoEqmL But when concentration wasincreased to 125 ng IsoEqmL OFI extract inhibited the NOproduction similarly or more potently than the isorhamnetinglycosides tested Kazłowska et al [34] reported thatPorphyradentata crude extract was more active inhibiting NO pro-duction compared to its pure compounds (catechol rutinand hesperidin) Harasstani et al [35] have demonstrated thatwhen flavonoids are combined as occurs in plant extractsthese can significantly increase their inhibitory effect uponNO production

In the ear edema inflammation model IGR andindomethacin showed the better effects The effect of IGRwas comparable with the 81 of inhibition induced by thetiliroside (flavonol acyl-glucoside) on an acute TPA-inducedear edema inflammation model [36] As observed in thein vitro experiments triglycoside IGRR showed loweranti-inflammatory effects compared with diglycosides andindomethacin The difference in hydrophobicity betweenisorhamnetin diglycosides and triglycosides alters the cell-membrane permeability which may be the reason for thebetter effect exerted by diglycosides It has been suggestedthat the compounds with a MW lt 500Da are suitablytransported across skin [37 38]

Different anti-inflammatory effects were observed amongisorhamnetin diglycosides IGR and IGP although they onlydiffered in the pentose methylation These results indicatedthat the type of sugar affected bioactivity contrary to thosereported by Sosa et al [39] who showed that quercetin-3-O-galactoside and quercetin-3-O-glucoside at 10 120583moLearexhibited similar inhibition in a mouse ear edema inducedby croton oil (52 and 51 resp) Erdemoglu et al [40]demonstrated that quercetin-3-O-galactoside and quercetin-3-O-rhamnoside tested at 05mgear inhibited differentlythe edema induced in mice by TPA (32 and 447 resp)suggesting that the presence of rhamnose could increase theanti-inflammatory potential

Although some reports indicated that flavonoid gly-cosylation reduced the anti-inflammatory effect isorham-netin (ISO) and IGP showed similar effects on the rat earedema inflammation model This trend had been previouslyreported for hesperetin and the hesperetin-7-rutinoside thathad similar anti-inflammatory effects (44 and 45 resp)in mice model of ear swelling induced by xylene [41]

The inhibitory effect of the OFI extract on ear edemawas comparable to that produced by the chloroform extractfrom Senna villosa leaves (579) in the TPA-induced earedema model and slightly greater (524) than the extract ofPistacia khinjuk L leaves which also was rich in glycosylatedflavonoids [42 43] Some isolated compounds can be morepotent anti-inflammatory drugs than their source extractMo et al [28] reported that the MeOH extracts of Lithraeamolleoides (Vell) Engl (Anacardiaceae) inhibited the earedema induced by TPA by 21 while the methyl gallateone of the main compounds in MeOH extract at an equiv-alent concentration produced 63 of ear edema inhibitionLikewise an ethanolic extract of Myagropsis myagroides(90 120583gear)markedly inhibited the phorbolmyristate acetate-(PMA-) induced ear edema with 668 inhibition (119875 lt005) one-third of 661015840-bieckol activity which was tested at30 120583gear [44] In this investigation although the OFI extractshowed an adequate inhibitory effect on ear edema it did notexceed the effects observed with isorhamnetin diglycosidesIGR and IGP Based on these results the order of anti-inflammatory potential against the ear edema was IGR =INDO gt IGP = ISO gt OFI extract gt IGRR

Tumor necrosis factor (TNF) is a potent mediator ofinflammation with a powerful proinflammatory capacityespecially within immune cells capable of producing a cas-cade of downstream cytokines and chemokines expressed bymonocytesmacrophages [45] Meanwhile IL-6 is an impor-tant factor for the synthesis of acute phase proteins whoseserum level is increased in acute and chronic inflammatorydiseases such as cancer [46 47] In skin inflammationcytokines such as TNF-120572 and IL-6 from resident cells ker-atinocytes bind to receptors on the vascular endotheliumactivating cellular signaling pathways and inducing expres-sion of vascular endothelial cell adhesion molecules [48]

Both IGR and OFI extract inhibited COX-2 activity andinflammatory cytokines IL-6 and TNF-120572 TNF-120572 activationstimulates the IL-6 synthesis in several cell types and in turnIL-6 inhibits the TNF-120572 production providing a negativefeedback thus limiting the acute inflammatory response [49]


IGR showed more pronounced effect on TNF-120572 than IL-6 or COX-2 activity indicating that its anti-inflammatoryeffect was mediated by the inhibition of proinflammatorycytokines Differences in the cytokine modulation indicatethat at least a part of the IL-6 production could be directedand not mediated by TNF-120572 activation [50] Mueller et al[51] observed that an apple extract containing 5 quercetinand 30 phloridzin (glycosylated phenolic compound) sup-pressedmore potently the TNF-120572 production (89) than thatof IL-6 (32) in a LPS-stimulated macrophage model

The OFI extract showed greater inhibition on COX-2activity than on TNF-120572 or IL-6 Inhibitory effects of OFIextract on TNF-120572 and IL-6 were similar to those observedfor quercetin (591 plusmn 33 and 579 plusmn 99 resp) measuredin human blood [52]

On the other hand Lee et al [53] proved that aflavonol-rich hexane fraction from Rhus verniciflua stokesand its major compound fisetin affected differently the TNF-120572 and IL-6 production in vitro The R verniciflua hexanefraction (1 120583gmL) which contained about 00827120583gmLof fisetin suppressed approximately 263 of the TNF-120572production in ear edema When pure fisetin was testedat 01 120583gmL the TNF-120572 inhibition increased Likewisea crude hydroalcoholic extract of Croton antisyphiliticusMart and its isolated glycosylated flavonoid (apigenin-120573-D-glucopyranosyl) showed similar suppressing effect on TNF-120572 production despite the differences among the concentra-tions tested (100mgKg and 10mgKg resp) [54] Resultspresented herein agree with previous reports for glycosylatedflavonoids such as the tiliroside wogonoside kaempferol-3-O-glucoside quercetin 3-O-glucoside and isorhamnetin-3-O-glucoside as important inhibitors of TNF-120572 and IL-6production [55ndash58]

Anti-inflammatory effects of flavonoids can occurthrough different molecular mechanisms such as inhibitionof histamine release from basophils inhibition of proinflam-matory eicosanoids production or by inhibiting theproduction of proinflammatory cytokines [23] In thisstudy it was demonstrated that isorhamnetin glycosides andthe OFI extract modulate the inflammatory response bysuppressing the COX-2 activity and also by the reduction inlevels of NO IL-6 and TNF-120572 both in vitro and in vivo

5 Conclusions

The Opuntia ficus-indica (OFI) extract and its isorham-netin glycosides exerted anti-inflammatory effects In vitroisorhamnetin diglycoside IGR and the OFI extract signifi-cantly reduced the nitric oxide production in RAW 2647macrophage cells without significantly affecting viabilitywhile in vivo IGR showed an efficacy comparable toindomethacin to reduce rat ear edema induced by croton oil

The attached sugar moiety affected the in vivo anti-inflammatory effects of the diglycosides IGP and IGRLikewise the glycosylation degree affected the bioactivitythe triglycoside IGRR being less bioactive compared to thediglycoside IGRThe topical anti-inflammatory effect of IGRwas mediated by the suppression of COX-2 activity and

inhibition on the production of proinflammatory cytokinessuch as TNF-120572 and IL-6

Further research is needed in order to determine thepermeability and absorption of these compounds and eval-uate their efficacy in dermatological preparations Additionalexperiments are required to validate themechanismof actionincluding iNOS expression and other genes involved in theinflammatory response

Abbreviations

IsoEq Isorhamnetin equivalentsOFI Opuntia ficus-indica

Conflict of Interests

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

Acknowledgments

The authors acknowledge the financial support from theConsejo Nacional de Ciencia y Tecnologıa (CONACYT-CBResearch Project 1168708 and Scholarship Program) and theTecnologico de Monterrey (Nutriomics Research Group)Also they acknowledge the donation of Opuntia ficus-indicapowder by Alimentos Funcionales S de RLMI

References

[1] D R Bickers andMAthar ldquoOxidative stress in the pathogenesisof skin diseaserdquo Journal of Investigative Dermatology vol 126no 12 pp 2565ndash2575 2006

[2] E Omori S Morioka K Matsumoto and J Ninomiya-TsujildquoTAK1 regulates reactive oxygen species and cell death inkeratinocytes which is essential for skin integrityrdquo The Journalof Biological Chemistry vol 283 no 38 pp 26161ndash26168 2008

[3] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer how are theylinkedrdquo Free Radical Biology and Medicine vol 49 no 11 pp1603ndash1616 2010

[4] C N Young J I Koepke L J Terlecky M S Borkin S L Boydand S R Terlecky ldquoReactive oxygen species in tumor necrosisfactor-120572-activated primary human keratinocytes implicationsfor psoriasis and inflammatory skin diseaserdquo Journal of Inves-tigative Dermatology vol 128 no 11 pp 2606ndash2614 2008

[5] A Chiricozzi E Guttman-YasskyM Suarez-Farıas et al ldquoInte-grative responses to IL-17 and TNF-120572 in human keratinocytesaccount for key inflammatory pathogenic circuits in psoriasisrdquoJournal of Investigative Dermatology vol 131 no 3 pp 677ndash6872011

[6] W-W Li T-Z Guo X-Q Li W S Kingery and J D ClarkldquoFracture induces keratinocyte activation proliferation andexpression of pro-nociceptive inflammatory mediatorsrdquo Painvol 151 no 3 pp 843ndash852 2010

[7] M Eddouks D Chattopadhyay and N A Zeggwagh ldquoAni-mal models as tools to investigate antidiabetic and anti-inflammatory plantsrdquo Evidence-Based Complementary andAlternativeMedicine vol 2012 Article ID 142087 14 pages 2012


[8] D Y Lee G Choi T Yoon M S Cheon B K Choo and H KKim ldquoAnti-inflammatory activity of Chrysanthemum indicumextract in acute and chronic cutaneous inflammationrdquo Journalof Ethnopharmacology vol 123 no 1 pp 149ndash154 2009

[9] S-B Yoon Y-J Lee S K Park et al ldquoAnti-inflammatory effectsof Scutellaria baicalensis water extract on LPS-activated RAW2647macrophagesrdquo Journal of Ethnopharmacology vol 125 no2 pp 286ndash290 2009

[10] E Fasano S Serini N Mondella et al ldquoAntioxidant andanti-inflammatory effects of selected natural compounds con-tained in a dietary supplement on two human immortalizedkeratinocyte linesrdquo BioMed Research International vol 2014Article ID 327452 11 pages 2014

[11] H A S Favacho B R Oliveira K C Santos et al ldquoAnti-inflammatory and antinociceptive activities of Euterpe oleraceaMart Arecaceae oilrdquo Revista Brasileira de Farmacognosia vol21 no 1 pp 105ndash114 2011

[12] M Antunes-Ricardo B E Moreno-Garcıa J A Gutierrez-Uribe D Araiz-Hernandez M M Alvarez and S O Serna-Saldivar ldquoInduction of apoptosis in colon cancer cells treatedwith isorhamnetin glycosides from Opuntia ficus-indica padsrdquoPlant Foods for Human Nutrition vol 69 no 4 pp 331ndash3362014

[13] G Ginestra M L Parker R N Bennett et al ldquoAnatomicalchemical and biochemical characterization of cladodes fromprickly pear [Opuntia ficus-indica (L)Mill]rdquo Journal of Agricul-tural and Food Chemistry vol 57 no 21 pp 10323ndash10330 2009

[14] L Santos-Zea J A Gutierrez-Uribe and S O Serna-SaldivarldquoComparative analyses of total phenols antioxidant activityand flavonol glycoside profile of cladode flours from differentvarieties of Opuntia spprdquo Journal of Agricultural and FoodChemistry vol 59 no 13 pp 7054ndash7061 2011

[15] S Aquila R M Giner M C Recio E D Spegazzini and J LRıos ldquoAnti-inflammatory activity of flavonoids fromCayaponiatayuya rootsrdquo Journal of Ethnopharmacology vol 121 no 2 pp333ndash337 2009

[16] J H Jin J S Kim S S Kang K H Son H W Chang andH P Kim ldquoAnti-inflammatory and anti-arthritic activity oftotal flavonoids of the roots of Sophora flavescensrdquo Journal ofEthnopharmacology vol 127 no 3 pp 589ndash595 2010

[17] M Hamalainen R Nieminen P Vuorela M Heinonen and EMoilanen ldquoAnti-inflammatory effects of flavonoids genisteinkaempferol quercetin and daidzein inhibit STAT-1 and NF-120581B activations whereas flavone isorhamnetin naringenin andpelargonidin inhibit only NF-120581B activation along with theirinhibitory effect on iNOS expression and NO production inactivated macrophagesrdquo Mediators of Inflammation vol 2007Article ID 45673 10 pages 2007

[18] A J Day J M Gee M S DuPont I T Johnson andG Williamson ldquoAbsorption of quercetin-3-glucoside andquercetin-41015840-glucoside in the rat small intestine the role oflactase phlorizin hydrolase and the sodium-dependent glucosetransporterrdquo Biochemical Pharmacology vol 65 no 7 pp 1199ndash1206 2003

[19] G M Huber H P Vasantha Rupasinghe and F ShahidildquoInhibition of oxidation of omega-3 polyunsaturated fatty acidsand fish oil by quercetin glycosidesrdquo Food Chemistry vol 117no 2 pp 290ndash295 2009

[20] C-T Yen P-WHsieh T-L Hwang Y-H Lan F-R Chang andW U Yang-Chang ldquoFlavonol glycosides from Muehlenbeckiaplatyclada and their anti-inflammatory activityrdquo Chemical ampPharmaceutical Bulletin vol 57 no 3 pp 280ndash282 2009

[21] G O de Melo D D C Malvar F A Vanderlinde et alldquoAntinociceptive and anti-inflammatory kaempferol glycosidesfrom Sedum dendroideumrdquo Journal of Ethnopharmacology vol124 no 2 pp 228ndash232 2009

[22] S-H Fang Y K Rao and Y-M Tzeng ldquoInhibitory effectsof flavonol glycosides from Cinnamomum osmophloeum oninflammatory mediators in LPSIFN-120574-activated murinemacrophagesrdquo Bioorganic amp Medicinal Chemistry vol 13 no 7pp 2381ndash2388 2005

[23] Z Benayad C Martinez-Villaluenga J Frias C Gomez-Cordoves andN E Es-Safi ldquoPhenolic composition antioxidantand anti-inflammatory activities of extracts from MoroccanOpuntia ficus-indica flowers obtained by different extractionmethodsrdquo Industrial Crops and Products vol 62 pp 412ndash4202014

[24] D-W Cho D-E Kim D-H Lee et al ldquoMetabolite profilingof enzymatically hydrolyzed and fermented forms of Opuntiaficus-indica and their effect on UVB-induced skin photoagingrdquoArchives of Pharmacal Research vol 37 no 9 pp 1159ndash11682014

[25] T E Moussa-Ayoub E S A A El-Hady H T Omran S KEl-Samahy L W Kroh and S Rohn ldquoInfluence of cultivar andorigin on the flavonol profile of fruits and cladodes from cactusOpuntia ficus-indicardquo Food Research International vol 64 pp864ndash872 2014

[26] B Kim Y-E Choi and H-S Kim ldquoEruca sativa and itsflavonoid components quercetin and isorhamnetin improveskin barrier function by activation of peroxisome proliferator-activated receptor (PPAR)-120572 and suppression of inflammatorycytokinesrdquo Phytotherapy Research vol 28 pp 1359ndash1366 2014

[27] T H Kim S-K Ku and J-S Bae ldquoAnti-inflammatory activi-ties of isorhamnetin-3-O-galactoside against HMGB1-inducedinflammatory responses in both HUVECs and CLP-inducedseptic micerdquo Journal of Cellular Biochemistry vol 114 no 2 pp336ndash345 2013

[28] JMo P Panichayupakaranant N Kaewnopparat A Nitiruang-jaras and W Reanmongkol ldquoTopical anti-inflammatory andanalgesic activities of standardized pomegranate rind extractin comparison with its marker compound ellagic acid in vivordquoJournal of Ethnopharmacology vol 148 no 3 pp 901ndash908 2013

[29] L H Jones D S P Abdalla and J C Freitas ldquoEffects of indole-3-acetic acid on croton oil- and arachidonic acid-inducedmouse ear edemardquo Inflammation Research vol 44 no 9 pp372ndash375 1995

[30] H-Y Lin and S-T Chang ldquoKaempferol glycosides from thetwigs of Cinnamomum osmophloeum and their nitric oxideproduction inhibitory activitiesrdquo Carbohydrate Research vol364 pp 49ndash53 2012

[31] H S Rho A K Ghimeray D S Yoo et al ldquoKaempferoland kaempferol rhamnosides with depigmenting and anti-inflammatory propertiesrdquo Molecules vol 16 no 4 pp 3338ndash3344 2011

[32] V A Vo J-W Lee J-E Chang et al ldquoAvicularin inhibitslipopolysaccharide-induced inflammatory response by sup-pressing ERK phosphorylation in RAW 2647 macrophagesrdquoBiomolecules ampTherapeutics (Seoul) vol 20 no 6 pp 532ndash5372012

[33] K H Kim Y D Park H Park et al ldquoSynthesis and bio-logical evaluation of a novel baicalein glycoside as an anti-inflammatory agentrdquo European Journal of Pharmacology vol744 pp 147ndash156 2014


[34] K Kazłowska T Hsu C-C Hou W-C Yang and G-J TsaildquoAnti-inflammatory properties of phenolic compounds andcrude extract from Porphyra dentatardquo Journal of Ethnopharma-cology vol 128 no 1 pp 123ndash130 2010

[35] O A Harasstani S Moin C L Tham et al ldquoFlavonoidcombinations cause synergistic inhibition of proinflamma-torymediator secretion from lipopolysaccharide-inducedRAW2647 cellsrdquo Inflammation Research vol 59 no 9 pp 711ndash7212010

[36] A Sala M C Recio G R Schinella et al ldquoAssessment of theanti-inflammatory activity and free radical scavenger activity oftilirosiderdquo European Journal of Pharmacology vol 461 no 1 pp53ndash61 2003

[37] C-F Lin Y-L Leu S A Al-Suwayeh M-C Ku T-L Hwangand J-Y Fang ldquoAnti-inflammatory activity and percutaneousabsorption of quercetin and its polymethoxylated compoundand glycosides the relationships to chemical structuresrdquo Euro-pean Journal of Pharmaceutical Sciences vol 47 no 5 pp 857ndash864 2012

[38] B C Finnin and T M Morgan ldquoTransdermal penetrationenhancers applications limitations and potentialrdquo Journal ofPharmaceutical Sciences vol 88 no 10 pp 955ndash958 1999

[39] S Sosa R Pace A Bornancin et al ldquoTopical anti-inflammatoryactivity of extracts and compounds fromHypericum perforatumLrdquo Journal of Pharmacy and Pharmacology vol 59 no 5 pp703ndash709 2007

[40] N Erdemoglu E K Akkol E Yesilada and I Calis ldquoBioassay-guided isolation of anti-inflammatory and antinociceptive prin-ciples from a folk remedy Rhododendron ponticum L leavesrdquoJournal of Ethnopharmacology vol 119 no 1 pp 172ndash178 2008

[41] A E Rotelli T Guardia A O Juarez N E de La Rocha andL E Pelzer ldquoComparative study of flavonoids in experimentalmodels of inflammationrdquo Pharmacological Research vol 48 no6 pp 601ndash606 2003

[42] A D C Susunaga-Notario S Perez-Gutierrez M A Zavala-Sanchez et al ldquoBioassay-guided chemical study of the anti-inflammatory effect of Senna villosa (Miller) HS Irwin ampBarneby (Leguminosae) in TPA-induced ear edemardquoMoleculesvol 19 no 7 pp 10261ndash10278 2014

[43] A Esmat F A Al-Abbasi M M Algandaby et al ldquoAnti-inflammatory activity of Pistacia khinjuk in different experi-mental models Isolation and characterization of its flavonoidsand galloylated sugarsrdquo Journal of Medicinal Food vol 15 no 3pp 278ndash287 2012

[44] E-J Joung M-S Lee J-W Choi et al ldquoAnti-inflammatoryeffect of ethanolic extract from Myagropsis myagroides onmurinemacrophages andmouse ear edemardquoBMCComplemen-tary and Alternative Medicine vol 12 article 171 2012

[45] L M Sedger and M F McDermott ldquoTNF and TNF-receptorsfrom mediators of cell death and inflammation to therapeuticgiantsmdashpast present and futurerdquo Cytokine amp Growth FactorReviews vol 25 no 4 pp 453ndash472 2014

[46] M F Neurath and S Finotto ldquoIL-6 signaling in autoimmunitychronic inflammation and inflammation-associated cancerrdquoCytokineampGrowth Factor Reviews vol 22 no 2 pp 83ndash89 2011

[47] K Taniguchi and M Karin ldquoIL-6 and related cytokines as thecritical lynchpins between inflammation and cancerrdquo Seminarsin Immunology vol 26 no 1 pp 54ndash74 2014

[48] J Y Ahn S E Choi M S Jeong et al ldquoEffect of taxifolinglycoside on atopic dermatitis-like skin lesions in NCNgamicerdquo Phytotherapy Research vol 24 no 7 pp 1071ndash1077 2010

[49] C A Feghali and TMWright ldquoCytokines in acute and chronicinflammationrdquo Frontiers in Bioscience vol 2 pp d12ndashd26 1997

[50] M Sironi M Gadina M Kankova et al ldquoDifferential sen-sitivity of in vivo TNF and IL-6 production to modulationby anti-inflammatory drugs in micerdquo International Journal ofImmunopharmacology vol 14 no 6 pp 1045ndash1050 1992

[51] M Mueller S Hobiger and A Jungbauer ldquoAnti-inflammatoryactivity of extracts from fruits herbs and spicesrdquo Food Chem-istry vol 122 no 4 pp 987ndash996 2010

[52] D Ribeiro M Freitas S M Tome et al ldquoFlavonoids inhibitCOX-1 and COX-2 enzymes and cytokinechemokine produc-tion in human whole bloodrdquo Inflammation 2014

[53] J-D Lee J-E Huh G Jeon et al ldquoFlavonol-rich RVHxRfrom Rhus verniciflua Stokes and its major compound fisetininhibits inflammation-related cytokines and angiogenic factorin rheumatoid arthritic fibroblast-like synovial cells and in vivomodelsrdquo International Immunopharmacology vol 9 no 3 pp268ndash276 2009

[54] G O dos Reis G Vicente F K de Carvalho et al ldquoCrotonantisyphiliticus Mart attenuates the inflammatory response tocarrageenan-induced pleurisy in micerdquo Inflammopharmacol-ogy vol 22 no 2 pp 115ndash126 2014

[55] R Velagapudi M Aderogba and O A Olajide ldquoTilirosidea dietary glycosidic flavonoid inhibits TRAF-6NF-120581Bp38-mediated neuroinflammation in activated BV2 microgliardquoBiochimica et Biophysica Acta vol 1840 no 12 pp 3311ndash33192014

[56] A R Kim Q Jin H G Jin H J Ko and E R Woo ldquoPhenoliccompounds with IL-6 inhibitory activity from Aster yomenardquoArchives of Pharmacal Research vol 37 no 7 pp 845ndash851 2014

[57] M S Kim and S H Kim ldquoInhibitory effect of astragalin onexpression of lipopolysaccharideinduced inflammatory medi-ators through NF-120581B in macrophagesrdquo Archives of PharmacalResearch vol 34 no 12 pp 2101ndash2107 2011

[58] Y-Z Yang Y-Z Tang and Y-H Liu ldquoWogonoside dis-plays anti-inflammatory effects throughmodulating inflamma-tory mediator expression using RAW2647 cellsrdquo Journal ofEthnopharmacology vol 148 no 1 pp 271ndash276 2013

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


on RAW 2647 macrophages it inhibited the expressionsof iNOS and COX-2 by 97 and 65 respectively [15]Likewise flavonoids contained in a flavonoid-enriched frac-tion (50 120583gmL) extracted from the rhizomes of Sophoraflavescens also showed in vitro inhibitory effects greater than50 on the NO production [16] Isorhamnetin kaempferoland quercetin inhibited iNOS protein andmRNA expressionwhen tested in macrophages stimulated with lipopolysac-charide (LPS) [17] Also these compounds inhibited theactivation of nuclear transcription factor-120581B (NF-120581B)

In nature flavonoids are generally attached to sugarresidues that modify the mechanism of absorption and theirability to enter cells or to interact with transporters andcellular lipoproteins [18] Therefore there are differencesamong the biological effects exerted by flavonoid glycosidesaccording to the type number and position of sugar moieties[19 20] de Melo et al [21] showed that kaempferol 3-O-120573-glucopyranoside-7-O-120572-rhamnopyranoside and kaempferol7-O-120572-rhamnopyranoside inhibited the croton oil-inducedear edema by 465 and 333 respectively Likewisekaempferol 3-O-120573-D-apiofuranosyl-(1rarr 2)-120572-L-arabinofur-anosyl-7-O-120572-L-rhamnopyranoside (10 120583M) showed betterinhibitory effects on TNF-120572 and IL-12 production thankaempferol 3-O-120573-D-apiofuranosy-(1rarr 4)-120572-L-rhamnopy-ranosyl-7-O-120572-L-rhamnopyranoside (40 120583M) [22]

Opuntia ficus-indica (OFI) has been used in Mexico as afood and as a remedy against different health disorders relatedto skin such as inflammation [23 24] This plant is a naturalsource of flavonoids mainly isorhamnetin glycosides [12ndash1425] Isorhamnetin and isorhamnetin-3-O-galactoside possessin vitro anti-inflammatory activity [26 27]

Considering all the above the aim of this research workwas to evaluate the in vitro and in vivo anti-inflammatoryeffects of an OFI extract and compare the bioactivity ofdifferent isorhamnetin glycosides isolated from this sourceOFI extract and isolated isorhamnetin diglycosides andtriglycosides were tested on RAW 2647 macrophage cellsin order to evaluate their effect on NO production Laterthese compoundswere tested in a rat ear edema inflammationmodel to evaluate their effect on COX-2 TNF-120572 and IL-6levels

2 Materials and Methods

21 Reagents Dulbeccorsquos Modified Eagle Medium NutrientMixture F-12 (DMEM-F12) ampicillinstreptomycin andphosphate-buffered saline (pH 74) were purchased fromGibco Invitrogen (Carlsbad CA) Trypsin-EDTA (025)and fetal bovine serumwere obtained fromHyCloneThermoScientific (Logan UT) Triton X-100 was acquired fromResearch Organics (Cleveland OH) Lipopolysaccharides(LPS) from Salmonella enterica serotype typhimurium L7261as well as croton oil indomethacin isorhamnetin stan-dard and formic acid solution HPLC grade were purchasedfrom Sigma-Aldrich (St Louis MO) Chromatography gradewater and methanol (VWR International LLC West ChesterPA) were used for high-pressure liquid chromatographequippedwith a photodiode array detector (HPLC-PDA) and

liquid chromatographmass selective detector time-of-flight(LCMSD TOF) analysis

22 Obtaining O ficus-indica Extract The taxonomic identi-fication of Opuntia ficus-indica was done by PhD RigobertoE Vazquez-Alvarado at the School of Agronomy of Uni-versidad Autonoma de Nuevo Leon (UANL) Mexico OFIcladodes were harvested at 7 months and grown in the regionof Montemorelos Nuevo Leon Mexico and then they wereprocessed into powder according to Santos-Zea et al [14]OFI extract was obtained by alkaline hydrolysis following themethod previously reported by Antunes-Ricardo et al [12]

23 Identification Quantification and Purification ofIsorhamnetin Glycosides in OFI Extract Identification andquantification of isorhamnetin glycosides were performedaccording to the method described by Antunes-Ricardoet al [12] using HPLC-PDA (Agilent 1100 Series SantaClara CA) Purification of isorhamnetin glycosides wasdone by semipreparative chromatography according to themethod described by Antunes-Ricardo et al [12] usinga semipreparative Zorbax SB-C18 (94 times 250mm 5120583m)column at 17∘C flow rate of 20mLmin and injectionvolume of 50 120583L Water with 01 formic acid (A) andmethanol 80 (B) were used as mobile phases startingwith 35 of B and increasing to 60 and 90 after 2 and19min respectively and then decreasing to 0 B for the next3min Percentages of purity of 90 or more were obtainedfor the isorhamnetin glycosides LCMSD TOF technology(Model G1969A Agilent 1100 Santa Clara CA) was usedto identify the isorhamnetin glycosides according to themethod described by Santos-Zea et al [14]

24 RAW 2647 Cell Culture RAW 2647 a murinemacrophage cell line was obtained from the AmericanType Culture Collection (ATCC Manassas VA) Cells werecultured in petri plates with Dulbeccorsquos Modified EagleMedium (DMEM) supplemented with 10 fetal bovineserum (FBS) and 1 antibiotic and incubated at 37∘C and 5of CO

2(NuAire Plymouth MN) To evaluate the effects of

an OFI extract and isolated isorhamnetin glycosides on nitricoxide production cells were plated in 96-well plate (5 times 104cellswell) and allowed to adhere for 4 h After that differentconcentrations (25 50 or 125 ng isorhamnetin equivalents(IsoEqmL) of the compounds were added and incubated foranother 24 h Following half of wells were stimulated withLPS at 10 120583gmL (final concentration) while the other halfwere used as control for each sample

25 Measurement of Nitrite Concentration Production ofnitric oxide (NO) was measured by nitrites determinationwhich is a stable and nonvolatile breakdown product of NOAfter incubation with the different treatments 100120583L ofsupernatant was transferred to another 96-well plate to mea-sure nitrites concentration using the Griess Reagent System(Promega Madison WI) at 550 nm Data was expressed aspercentage of inhibition of NO production The remaining







IGRR IGRP

IGP IGR

Triglycosides

Diglycosides


0255075

100125150175200225

(mAU

)

250




3 Results







[M+H]mz References

IGRR

OOH

HO

OH

O-GLU-RHA-RHA

OCH3

O 2276 plusmn 75 253 353 771 [12ndash14]

IGRP

OOH

HO

OH

OCH3

O-GLU-RHA-PEN

O2202 plusmn 64 253 354 757 [12ndash14]

IGP

OOH

HO

OH

O

O-GLU-PEN

OCH3

931 plusmn 33 253 353 611 [12ndash14]

IGR

OOH

HO

OH

OCH3

O

O-GLU-RHA

951 plusmn 59 254 354 625 [12ndash14]




























abbc

bca

dc

0

20

40

60

80

100


Ear e

dem

a inh

ibiti

on (

) lowast lowast

OFIextract


c

a

b

c

ba

b ba

0

20

40

60

80

100


IL-6COX-2

Inhi

bitio

n (

of co

ntro

l)

TNF-120572


4 Discussion



















5 Conclusions





Abbreviations




Acknowledgments


References

































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







IGRR IGRP

IGP IGR

Triglycosides

Diglycosides


0255075

100125150175200225

(mAU

)

250




3 Results







[M+H]mz References

IGRR

OOH

HO

OH

O-GLU-RHA-RHA

OCH3

O 2276 plusmn 75 253 353 771 [12ndash14]

IGRP

OOH

HO

OH

OCH3

O-GLU-RHA-PEN

O2202 plusmn 64 253 354 757 [12ndash14]

IGP

OOH

HO

OH

O

O-GLU-PEN

OCH3

931 plusmn 33 253 353 611 [12ndash14]

IGR

OOH

HO

OH

OCH3

O

O-GLU-RHA

951 plusmn 59 254 354 625 [12ndash14]




























abbc

bca

dc

0

20

40

60

80

100


Ear e

dem

a inh

ibiti

on (

) lowast lowast

OFIextract


c

a

b

c

ba

b ba

0

20

40

60

80

100


IL-6COX-2

Inhi

bitio

n (

of co

ntro

l)

TNF-120572


4 Discussion



















5 Conclusions





Abbreviations




Acknowledgments


References

































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





[M+H]mz References

IGRR

OOH

HO

OH

O-GLU-RHA-RHA

OCH3

O 2276 plusmn 75 253 353 771 [12ndash14]

IGRP

OOH

HO

OH

OCH3

O-GLU-RHA-PEN

O2202 plusmn 64 253 354 757 [12ndash14]

IGP

OOH

HO

OH

O

O-GLU-PEN

OCH3

931 plusmn 33 253 353 611 [12ndash14]

IGR

OOH

HO

OH

OCH3

O

O-GLU-RHA

951 plusmn 59 254 354 625 [12ndash14]




























abbc

bca

dc

0

20

40

60

80

100


Ear e

dem

a inh

ibiti

on (

) lowast lowast

OFIextract


c

a

b

c

ba

b ba

0

20

40

60

80

100


IL-6COX-2

Inhi

bitio

n (

of co

ntro

l)

TNF-120572


4 Discussion



















5 Conclusions





Abbreviations




Acknowledgments


References

































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






















abbc

bca

dc

0

20

40

60

80

100


Ear e

dem

a inh

ibiti

on (

) lowast lowast

OFIextract


c

a

b

c

ba

b ba

0

20

40

60

80

100


IL-6COX-2

Inhi

bitio

n (

of co

ntro

l)

TNF-120572


4 Discussion



















5 Conclusions





Abbreviations




Acknowledgments


References

































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

















5 Conclusions





Abbreviations




Acknowledgments


References

































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






5 Conclusions





Abbreviations




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

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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