Anti-inflammatory activity of flavonoids from Cayaponia tayuya roots

Journal of Ethnopharmacology 121(2009)333–337

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Journal of

Ethnopharmacology

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j e t h p h a r

m

Ethnopharmacological communication

Anti-in?ammatory activity of ?avonoids from Cayaponia tayuya roots

Silvia Aquila a ,b ,Rosa M.Giner a ,María C.Recio a ,Etile D.Spegazzini b ,JoséLuis Ríos a ,?

a Departament de Farmacologia,Facultat de Farmàcia,Universitat de València,Spain

b

Departamento de Ciencias Biológicas,Facultad de Ciencias Exactas,Universidad Nacional de La Plata,Argentina

a r t i c l e i n f o Article history:

Received 13June 2008

Received in revised form 21October 2008Accepted 2November 2008

Available online 8November 2008Keywords:

Cayaponia tayuya Cucurbitaceae Flavonoids

Anti-in?ammatory activity RAW 264.7cells Cyclooxygenase

Nitric oxide synthase

a b s t r a c t

Ethnopharmacological relevance:Taiuiáor tayuya (Cayaponia tayuya ,Cucurbitaceae)is a climbing,lig-ni?ed plant with a large swollen root that has traditionally been used as an anti-in?ammatory and anti-rheumatic agent in the folk medicine of Brazil,Peru,and Colombia.

The aim of the study:We have assayed the pharmacological properties of a ?avonoid fraction obtained from the butanol extract of Cayaponia tayuya roots using two models of topical mouse ear oedema,paying special attention to its in?uence on the induction on pro-in?ammatory enzymes and peptidic mediators.Material and methods:The in vivo experiments involved both the acute oedema induced by a single appli-cation of TPA and the subchronic in?ammation brought on by repeated applications of TPA.The effects on the induction of pro-in?ammatory enzymes and peptidic mediators in RAW 264.7macrophages were analyzed with the aid of Western blot analysis.

Results:The extract was identi?ed as a mixture of ?avonoids in which vicenin-2,spinosin,isovitexin,and a mixture of swertisin and isoswertisin were found.In acute TPA-induced oedema in mouse ears,the ?avonoid-enriched fraction (at a dose of 0.5mg/ear)inhibited the oedema by 66%(4.2±0.6mg vs.12.3±1.4mg,P <0.01)while in the subchronic model,the inhibition reached 37%at a dose of 0.5mg/ear ×7applications (7.5±0.6mg vs.11.9±1.3mg,P <0.05).When assayed in vitro ,the ?avonoid showed no tox-icity at 33.45?g/mL on RAW 264.7macrophages.Although the nitric oxide production in these cells was moderately reduced (42%)at 33.45?g/mL,the ?avonoid-enriched fraction had no effect on TNF-?pro-duction.In addition,at 22.30?g/mL,the test sample inhibited both iNOS and COX-2expression by 98%and 49%,respectively.

Conclusion:These results indicate that the anti-in?ammatory activity of ?avonoids from tayuya roots most likely stems from their inhibition of the induction of the enzymes COX-2and iNOS.

?2008Elsevier Ireland Ltd.All rights reserved.

1.Introduction

Taiuiáor tayuya (Cayaponia tayuya (Well.)Cogn.,Cucurbitaceae)is a climbing,ligni?ed plant with a large swollen root.It was ?rst recorded in the Brazilian Pharmacopoeia as an of?cial herbal drug in 1929(Brand?o et al.,2008).This document mentions the syrup “Caroba composto”obtained from tayuya roots along with other natural extracts.It has been shown to possess diverse pharmacolog-ical properties,acting as an analgesic,diuretic,anti-in?ammatory,tonic,blood puri?er and detoxi?er,and has been used in folk

Abbreviations:COX,cyclooxygenase;IL-1?,interleukin-1?;LPS,lipopolysaccha-ride;MTT,3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide;NOS,nitric oxide synthase;TNF-?,tumour necrosis factor-?;TPA,12-O -tetradecanoylphorbol 13-acetate.

?Corresponding author at:Departament de Farmacología,Facultat de Farmàcia,Universitat de València,Av.Vicent Andrés Estellés s/n,46100Burjassot,Valencia,Spain.Tel.:+34963544973;fax:+34963544498.

E-mail address:riosjl@uv.es (J.L.Ríos).medicine for treating various pathologies,including skin disorders,arthritis,and rheumatism.It is also commonly employed as a gen-eral analgesic for many conditions (Brand?o et al.,2008).Tayuya root is traditionally prepared in infusions with water,but can also be prepared by stirring 1or 2g of powdered root into juice,water,or food and taken in one cup doses two to three times daily (Taylor,2005).Ethnopharmacological studies on this root have entailed the oral administration of infusions of either the dried (10%)or fresh plant (20%)corresponding to 1or 2g/kg (Ruppelt et al.,1991),as well as assays on the activities of its chloroform (dose of extract equivalent to 2.5g of root per kg)and methanol (dose of extract equivalent to 1.0g of root per kg)extracts (Ríos et al.,1990).

In 1985,Bauer et al.identi?ed seven ?avonoids and seven cucur-bitacins from the roots of this plant while in 1990,Ríos et al.demonstrated the anti-in?ammatory activity of the chloroform and methanol extracts obtained from them.Several years later,these same researchers isolated the two major compounds from the chloroform extract,namely 23,24-dihydrocucurbitacin B and cucurbitacin R (Recio et al.,2004).Both compounds were assayed

0378-8741/$–see front matter ?2008Elsevier Ireland Ltd.All rights reserved.doi:10.1016/j.jep.2008.11.002

334S.Aquila et al./Journal of Ethnopharmacology 121(2009)333–337

and found to be anti-in?ammatory agents in various experimental types of in?ammation,including acute,chronic,systemic,topical,dermatological,and arthritic models.Their effects on the differ-ent mediators and enzymes implicated in in?ammation were also examined (Recio et al.,2004;Escandell et al.,2006,2007a,b ).

Because the effects of cucurbitacins present in the chloroform extract and tayuya roots have already been demonstrated,we chose to study the effects of the ?avonoids present in the butanol extract,which has previously been reported to possess anti-in?ammatory properties.This extract contains various cucurbitacins in glyco-sidic form;therefore,our ?rst task was to purify and separate the ?avonoids present in the extract.The ?avonoid-enriched fraction thus obtained was assayed in two experimental models of in?am-mation.Both the extract’s anti-in?ammatory effects and in?uence on the induction of pro-in?ammatory enzymes and peptidic medi-ators were analyzed and are presented herein.2.Materials and methods 2.1.Chemicals and biochemicals

All chemicals and biochemicals were purchased from Sigma-Aldrich (St.Louis,MO,USA)or Panreac (Barcelona,Spain).Silica gel and solvents for mobile phases were purchased from Merck (Darm-stadt,Germany);polyamide was obtained from Macherey-Nagel (Düren,Germany).

2.2.Preparation of the ?avonoid-enriched fraction and identi?cation of major compounds

The powdered dry roots (100g)were exhaustively extracted with chloroform (500mL),yielding 5g of solid after evaporation.The marc was then extracted with methanol (500mL),yielding 29g of yellow residue after evaporation.The methanol extract was sus-pended in water and successively extracted with chloroform,ethyl acetate,and butanol (4g).The butanol extract was then subjected to both thin-layer chromatography (TLC:EtOAc-HCOOH-HOAc-H 2O,100:11:11:27;silica gel 60Merck)and high-performance liquid chromatography (HPLC:Column LiChrospher RP-18(5m)250-4(Merck)and;eluent CH 3CN-H 2O-HOAc (18:82:1);?ow rate:1.0mL/min;detection wavelength:340nm).Thus,for the present study,the butanol extract (2g)was dissolved in methanol and mixed with silica gel 60(0.2–0.5mm).The resulting mix-ture was dried and introduced into a column with silica gel 60(0.063–0.2mm,60cm ×3.5cm)which was then eluted with mix-tures of EtOAc-MeOH.From fractions 85–108eluted with a 90:10mixture we obtained a ?avonoid-enriched residue (276mg),which was used for this study and represents 13.9%of the total butanol extract.For its part,TLC on polyamide and silica gel 60indicated the presence of four compounds,which were identi?ed through com-parisons with TLC and HPLC (Fig.1)data reported previously by Bauer et al.(1985)as vicenin-2,spinosin,isovitexin and swertisin (Fig.2).However,TLC in polyamide showed that the last compound was actually a mixture of two similar compounds.To identify them unequivocally,20mg of the ?avonoid-enriched residue were puri-?ed with the aid of vacuum liquid chromatography (5cm ×4.3cm)on MN-polyamide SC 6(<0.07mm)eluted with a mixture of MeOH-H 2O (1:2).This led to a pure compound (fraction 13–23),which was identi?ed by means of 13C NMR and through comparisons with bibliographic data (Agrawal,1989)as swertisin (5mg,Fig.2).Isoswertisin (Fig.2)was identi?ed in the mixture with swertisin through comparisons with previously published 13C NMR data (Agrawal,1989).We then tested the ?avonoid-enriched fraction with the ?ve aforementioned compounds for anti-in?ammatory

activity.

Fig.1.HPLC separation of the ?avonoid-enriched fraction from Cayaponia tayuya roots.(1)vicenin-2(t r =2.05);(2)spinosin (t r =10.72);(3)isovitexin (t r =12.59),and (4)swerticin +isoswerticin (t r =14.35).Column LiChrospher RP-18(5?m)250-4(Merck);eluent CH 3CN-H 2O-HOAc (18:82:1);?ow rate:1.0mL/min;detection wavelength:340nm.t r =retention time (min).Ratios of each ?avonoid in the mix-ture are:13.5(1):4.1(2):1(3):3.7(4).

2.3.Animals

For in vivo experiments,groups of ?ve Swiss female mice weighing 25–30g (8weeks old)from Harlan Interfauna Iberica (Barcelona,Spain)were used.All animals were fed a standard diet ad libitum .Housing conditions and all in vivo experiments were approved by the Institutional Ethics Committee of the University of Valencia (Spain)in accordance with the guidelines established by the European Union on Animal Care (CEE Council 86/609).2.4.12-O-Tetradecanoylphorbol 13-acetate (TPA)-induced ear oedema in mice

Oedema was induced through topical application of 2.5?g of TPA (Sigma-Aldrich).At the same time,the ?avonoid-enriched frac-tion (0.5mg/ear)and indomethacin (Sigma-Aldrich,0.5mg/ear)dissolved in acetone (Panreac)were also applied topically to the ears (20?L/ear,10?L in each side).After 4h,the mice were killed (cervical dislocation)and two ear punches from each animal

were

Fig.2.Flavonoids from Cayaponia tayuya roots.

S.Aquila et al./Journal of Ethnopharmacology121(2009)333–337335

taken.The swelling was assessed in terms of the mean weight increase of each ear,while inhibition of the swelling was expressed as weight reduction in comparison to the control group treated with TPA only.Details of this protocol have been described by Giner et al.(2000).

2.5.Mouse ear in?ammation induced by multiple applications of TPA

In?ammation was induced through topical application of2?g of TPA dissolved in acetone(20?L)on days1,3,5,8and10to the inner and outer surfaces of each ear with the aid of a micropipette. The?avonoid-enriched fraction(0.5mg/ear,in acetone,20?L/ear, 10?L in each side,topically)was administered twice daily for four consecutive days(8,9,10and11)while dexamethasone(Sigma-Aldrich,0.025mg/ear,in acetone,20?L/ear,10?L in each side, topically)was applied only once a day in the morning for four con-secutive days.On the last day,the test sample was applied only in the morning.The blank group was given topical applications of ace-tone in the same regimen as described above and control group was treated with TPA only.The mice were then killed(cervical disloca-tion)and two ear punches from each animal were taken.Swelling was assessed in terms of the mean weight increase of each ear while inhibition of swelling was expressed as the weight reduction in comparison with the control group.Details of the protocol have been previously described by Giner et al.(2000).

2.6.Cell culture

Murine RAW264.7macrophages were cultured in Dulbecco’s modi?ed Eagle’s medium(DMEM)supplemented with10%foetal bovine serum,100IU/mL penicillin,and100?g/mL streptomycin, all from Invitrogen(Langley,OK,USA),in an incubator at37?C in a humidi?ed atmosphere of95%air and5%CO2.Macrophages were removed from the tissue culture?ask with the aid of a cell scraper. Cells were then cultured in dishes at a density of5×105cells/mL and,when sub-con?uent,the medium was substituted by DMEM supplemented with0.5%foetal bovine serum.The cells were then subjected to the treatment described below.

2.7.Cytotoxicity

The cytotoxicity of the?avonoid-enriched fraction was mea-sured with the aid of Mosmann’s colorimetric assay(Mosmann, 1983).First,RAW264.7cells(5×105per mL)were exposed to the test fraction(?nal concentration44.6–0.5?g/mL;?nal volume 200?L)in a96-well microplate for24h.The culture medium was then eliminated and100?L per well of a0.5mg/mL solution of3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide(MTT, Sigma-Aldrich)were added.The microplates were then incu-bated at37?C until blue deposits were visible.The supernatant was eliminated and the coloured metabolite was dissolved in dimethylsulfoxide(Sigma-Aldrich,100?L per well).This reaction was performed in triplicate.Absorbance was measured at570nm with the aid of a Labsystems Multiskan MCC/340plate reader (Helsinki,Finland).

2.8.Nitric oxide production in RAW264.7macrophages

RAW264.7murine macrophages were cultured in DMEM medium containing100U/mL penicillin,100?g/mL streptomycin, and10%foetal bovine serum.Cells were removed from the tissue culture?ask with a cell scraper and resuspended until a?nal relation of5×105cells/mL in the culture medium was reached.Macrophages(RAW264.7)were then co-incubated in a96-well culture plate(200?L)with1?g/mL(?nal concentra-tion)of lipopolysaccharide(LPS,Sigma-Aldrich)at37?C for24h in the presence of the test fraction(0.5–44.6?g/mL),dexametha-sone(1.8?g/mL=5?M),or vehicle.The presence of nitrites was then determined in the culture supernatant with the aid of Griess reagent(Sigma-Aldrich).Nitrite production was assessed as the index of nitric oxide generation in the induction phase.

2.9.Western blot analysis

RAW264.7macrophages(1.5×106cells per well)were co-incubated in a6-well culture plate(1mL)with1?g/mL(?nal concentration)LPS at37?C for24h in the presence of the test frac-tion(22.30and33.45?g/mL),dexamethasone(1.8?g/mL=5?M), or vehicle(culture medium).Cellular lysates were obtained accord-ing to the methodology described by Escandell et al.(2007a).The membranes were incubated with anti-NOS-2(1:1000dilution)and anti-COX-2(1:2000dilution)polyclonal antibodies(Cayman,Ann Arbor,MI,USA).The blots were then washed and incubated with peroxidase-conjugate goat anti-rabbit immunoglobulin G(1:12000 dilution).The immunoreactive bands were visualized with a LAS 3000mini system(Fuji?lm,Tokyo,Japan).Western blot quanti?ca-tion was carried out with Multi Gauge software(Fuji?lm).

2.10.Determination of TNF-?production in RAW264.7 macrophages

RAW264.7cells(5×105cells/mL)were co-incubated in a96-well culture plate(200?L)with1?g/mL(?nal concentration)of LPS at37?C for24h in the presence of the?avonoid-enriched fraction at22.30and33.45?g/mL.The supernatants were then collected and assayed for TNF-?production with the aid of a spe-ci?c enzyme immunoassay from eBioscience(San Diego,CA,USA), used according to the manufacturer’s instructions.Details of this protocol have been previously described by Escandell et al.(2007a).

2.11.Statistics

For statistical evaluation,a one-way analysis of variance (ANOVA)was carried out followed by Dunnett’s t-test for multi-ple comparisons of unpaired data.In comparisons with the control group,values of P less than0.05were considered signi?cant.Inhibi-tion percentages(%I)were calculated from the differences between the drug-treated group and the control animal,which was treated with the in?ammatory agent only.For each group,n=5animals.

3.Results and discussion

A?avonoid-enriched fraction had previously been obtained from tayuya roots by Ríos et al.(1990),who identi?ed two com-pounds in the butanol fraction,namely vicenin-2and spinosin. These researchers went on to determine the in vivo toxicity of the different extracts as well as the anti-in?ammatory activity in a carrageenan-induced paw oedema assay of the extracts and two isolated?avonoids.The toxicity for the butanol extract was higher than2000mg/kg for both intraperitoneal and oral adminis-tration(Ríos et al.,1990).In the present study,we have examined a?avonoid-enriched fraction obtained from the anti-in?ammatory butanol extract and studied its pharmacological properties in two models of topical oedema:acute TPA-induced oedema and the sub-chronic in?ammation induced by repeated applications of TPA, both in mouse ears.This fraction represents13.8%of the total butanol extract,whereas the remaining fractions are complex mix-tures of?avonoids and cucurbitacin glycosides.

336S.Aquila et al./Journal of Ethnopharmacology 121(2009)333–337

Table 1

Effects of ?avonoid-enriched fraction from Cayaponia tayuya roots on mouse ear oedema induced by TPA.Acute ear oedema:effects of topically admin-istered ?avonoids and indomethacin at 0.5mg/ear.In?ammation induced by repeated application of TPA:effects of ?avonoid-enriched fraction (7×0.5mg/ear,2times/day)and dexamethasone (4×0.025mg/ear,1time/day)administered top-ically on days 8,9,10,and 11after challenge.Increase in ear weight in mg was calculated with respect to a group which received acetone only (blank).n =5animals.

Acute ear oedema Subchronic ear oedema W ±S.E.M.

%I W ±S.E.M.%I Control (TPA only)12.3±1.4–11.9±1.3–Flavonoid-enriched fraction

4.2±0.6**667.5±0.6*37Indomethacin (0.5mg/ear) 1.4±0.4**89–

–Dexamethasone (0.025mg/ear)

–

–

0.9±0.4**

92

Statistically signi?cant difference with respect to the control group is expressed as **P <0.01,*P <0.05(Dunnett’s t -test).Percentages of inhibition were expressed as the reduction of increase in ear weight with respect to the control group.

The test sample,identi?ed as a mixture of the ?avonoids vicenin-2,spinosin,isovitexin,swertisin,and isoswertisin (Fig.2),was ?rst studied for its anti-in?ammatory properties.In the acute model,the ?avonoid-enriched fraction at 0.5mg/ear inhib-ited the TPA-induced mouse ear oedema by 66%(4.2±0.6mg vs.12.3±1.4mg,P <0.01)whereas in the subchronic model,the reduc-tion was about 37%(7.5±0.6mg vs.11.9±1.3mg,P <0.05)after 7applications of 0.5mg/ear (Table 1).When assayed in vitro ,the test fraction at 33.45?g/mL showed no toxicity against RAW 264.7macrophages.The nitrite production in these cells was moder-ately reduced (42%)at a test fraction concentration of 33.45?g/mL,but there was no effect on TNF-?production (data not shown).In addition,at a concentration of 22.30?g/mL the ?avonoid-enriched fraction inhibited iNOS and COX-2expression by 97%and 63%,respectively (Fig.3).

Different ?avonoids have been found to be anti-in?ammatory agents due to their ability to inhibit the arachidonic acid metabolism (Middleton et al.,2000;Chi et al.,2001;Havsteen,2002).In addition,some

?avones,such as apigenin,may inhibit NOS-2induction at the level of NOS-2gene transcription while concomitantly increasing endothelial NOS-3activity (Olszanecki et al .,2002).Recently,Bas et al.(2006,2007)demonstrated that the polymethoxy?avone demethylnobiletin exhibits anti-in?ammatory activity in which the inhibition of both nitric oxide production and the arachidonate metabolism is implicated,but

Fig.3.Inhibition of COX-2and iNOS expression by the ?avonoid-enriched fraction of tayuya roots in RAW 264.7macrophages as detected by Western blot analysis.Cells were stimulated with LPS and test fraction was used at 33.45and 22.30?g/mL,and the reference drug dexamethasone at 1.8?g/mL (5?M).Figures are representative at least of ?ve similar experiments performed with similar results.Optical density (O.D.)values represent (A–B)/mm 2,A =O.D.of problem,and B =O.D.of background.Percentage of inhibition (%I )is referred to the control group minus blank.

it has no noticeable effect on enzyme expression.In contrast,at 50?M (19.4?g/mL),the compound’s methyl derivative nobiletin has been found to suppress the expression of iNOS and COX-2by 52%and 42%,respectively (Murakami et al .,2003).In our study,the ?avone inhibited the induction of both enzymes in RAW 264.7,probably acting in the same fashion as the ?avones studied and cited above.Inhibition of COX-2and iNOS expression reduces the in?ammatory response caused by the application of TPA,which is known to induce the expression of both enzymes through the activation of protein kinase C (Lin,2007)in different tissues and pro-in?ammatory cells.

Pan et al.(2006)demonstrated that acacetin (4 -methylapigenin),a structurally related compound,has properties similar to those of swertisin (7-methylapigenin 6-C-glucoside),the major constituent of this mixture,and is able to inhibit the induction of NOS and COX-2in RAW 264.7cells activated with LPS.Moreover,they demonstrated that acacetin actively inhibited the transcriptional activation of iNOS and COX-2blocking protein as well as mRNA expression of both enzymes in these cells.Finally,they found that this ?avone reduces both the translocation of the nuclear factor-?B (NF-?B)subunit and the dependent tran-scriptional activity of NF-?B.The activation of NF-?B seems to be inhibited by preventing the degradation of inhibitor-?B (I ?B).

Taken together,these data lead to the hypothesis that ?avonoids from Cayaponia tayuya may exert their anti-in?ammatory activity through the inhibition of COX and NOS induction,but further stud-ies need to be performed to determine the precise mechanism of action.

Acknowledgements

This study was supported by grant SAF2006-06726(from the Spanish Government through the Ministerio de Educación y Ciencia and FEDER).Ms Aquila thanks the University of Valencia for the grant received (beca jóvenes investigadores de países en vías de desarrollo).References

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