Research ArticleStudy of the Gastroprotective Effect of Extractsand Semipurified Fractions of Chresta martii DCand Identification of Its Principal Compounds

E S Franco1 M E B Meacutelo2 B J A Jatobaacute1 A L B D Santana3 A A R Silva4

T G Silva5 M S Nascimento3 and M B S Maia1

1Department of Physiology and Pharmacology Laboratory of Pharmacology of Bioactive ProductsFederal University of Pernambuco (UFPE) Recife PE Brazil2Department of Parasitology Laboratory of Mutagenesis and Research Center Aggeu Magalhaes Fiocruz Recife PE Brazil3Department of Antibiotics Laboratory of Chemistry of Natural Products UFPE Recife PE Brazil4Sobral Laboratory of Pharmacology Federal University of Ceara (UFC) CE Brazil5Department of Antibiotics Laboratory of Bioassays for Research on Drugs UFPE Recife PE Brazil

Correspondence should be addressed to M B S Maia mbsmufpebr

Received 29 November 2014 Accepted 3 March 2015

Academic Editor Olumayokun A Olajide

Copyright copy 2015 E S Franco et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chresta martii (Asteraceae) is a species widely used by the population of the Xingu region of Sergipe Brazil in the form of adecoction (aerial parts) for the treatment of gastrointestinal diseasesThe study aims to assess the gastroprotective activity of organicextracts and semipurified fractions and identify the principal compounds present in C martii responsible for such activity Theorganic extracts (cyclohexane ECCm ethyl acetate EACm and ethanol EECm) were obtained from the dried aerial parts (500 g)ofCmartii For evaluation of the gastroprotective activity of extracts (50 100 or 200mgkg po)male SwissWebstermice (25ndash30 g)were used which had gastric ulcers induced by indomethacin (40mgkg sc) or ethanol (02mLanimal po) Among the extractsevaluated EACm exhibited significant (119875 lt 005) gastroprotective activity in the models used The fractionation of EACm wasperformed in a silica gel column 60 eluted with the following compounds [chloroformmdashF1 yield (10)] [chloroformethyl acetate(11)mdashF2 yield (6)] [ethyl acetatemdashF3 yield (8)] and [ethylmethanol acetate (11)mdashF4 yield (5)] Of the fractions describedabove the F1 (25mgkg po) had greater gastroprotective activity (119875 lt 005) than that displayed by ranitidine (80mgkg po)in the ethanol-induced ulcer model The refractionation of F1 produced 23 subfractions and from these two yellow amorphouscompounds were obtained by recrystallization Rf 046 and 031 (ethyl acetate chloroform 5 5) The compounds isolated werecharacterized by nuclear magnetic resonance spectroscopy (1H-NMR and 13C-NMR) and identified as flavones chrysoeriol (yield043) and 3101584041015840-dimethoxyluteolin (yield 058)Conclusion Flavone 3101584041015840-dimethoxyluteolin is the principal compound presentin the species C martii and is probably responsible for gastroprotective activity observed in this species

1 Introduction

Gastric and duodenal ulcers are associated with the imbal-ance between the protective and aggressive factors ofthe gastric mucosa [1] Among the protective factors areprostaglandins nitric oxide mucus and bicarbonate [2 3]However the aggressive factors which predispose the emer-gence of gastric ulcers are the following nonsteroidal anti-inflammatory medications (NSAIDs) Helicobacter pylori

infections and the chronic use of alcoholic beverages [45] It is known that this imbalance can significantly impactcontrol of hydrochloric acid secretion by the stomach whoseproduction has hormonal and neural influences Hormonalcontrol (endocrine and paracrine) occurs in a complex man-ner via at least three different cell types enterochromaffin-like cells producing histamine G cells producing gastrin andD cells secreting somatostatin Neural control is linked tovagal cholinergic control which acts on all these cells and

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 576495 7 pageshttpdxdoiorg1011552015576495

2 Evidence-Based Complementary and Alternative Medicine

parietal cells Histamine gastrin and acetylcholine the latterreleased by the vagus directly and positively influence theproduction of HCl by parietal cells Somatostatin acts inreverse inhibiting the release of histamine and gastrin andconsequently the production of acid [6]

This pathology is the most common chronic illnessamong adults affecting 5 to 10 of the worldrsquos populationIn children from four to seven new cases of gastric ulcersare registered per year in the major pediatric centers [7]In both cases the treatment is carried out using drugswhich act by inhibiting the proton pump neutralizing theacid secretion (antacids) or blocking the histamine receptors(H2 antihistamines) [8] Although these medications canproduce serious adverse reactions including hypersensitivityarrhythmia and impotence [9] they are commonly used forthe treatment of gastric ulcers [5]

From this perspective investigations into the use ofmedicinal plants are supported by the richness of the Brazil-ian flora and the search for new antiulcerogenic agents mostnotably with regard to the identification of their principalcompounds elucidation of structure and pharmacologicalactivity (safety and efficacy) has been the target of constantresearch [10]

Chresta martii (Asteraceae) is popularly known as muri-cica In the Xingu region Sergipe Brazil this species is widelyused in the form of a decoction of aerial parts (plusmn10 gL) forthe treatment of gastrointestinal diseases [11 12] Until nowonly two studies using an experimental model of gastriculcers have been published reporting the gastroprotectiveactivity of a hydroalcoholic extract of C martii One ofthe studies identified a compound belonging to the class ofterpenes (sesquiterpene lactone) [13 14] Thus the presentstudy aimed to investigate the gastroprotective activity ofdifferent organic extracts and semipurified fractions of Cmartii in experimental models of gastric ulcers induced byindomethacin or ethanol in Swiss Webster mice and identifythe class and chemical structure of the principal compoundscoming from the product with the best gastroprotectiveactivity

2 Materials and Methods

21 Botanical Material Chresta martii (DC) H Rob wascollected in the Xingu region located in Sergipe Brazil atcoordinates ranging from minus95563 to minus95548 latitude 37940longitude and an altitude of 130 meters in January 2011Samples of thematerial collected were identified byDr NadiaRoque (Institute of Biology in the Department of Botany atthe Federal University of Bahia Brazil) and plant specimenswere deposited at the herbarium HUVA (Sobral CearaBrazil) under number 14602

22 Animal Model Male Swiss Webster mice (25ndash30 g) werekept at 22 plusmn 2∘C under a lightdark cycle of 1212 h withwater and food ad libitum Before the experiment (24 h)the animals were fed with a 5 glucose solution and waterad libitum All treatments and protocols were carried outaccording to the ldquoPractical Manual on the Ethical Use and

Care of Laboratory Animalsrdquo of the Brazilian Society ofLaboratory Animal Science (SBCAL) The experiments arein accordance with the Guidelines for the Care and Use ofLaboratory AnimalsThe research was approved and licensed(number 230760152072012-42) by theCommission of Ethicson the Use of Animals (CEUA) of the Federal University ofPernambuco Brazil

23 Preparation of Organic Extracts and Semipurified Frac-tions To obtain the organic extracts aerial parts of C martiiwere used which had been dried in a convection ovenat 50∘C and pulverized in a knife mill Subsequently thematerial (500 g) was submitted to extraction by macerationat room temperature using organic solvents of increasingpolarity cyclohexane (ECCm) ethyl acetate (EACm) and96 ethanol (EECm) at a proportion of approximately10 g100mL of solvent Each extraction cycle lasted 48 hoursusing the residue of the former to perform the next extrac-tion with three repetitions for each solvent [15] The extractswere concentrated in a rotary evaporator with reducedpressure at 50∘C and 90RPM andor lyophilized After thedetermination of yield the extracts were stored in amberbottles and kept at a temperature of minus20∘C (freezer) until themoment of their use at which time the material was dilutedin saline (09 NaCl)

The EACm extract (10 g) was submitted to fraction-ation giving rise to four semipurified fractions (FIndashF4)using silica gel column chromatography (Vetec 0063ndash020mm) eluted with chloroform [Fraction ImdashF1 yield(10)] chloroformethyl acetate (1 1) [Fraction IImdashF2 yield(6)] ethyl acetate [Fraction IIImdashF3 yield (8)] and ethylacetatemethanol (1 1) [Fraction IVmdashF4 yield (5)] Thesemipurified fractions were concentrated in a rotary evapo-rator with reduced pressure at 50∘C and 90RPM and storedin amber bottles at a temperature of minus20∘C (freezer) until themoment of their use at which time the material was dilutedin a saline solution (09 NaCl)

24 Isolation and Identification of the Principal Compound(s)of Fraction (F1) TheF1 (3 g) underwent refractionation usingsilica gel column chromatography (Vetec 0063ndash020mm)eluted with chloroformethyl acetate initially (91) with agradual increase in polarityThis procedure resulted in a totalof 23 subfractions arising from the mixing of similar samplesas verified by thin-layer chromatography (TLC) silica gel60 F254

(precoated sheets of aluminum Macherey Nagel)revealed with anisaldehydesulfuric acid The subfractionswere dried at room temperature for crystallization of thecompounds Then the residue was resuspended in acetonefor recrystallization [16] and filtered to obtain pure crystalsThese crystals were identified by nuclear magnetic resonance(NMR) spectroscopy

25 Magnetic Resonance Imaging (MRI) The spectra of 1H-and 13C-NMR were recorded using the Varian Unity Plusmodel 300MHz for 1H and 75MHz for 13C in the AnalyticalCenter of the Fundamental Chemistry Department UFPEThe chemical shifts (120575) were reported in ppm and the

Evidence-Based Complementary and Alternative Medicine 3

coupling constants were reported inHertz (Hz) Tetramethyl-silane (TMS) was used as an internal reference standard Thecrystals were solubilized in DMSO-d

6

26 Study of the Gastroprotective Effect of the Organic Extractsand Semipurified Fraction of C martii

261 Gastric Lesions Induced by Indomethacin Gastric ulcer-ations were induced in Swiss Webster mice (119899 = 6 animalsper group) held under fasting (24 h) by the administrationof indomethacin (Sigma) (40mgkg sc) The animals werepretreated with extracts (ECCm EACm or EECm) eachin doses of 50 100 or 200mgkg (po) with a positivecontrol of omeprazole (30mgkg po) or a negative controlof saline (5mLkg po) 1 h before the administration ofindomethacinA group containingmice (119899 = 3)without ulcerinduction received only saline (5mLkg po) to be used asthe default physiological group The doses of extracts werechosen taking into consideration the prior determinationof LD

50(results not presented) and by comparison with

the dose used by Silva et al [13 14] for the same plantspecies and model of ulcer The animals were euthanizedsix hours after the ulcerogenic procedure Their stomachswere removed and opened along the greater curvature Theulcer index was evaluated using the quantitative method forgauging the extent of erosion and experimental gastric ulcersas described by Szabo et al [17] The percentage of inhibitionwas calculated in relation to the saline group according to thefollowing formula inhibition = UItUIs times 100 where UItand UIs correspond to the ulcer indices of the treated andsaline groups respectively

262 Ethanol-Induced Gastropathy The gastric damage wasinduced by the administration of 999 ethanol (02mLanimal) in Swiss Webster mice (119899 = 6 animals per group)kept under fasting (24 h) [18] The animals were treated anhour before induction with EACm (50 100 or 200mgkgpo) or semipurified fractions F1 F2 F3 and F4 (50mgkgpo) or only F1 (125 25 or 50mgkg po)The control groupswere treated with ranitidine (80mgkg po) as a positivecontrol or saline (5mLkg po) as a negative control A groupcontaining mice (119899 = 3) without ulcer induction receivedsaline (5mLkg po) to be used as a physiological parametergroup All the animals were euthanized in a CO

2chamber

30min after the injurious procedure Their stomachs wereremoved and opened along the greater curvature washed insaline fixed between Petri dishes and photographed (SonyCyber-Shot Dsc-h2) at 72 dpi resolution (2816 times 2112 pixels)Hemorrhagic or ulcerative lesions were measured and com-pared to the total area of each stomach through computerizedplanimetry using the Image J program (National Institutesof Health 9000 Rockville Pike Bethesda Maryland USA)The percentage of inhibition was calculated in relation to thesaline group according to the following formula inhibition= UItUIs times 100 where UIt and UIs correspond to the indicesof ulcers in the treated and saline groups respectively [18]

0

5

10

15

20

25

30

35

40

Ulc

er in

dex

Salin

e

Om

epra

zole

(30

mg

kg)

ECCm

(50

mg

kg)

ECCm

(100

mg

kg)

ECCm

(200

mg

kg)

EACm

(50

mg

kg)

EACm

(100

mg

kg)

EACm

(200

mg

kg)

EECm

(50

mg

kg)

EECm

(100

mg

kg)

EECm

(200

mg

kg)

aa a

a

b

c c

b

d dd

Figure 1 Gastroprotective effect of cyclohexane extract of C martii(ECCm) ethyl acetate extract of C martii (EACm) and ethanolicextract of C martii (EECm) at doses of (50 100 or 200mgkgvo) on gastric lesions induced by indomethacin in mice (119899 = 6)Different letters indicateminimal significance (119875 lt 005)Bonferronitest

27 Statistical Analysis All values were expressed as mean plusmnSD For the ulcerativehemorrhagic area ANOVA followedby the Bonferroni test formultiple comparisons was usedThedifferences were considered significant when 119875 lt 005

3 Results and Discussion

The gastroprotective effect of organic extracts (ECCmEACm and EECm) on gastric lesions induced by indometha-cin is as shown in Figure 1

As can be observed the EACmwas the extract which con-ferred the best gastroprotective activityThe activity observedin the group treated with EACm (50mgkg) was significantly(119875 lt 005) different from the control group (saline) andcomparable (119875 gt 005) to that observed in the grouptreated with omeprazole (30mgkg po) A previous studyusing a hydroalcoholic extract of aerial parts of C martii(100 or 400mgkg po) showed significant gastroprotectiveactivity in an ulcer model induced by indomethacin withinhibition indices of 5466 and 8130 respectively [14] Ourresults demonstrate superior gastroprotective activity withEACm (100 or 200mgkg po) (7989 and 8083 resp)taking into consideration the fact that the second dose ofEACm was 50 less than the highest dose used by Silva etal [14] The best performance observed in our work was dueto the extractive process and choice of solvent These twofactors favored the best extraction of substances present in Cmartii resulting in significant gastroprotective activity Theability of indomethacin to induce ulcers experimentally andin humans has been well documented in the literature [19]because it is a potent inhibitor of cyclooxygenases preventing

4 Evidence-Based Complementary and Alternative Medicine

0

10

20

30

40

50

60

70

80

d

ca

b b

Ulc

er in

dex

Salin

e

Rani

tidin

e (80

mg

kg)

EACm

(50

mg

kg)

EACm

(100

mg

kg)

EACm

(200

mg

kg)

Figure 2 Gastroprotective effect of an ethyl acetate extract of Cmartii (EACm) (50 100 or 200mgkg vo) in ethanol-inducedgastric lesions in mice (119899 = 6) Different letters indicate minimalsignificance (119875 lt 005) Bonferroni test

the biosynthesis of prostaglandins [20] The reduction inbiosynthesis of prostanoids would result in decreased resis-tance of the gastric mucosa to the action of HCl and pepsin asit induces an imbalance between the protective and aggressivefactors of the mucosa [1] taking into account the previousknowledge of the mechanism of action of indomethacin onthe gastric mucosa The likely mechanism of action assignedto EACm can be deduced to involve the biosynthesis ofprostaglandins or mimetic action the same as that whichoccurs with synthetic analogues of PGE1 (eg misoprostol)This mimetic effect can act through stimulation of secretionof mucus and bicarbonate andor increased blood flow to thestomach

Similar to what was verified with the model of gas-tric injury induced by indomethacin EACm (50mgkgpo) also presented a cytoprotective effect against ethanol-induced gastric lesions The gastroprotective effect was dose-dependent and in all doses tested superior to that verifiedwith ranitidine (Figure 2)

Ethanol-induced gastric lesions involve different mech-anisms such as a reduction in bicarbonate secretion areduction in mucus production damage to gastric bloodflow and direct injury to mucosal cells [21 22] Theselesions are associated with the excessive production of freeradicals which attack essential cellular constituents such asnucleic acids proteins and lipids [23] The increase of thecontent of lipid peroxides and oxygen-derived free radicalsresults in significant changes at the cellular level and causesdamage to membranes cell death exfoliation and epithelialerosion [21] Studies have demonstrated the presence offlavonoids in species belonging to the family Asteraceaeand this chemical class has been shown to have importantantioxidant properties This could be one of the forms of

action of the compounds present in EACm in providingsignificant cytoprotection given that free radicals are one ofthe harmful factors of the gastric mucosa induced by ethanol

Evaluation of the semipurified fractions (F1 F2 F3 andF4) (50mgkg po) against the ethanol-induced ulcer modelrevealed that animals treated with F1 and F2 showed lowerrates of ulcers when compared to the ranitidine controlHowever when compared with each other F1 presented asignificantly lower ulcer index (Figure 3(a)) Additionally thegastroprotective effect presented by F1 was dose-dependent(Figure 3(b))

After verification of the significant gastroprotective activ-ity of F1 it was refractioned giving rise to 23 subfractionsin which the presence of bands was determined throughTLC revealing anisaldehydesulfuric acid characteristics offlavonoids (yellow) steroids (lilac) and terpenoids (blue)(Figure 4) The existence of a larger number of bands withcolors ranging from orange to yellow can be seen Two puresubstances F11 (Rf 031) and F12 (Rf 048) were recrystal-lized showing a yellow amorphous physical appearance

The NMR (1H and 13C) presented peaks from which itwas possible to calculate the chemical shift (120575) in ppm andthe coupling constants in Hertz (Table 1)

From the interpretation of the spectra two flavones wereidentified chrysoeriol (F11) and 3101584041015840-dimethoxyluteolin(F12) (Figures 5(a) and 5(b)) The yield of 3101584041015840-dimethoxyluteolin was 058 and was higher than thatobserved for chrysoeriol at 043 It was verified in thisstudy that the principal compounds present in EACm areflavones

This study is a pioneer in the identification of two flavonesof Chresta martii According to Van and Lea [24] flavonespresent at least four important physiological mechanismsthey (1) bind to enzymes in the cell membrane (2) bindto heavy metal ions (3) participate in electron transfer ofenzymatic systems and (4) sequester free radicals

The flavone (3101584041015840-dimethoxyluteolin) among otherflavonoids was assessed as to its gastroprotective and anti-inflammatory potential (in different experimental modelsof gastric ulcers and inflammation using rodents) Themechanism of gastroprotective action proposed for thesecompounds has been reported to be the metabolic activationof arachidonic acid via cyclooxygenase activation leadingto biosynthesis of prostaglandins such as PGE2 and PGI2both with important protective function of the gastricmucosa The compounds were furthermore able to inhibitthe activation of 5-lipoxygenase (leukotrienes) which isan important inflammatory mediator besides acting in thesequestration of free radicals These results have given rise tosome patents by Yoo et al [25] (EPO 004541) Yoo et al [26](US 6025387) and Yoo et al [27] (EPO 0915864B1) SimilarlySadik et al [28] verified potent antioxidant and inhibitoryactivity of lipoxygenase in a culture of rabbit reticulocyteswhen different flavonoids were used including luteolinwhose structure is very similar to 3101584041015840-dimethoxyluteolin

However the fraction (F1) is a mixture (terpenesflavonoids and steroids) and these compounds can act

Evidence-Based Complementary and Alternative Medicine 5

0

10

20

30

40

50

60

70

80

a

b

cd

ef

Ulc

er in

dex

Salin

e (5

mL

kg)

Rani

tidin

e (80

mg

kg)

F1(50

mg

kg)

F2(50

mg

kg)

F3(50

mg

kg)

F4(50

mg

kg)

(a)

0

10

20

30

40

50

60

70

80

a

bc

d d

Ulc

er in

dex

Salin

e (5

mL

kg)

Rani

tidin

e (80

mg

kg)

F1(125

mg

kg)

F1(25

mg

kg)

F1(50

mg

kg)

(b)

Figure 3 Gastroprotective effect of fractions (F1 F2 F3 and F4) (50mgkg vo) (a) and the fraction (F1) in isolation (50 25 or 125mgkgvo) (b) obtained from the ethyl acetate extract of C martii on ethanol-induced gastric lesions in mice (119899 = 6) Different letters indicateminimal significance (119875 lt 005) Bonferroni test

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Figure 4 Chromatographic profile by TLC of the eluded fraction (F1) (chloroform ethyl acetate 8 2) revealed with anisaldehydesulfuricacid

HO O

O

OMe

OH

OH

(a)

O

O

HO

OMe

OMe

OH

(b)

Figure 5 Chemical structure of chrysoeriol (a) and 3101584041015840-dimethoxyluteolin (b) isolated from Chresta martii

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Data of 1H- and 13C-NMR of flavonoids (F11 and F12) isolated from the F1 of C martii (300MHz) in DMSO-d6

Flavonoid (F11) Flavonoid (F12)Positions Chrysoeriol 3101584041015840-Dimethoxyluteolin

1205751H (119869 Hz) 120575

13C 1205751H (119869 Hz) 120575

13C2 1637 16393 688 (s) 1041 697 (s) 10604 1818 18155 1620 16306 619 (d 18) 980 621 (d 21) 9827 1642 16478 652 (d 18) 940 653 (d 18) 9409 1574 157910 1040 104011015840 1210 120621015840 756 (d 24) 1100 756 (d 21) 109831015840 1481 148041015840 1508 151751015840 694 (dd 84 24) 1160 713 (d 84) 116261015840 755 (dd 84 24) 1220 768 (dd 96 18) 12165-OH 12978-OH 107941015840-OH 99631015840-OCH3 389 (s) 560 385 (s) 56041015840-OCH3 388 (s) 560

synergistically with regard to the gastroprotective potentialpresented here possibly involving other mechanisms ofaction beyond those cited by Yoo et al [25ndash27] such asactivation of alpha-2 adrenergic receptors as proposed bySilva et al [13] when evaluating the hydroalcoholic extract ofC martii in an ethanol-induced ulcer model

There is a scarcity of studies that assess the phytochemicalprofile at the level of identifying the principal compoundsfor C martii Such work was pioneered by Silva et al[13 14] in identifying two sesquiterpene lactones directlyfrom a hydroalcoholic extract The present study docu-ments for the first time identification of two flavones (3101584041015840-dimethoxyluteolin and chrysoeriol) from the genus Chrestawith relevant evidence for them being the agents responsiblefor the gastroprotective effect seen inCmartii given that thisinvestigation occurred through direct monitoring betweengastroprotective pharmacological activity and processes ofextraction purification and identification of compoundsFinally the results presented here provide rational supportfor the purported use of the species in the treatment of gas-trointestinal disorders according to ethnopharmacologicalinformation

Conflict of Interests

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

References

[1] HD S Falcao J A Leite JM Barbosa-Filho et al ldquoGastric andduodenal antiulcer activity of alkaloids a reviewrdquoMolecules vol13 no 12 pp 3198ndash3223 2008

[2] C E P Araujo R F R Rodrigues and A F OliveiraldquoAnalise preliminar da atividade antiulcerogenica do extratohidroalcoolico de Solanum cernuum Vellrdquo Acta FarmaceuticaBonaerense vol 21 no 4 pp 283ndash286 2002

[3] R S Donatini T Ishikawa S B M Barros and E M BacchildquoAtividade antiulcera e antioxidante do extrato de folhas deSyzygium jambos (L) Alston (Myrtaceae)rdquo Revista Brasileira deFarmacognosia vol 19 no 1 pp 89ndash94 2009

[4] A B A De Almeida A Luiz-Ferreira M Cola et al ldquoAnti-ulcerogenic mechanisms of the sesquiterpene lactoneonopordopicrin-enriched fraction from Arctium lappa L(Asteraceae) role of somatostatin gastrin and endogenoussulfhydryls and nitric oxiderdquo Journal of Medicinal Food vol 15no 4 pp 378ndash383 2012

[5] L C Ddine C C Ddine C C R Rodrigues V R Kirsten andE Colpo ldquoFactors associated with chronic gastritis in patientswith presence and absence of Helicobacter pylorirdquo ArquivosBrasileiros de Cirgia Digestiva vol 25 no 2 pp 96ndash100 2012

[6] G Tobin D Giglio and O Lundgren ldquoMuscarinic receptorsubtypes in the alimentary tractrdquo Journal of Physiology andPharmacology vol 60 no 1 pp 3ndash21 2009

[7] A S T Carvalho ldquoUlcera pepticardquo Jornal de Pediatria vol 76no 2 pp 127ndash134 2000

[8] M W Mulholland and H T Debas ldquoRecent advances in thetreatment of duodenal ulcer disease A surgical perspectiverdquoTheWestern Journal of Medicine vol 147 no 3 pp 301ndash308 1987

Evidence-Based Complementary and Alternative Medicine 7

[9] P Malfertheiner F K Chan and K E McColl ldquoPeptic ulcerdiseaserdquoThe Lancet vol 374 no 9699 pp 1449ndash1461 2009

[10] Agencia Nacional de Vigilancia Sanitaria Formulario deFitoterapicos da Farmacopeia Brasileira ANVISA Rio deJaneiro Brzil 2011

[11] C F C B R de Almeida T C de Lima E Silva E L C deAmorim M B D S Maia and U P de Albuquerque ldquoLifestrategy and chemical composition as predictors of the selectionof medicinal plants from the caatinga (Northeast Brazil)rdquoJournal of Arid Environments vol 62 no 1 pp 127ndash142 2005

[12] C D F C B R Almeida E L C de Amorim U P de Albu -querque andM B S Maia ldquoMedicinal plants popularly used inthe Xingo regionmdasha semi-arid location in Northeastern BrazilrdquoJournal of Ethnobiology and Ethnomedicine vol 2 article 152006

[13] AA R SilvaMMarques BezerraHVasconcelosChaves et alldquoProtective effect of Chresta martii extract on ethanol-inducedgastropathy depends on alpha-2 adrenoceptors pathways butnot on nitric oxide prostaglandins or opioidsrdquo Journal ofEthnopharmacology vol 142 no 1 pp 206ndash212 2012

[14] A A R Silva M M Bezerra H V Chaves et al ldquoProtectiveeffect of Chresta martii extract against indomethacin-inducedgastric lesions inmicerdquo Journal of Natural Medicines vol 67 no1 pp 143ndash151 2013

[15] J Singh ldquoMaceration percolation and infusion techniques forthe extraction of medicinal and aromatic plantsrdquo in ExtractionTechnologies for Medicinal and Aromatic Plants p 259 UnitedNations Industrial Development Organization and the Interna-tional Centre for Science and High Technology Triseste Italy2008

[16] A F Costa Farmacognosia vol 2 Fundacao Calouste Gul-benkian Lisbon Portugal 4th edition 1994

[17] S Szabo J S Trier A Brown J Schnoor H D Homanand J C Bradford ldquoA quantitative method for assessing theextent of experimental gastric erosions and ulcersrdquo Journal ofPharmacological Methods vol 13 no 1 pp 59ndash66 1985

[18] Y Morimoto K Shimohara S Oshima and T SukamotoldquoEffects of the new anti-ulcer agent KB-5492 on experimentalgastric mucosal lesions and gastric mucosal defensive factorsas compared to those of teprenone and cimetidinerdquo JapaneseJournal of Pharmacology vol 57 no 4 pp 495ndash505 1991

[19] F Borrelli and A A Izzo ldquoThe plant kingdom as a source ofanti-ulcer remediesrdquo Phytotherapy Research vol 14 no 8 pp581ndash591 2000

[20] J R Vane ldquoInhibition of prostaglandin synthesis as a mecha-nism of action for aspirin-like drugsrdquo Nature New Biology vol231 no 25 pp 232ndash235 1971

[21] F M Birdane M Cemek Y O Birdane I Gulcin and EBuyukokuroglu ldquoBeneficial effects of Foeniculum vulgare onethanol-induced acute gastric mucosal injury in ratsrdquo WorldJournal of Gastroenterology vol 13 no 4 pp 607ndash611 2007

[22] E Marhuenda M J Martin and C A de La Lastra ldquoAntiul-cerogenic activity of aescine in different experimental modelsrdquoPhytotherapy Research vol 7 no 1 pp 13ndash16 1993

[23] C la-Casa I Villegas C Alarcon de la Lastra V Motilva andM J Martın Calero ldquoEvidence for protective and antioxidantproperties of rutin a natural flavone against ethanol inducedgastric lesionsrdquo Journal of Ethnopharmacology vol 71 no 1-2pp 45ndash53 2000

[24] S C F Van and P J Lea The Biochemistry of Plant PhenolicsProceedings of the Phytochemical Society of Europe OxfordUniversity Press Oxford UK 1985

[25] M Yoo M W Son I K Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 004541 1998

[26] M Yoo M W Son I Y Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo US Patent no 6025387 2000

[27] M Yoo M W Son I Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 0915864 B104092002

[28] C D Sadik H Sies and T Schewe ldquoInhibition of 15-lipoxygen-ases by flavonoids structure-activity relations and mode ofactionrdquo Biochemical Pharmacology vol 65 no 5 pp 773ndash7812003

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Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

parietal cells Histamine gastrin and acetylcholine the latterreleased by the vagus directly and positively influence theproduction of HCl by parietal cells Somatostatin acts inreverse inhibiting the release of histamine and gastrin andconsequently the production of acid [6]

This pathology is the most common chronic illnessamong adults affecting 5 to 10 of the worldrsquos populationIn children from four to seven new cases of gastric ulcersare registered per year in the major pediatric centers [7]In both cases the treatment is carried out using drugswhich act by inhibiting the proton pump neutralizing theacid secretion (antacids) or blocking the histamine receptors(H2 antihistamines) [8] Although these medications canproduce serious adverse reactions including hypersensitivityarrhythmia and impotence [9] they are commonly used forthe treatment of gastric ulcers [5]

From this perspective investigations into the use ofmedicinal plants are supported by the richness of the Brazil-ian flora and the search for new antiulcerogenic agents mostnotably with regard to the identification of their principalcompounds elucidation of structure and pharmacologicalactivity (safety and efficacy) has been the target of constantresearch [10]

Chresta martii (Asteraceae) is popularly known as muri-cica In the Xingu region Sergipe Brazil this species is widelyused in the form of a decoction of aerial parts (plusmn10 gL) forthe treatment of gastrointestinal diseases [11 12] Until nowonly two studies using an experimental model of gastriculcers have been published reporting the gastroprotectiveactivity of a hydroalcoholic extract of C martii One ofthe studies identified a compound belonging to the class ofterpenes (sesquiterpene lactone) [13 14] Thus the presentstudy aimed to investigate the gastroprotective activity ofdifferent organic extracts and semipurified fractions of Cmartii in experimental models of gastric ulcers induced byindomethacin or ethanol in Swiss Webster mice and identifythe class and chemical structure of the principal compoundscoming from the product with the best gastroprotectiveactivity

2 Materials and Methods

21 Botanical Material Chresta martii (DC) H Rob wascollected in the Xingu region located in Sergipe Brazil atcoordinates ranging from minus95563 to minus95548 latitude 37940longitude and an altitude of 130 meters in January 2011Samples of thematerial collected were identified byDr NadiaRoque (Institute of Biology in the Department of Botany atthe Federal University of Bahia Brazil) and plant specimenswere deposited at the herbarium HUVA (Sobral CearaBrazil) under number 14602

22 Animal Model Male Swiss Webster mice (25ndash30 g) werekept at 22 plusmn 2∘C under a lightdark cycle of 1212 h withwater and food ad libitum Before the experiment (24 h)the animals were fed with a 5 glucose solution and waterad libitum All treatments and protocols were carried outaccording to the ldquoPractical Manual on the Ethical Use and

Care of Laboratory Animalsrdquo of the Brazilian Society ofLaboratory Animal Science (SBCAL) The experiments arein accordance with the Guidelines for the Care and Use ofLaboratory AnimalsThe research was approved and licensed(number 230760152072012-42) by theCommission of Ethicson the Use of Animals (CEUA) of the Federal University ofPernambuco Brazil

23 Preparation of Organic Extracts and Semipurified Frac-tions To obtain the organic extracts aerial parts of C martiiwere used which had been dried in a convection ovenat 50∘C and pulverized in a knife mill Subsequently thematerial (500 g) was submitted to extraction by macerationat room temperature using organic solvents of increasingpolarity cyclohexane (ECCm) ethyl acetate (EACm) and96 ethanol (EECm) at a proportion of approximately10 g100mL of solvent Each extraction cycle lasted 48 hoursusing the residue of the former to perform the next extrac-tion with three repetitions for each solvent [15] The extractswere concentrated in a rotary evaporator with reducedpressure at 50∘C and 90RPM andor lyophilized After thedetermination of yield the extracts were stored in amberbottles and kept at a temperature of minus20∘C (freezer) until themoment of their use at which time the material was dilutedin saline (09 NaCl)

The EACm extract (10 g) was submitted to fraction-ation giving rise to four semipurified fractions (FIndashF4)using silica gel column chromatography (Vetec 0063ndash020mm) eluted with chloroform [Fraction ImdashF1 yield(10)] chloroformethyl acetate (1 1) [Fraction IImdashF2 yield(6)] ethyl acetate [Fraction IIImdashF3 yield (8)] and ethylacetatemethanol (1 1) [Fraction IVmdashF4 yield (5)] Thesemipurified fractions were concentrated in a rotary evapo-rator with reduced pressure at 50∘C and 90RPM and storedin amber bottles at a temperature of minus20∘C (freezer) until themoment of their use at which time the material was dilutedin a saline solution (09 NaCl)

24 Isolation and Identification of the Principal Compound(s)of Fraction (F1) TheF1 (3 g) underwent refractionation usingsilica gel column chromatography (Vetec 0063ndash020mm)eluted with chloroformethyl acetate initially (91) with agradual increase in polarityThis procedure resulted in a totalof 23 subfractions arising from the mixing of similar samplesas verified by thin-layer chromatography (TLC) silica gel60 F254

(precoated sheets of aluminum Macherey Nagel)revealed with anisaldehydesulfuric acid The subfractionswere dried at room temperature for crystallization of thecompounds Then the residue was resuspended in acetonefor recrystallization [16] and filtered to obtain pure crystalsThese crystals were identified by nuclear magnetic resonance(NMR) spectroscopy

25 Magnetic Resonance Imaging (MRI) The spectra of 1H-and 13C-NMR were recorded using the Varian Unity Plusmodel 300MHz for 1H and 75MHz for 13C in the AnalyticalCenter of the Fundamental Chemistry Department UFPEThe chemical shifts (120575) were reported in ppm and the

Evidence-Based Complementary and Alternative Medicine 3

coupling constants were reported inHertz (Hz) Tetramethyl-silane (TMS) was used as an internal reference standard Thecrystals were solubilized in DMSO-d

6

26 Study of the Gastroprotective Effect of the Organic Extractsand Semipurified Fraction of C martii

261 Gastric Lesions Induced by Indomethacin Gastric ulcer-ations were induced in Swiss Webster mice (119899 = 6 animalsper group) held under fasting (24 h) by the administrationof indomethacin (Sigma) (40mgkg sc) The animals werepretreated with extracts (ECCm EACm or EECm) eachin doses of 50 100 or 200mgkg (po) with a positivecontrol of omeprazole (30mgkg po) or a negative controlof saline (5mLkg po) 1 h before the administration ofindomethacinA group containingmice (119899 = 3)without ulcerinduction received only saline (5mLkg po) to be used asthe default physiological group The doses of extracts werechosen taking into consideration the prior determinationof LD

50(results not presented) and by comparison with

the dose used by Silva et al [13 14] for the same plantspecies and model of ulcer The animals were euthanizedsix hours after the ulcerogenic procedure Their stomachswere removed and opened along the greater curvature Theulcer index was evaluated using the quantitative method forgauging the extent of erosion and experimental gastric ulcersas described by Szabo et al [17] The percentage of inhibitionwas calculated in relation to the saline group according to thefollowing formula inhibition = UItUIs times 100 where UItand UIs correspond to the ulcer indices of the treated andsaline groups respectively

262 Ethanol-Induced Gastropathy The gastric damage wasinduced by the administration of 999 ethanol (02mLanimal) in Swiss Webster mice (119899 = 6 animals per group)kept under fasting (24 h) [18] The animals were treated anhour before induction with EACm (50 100 or 200mgkgpo) or semipurified fractions F1 F2 F3 and F4 (50mgkgpo) or only F1 (125 25 or 50mgkg po)The control groupswere treated with ranitidine (80mgkg po) as a positivecontrol or saline (5mLkg po) as a negative control A groupcontaining mice (119899 = 3) without ulcer induction receivedsaline (5mLkg po) to be used as a physiological parametergroup All the animals were euthanized in a CO

2chamber

30min after the injurious procedure Their stomachs wereremoved and opened along the greater curvature washed insaline fixed between Petri dishes and photographed (SonyCyber-Shot Dsc-h2) at 72 dpi resolution (2816 times 2112 pixels)Hemorrhagic or ulcerative lesions were measured and com-pared to the total area of each stomach through computerizedplanimetry using the Image J program (National Institutesof Health 9000 Rockville Pike Bethesda Maryland USA)The percentage of inhibition was calculated in relation to thesaline group according to the following formula inhibition= UItUIs times 100 where UIt and UIs correspond to the indicesof ulcers in the treated and saline groups respectively [18]

0

5

10

15

20

25

30

35

40

Ulc

er in

dex

Salin

e

Om

epra

zole

(30

mg

kg)

ECCm

(50

mg

kg)

ECCm

(100

mg

kg)

ECCm

(200

mg

kg)

EACm

(50

mg

kg)

EACm

(100

mg

kg)

EACm

(200

mg

kg)

EECm

(50

mg

kg)

EECm

(100

mg

kg)

EECm

(200

mg

kg)

aa a

a

b

c c

b

d dd

Figure 1 Gastroprotective effect of cyclohexane extract of C martii(ECCm) ethyl acetate extract of C martii (EACm) and ethanolicextract of C martii (EECm) at doses of (50 100 or 200mgkgvo) on gastric lesions induced by indomethacin in mice (119899 = 6)Different letters indicateminimal significance (119875 lt 005)Bonferronitest

27 Statistical Analysis All values were expressed as mean plusmnSD For the ulcerativehemorrhagic area ANOVA followedby the Bonferroni test formultiple comparisons was usedThedifferences were considered significant when 119875 lt 005

3 Results and Discussion

The gastroprotective effect of organic extracts (ECCmEACm and EECm) on gastric lesions induced by indometha-cin is as shown in Figure 1

As can be observed the EACmwas the extract which con-ferred the best gastroprotective activityThe activity observedin the group treated with EACm (50mgkg) was significantly(119875 lt 005) different from the control group (saline) andcomparable (119875 gt 005) to that observed in the grouptreated with omeprazole (30mgkg po) A previous studyusing a hydroalcoholic extract of aerial parts of C martii(100 or 400mgkg po) showed significant gastroprotectiveactivity in an ulcer model induced by indomethacin withinhibition indices of 5466 and 8130 respectively [14] Ourresults demonstrate superior gastroprotective activity withEACm (100 or 200mgkg po) (7989 and 8083 resp)taking into consideration the fact that the second dose ofEACm was 50 less than the highest dose used by Silva etal [14] The best performance observed in our work was dueto the extractive process and choice of solvent These twofactors favored the best extraction of substances present in Cmartii resulting in significant gastroprotective activity Theability of indomethacin to induce ulcers experimentally andin humans has been well documented in the literature [19]because it is a potent inhibitor of cyclooxygenases preventing

4 Evidence-Based Complementary and Alternative Medicine

0

10

20

30

40

50

60

70

80

d

ca

b b

Ulc

er in

dex

Salin

e

Rani

tidin

e (80

mg

kg)

EACm

(50

mg

kg)

EACm

(100

mg

kg)

EACm

(200

mg

kg)

Figure 2 Gastroprotective effect of an ethyl acetate extract of Cmartii (EACm) (50 100 or 200mgkg vo) in ethanol-inducedgastric lesions in mice (119899 = 6) Different letters indicate minimalsignificance (119875 lt 005) Bonferroni test

the biosynthesis of prostaglandins [20] The reduction inbiosynthesis of prostanoids would result in decreased resis-tance of the gastric mucosa to the action of HCl and pepsin asit induces an imbalance between the protective and aggressivefactors of the mucosa [1] taking into account the previousknowledge of the mechanism of action of indomethacin onthe gastric mucosa The likely mechanism of action assignedto EACm can be deduced to involve the biosynthesis ofprostaglandins or mimetic action the same as that whichoccurs with synthetic analogues of PGE1 (eg misoprostol)This mimetic effect can act through stimulation of secretionof mucus and bicarbonate andor increased blood flow to thestomach

Similar to what was verified with the model of gas-tric injury induced by indomethacin EACm (50mgkgpo) also presented a cytoprotective effect against ethanol-induced gastric lesions The gastroprotective effect was dose-dependent and in all doses tested superior to that verifiedwith ranitidine (Figure 2)

Ethanol-induced gastric lesions involve different mech-anisms such as a reduction in bicarbonate secretion areduction in mucus production damage to gastric bloodflow and direct injury to mucosal cells [21 22] Theselesions are associated with the excessive production of freeradicals which attack essential cellular constituents such asnucleic acids proteins and lipids [23] The increase of thecontent of lipid peroxides and oxygen-derived free radicalsresults in significant changes at the cellular level and causesdamage to membranes cell death exfoliation and epithelialerosion [21] Studies have demonstrated the presence offlavonoids in species belonging to the family Asteraceaeand this chemical class has been shown to have importantantioxidant properties This could be one of the forms of

action of the compounds present in EACm in providingsignificant cytoprotection given that free radicals are one ofthe harmful factors of the gastric mucosa induced by ethanol

Evaluation of the semipurified fractions (F1 F2 F3 andF4) (50mgkg po) against the ethanol-induced ulcer modelrevealed that animals treated with F1 and F2 showed lowerrates of ulcers when compared to the ranitidine controlHowever when compared with each other F1 presented asignificantly lower ulcer index (Figure 3(a)) Additionally thegastroprotective effect presented by F1 was dose-dependent(Figure 3(b))

After verification of the significant gastroprotective activ-ity of F1 it was refractioned giving rise to 23 subfractionsin which the presence of bands was determined throughTLC revealing anisaldehydesulfuric acid characteristics offlavonoids (yellow) steroids (lilac) and terpenoids (blue)(Figure 4) The existence of a larger number of bands withcolors ranging from orange to yellow can be seen Two puresubstances F11 (Rf 031) and F12 (Rf 048) were recrystal-lized showing a yellow amorphous physical appearance

The NMR (1H and 13C) presented peaks from which itwas possible to calculate the chemical shift (120575) in ppm andthe coupling constants in Hertz (Table 1)

From the interpretation of the spectra two flavones wereidentified chrysoeriol (F11) and 3101584041015840-dimethoxyluteolin(F12) (Figures 5(a) and 5(b)) The yield of 3101584041015840-dimethoxyluteolin was 058 and was higher than thatobserved for chrysoeriol at 043 It was verified in thisstudy that the principal compounds present in EACm areflavones

This study is a pioneer in the identification of two flavonesof Chresta martii According to Van and Lea [24] flavonespresent at least four important physiological mechanismsthey (1) bind to enzymes in the cell membrane (2) bindto heavy metal ions (3) participate in electron transfer ofenzymatic systems and (4) sequester free radicals

The flavone (3101584041015840-dimethoxyluteolin) among otherflavonoids was assessed as to its gastroprotective and anti-inflammatory potential (in different experimental modelsof gastric ulcers and inflammation using rodents) Themechanism of gastroprotective action proposed for thesecompounds has been reported to be the metabolic activationof arachidonic acid via cyclooxygenase activation leadingto biosynthesis of prostaglandins such as PGE2 and PGI2both with important protective function of the gastricmucosa The compounds were furthermore able to inhibitthe activation of 5-lipoxygenase (leukotrienes) which isan important inflammatory mediator besides acting in thesequestration of free radicals These results have given rise tosome patents by Yoo et al [25] (EPO 004541) Yoo et al [26](US 6025387) and Yoo et al [27] (EPO 0915864B1) SimilarlySadik et al [28] verified potent antioxidant and inhibitoryactivity of lipoxygenase in a culture of rabbit reticulocyteswhen different flavonoids were used including luteolinwhose structure is very similar to 3101584041015840-dimethoxyluteolin

However the fraction (F1) is a mixture (terpenesflavonoids and steroids) and these compounds can act

Evidence-Based Complementary and Alternative Medicine 5

0

10

20

30

40

50

60

70

80

a

b

cd

ef

Ulc

er in

dex

Salin

e (5

mL

kg)

Rani

tidin

e (80

mg

kg)

F1(50

mg

kg)

F2(50

mg

kg)

F3(50

mg

kg)

F4(50

mg

kg)

(a)

0

10

20

30

40

50

60

70

80

a

bc

d d

Ulc

er in

dex

Salin

e (5

mL

kg)

Rani

tidin

e (80

mg

kg)

F1(125

mg

kg)

F1(25

mg

kg)

F1(50

mg

kg)

(b)

Figure 3 Gastroprotective effect of fractions (F1 F2 F3 and F4) (50mgkg vo) (a) and the fraction (F1) in isolation (50 25 or 125mgkgvo) (b) obtained from the ethyl acetate extract of C martii on ethanol-induced gastric lesions in mice (119899 = 6) Different letters indicateminimal significance (119875 lt 005) Bonferroni test

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Figure 4 Chromatographic profile by TLC of the eluded fraction (F1) (chloroform ethyl acetate 8 2) revealed with anisaldehydesulfuricacid

HO O

O

OMe

OH

OH

(a)

O

O

HO

OMe

OMe

OH

(b)

Figure 5 Chemical structure of chrysoeriol (a) and 3101584041015840-dimethoxyluteolin (b) isolated from Chresta martii

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Data of 1H- and 13C-NMR of flavonoids (F11 and F12) isolated from the F1 of C martii (300MHz) in DMSO-d6

Flavonoid (F11) Flavonoid (F12)Positions Chrysoeriol 3101584041015840-Dimethoxyluteolin

1205751H (119869 Hz) 120575

13C 1205751H (119869 Hz) 120575

13C2 1637 16393 688 (s) 1041 697 (s) 10604 1818 18155 1620 16306 619 (d 18) 980 621 (d 21) 9827 1642 16478 652 (d 18) 940 653 (d 18) 9409 1574 157910 1040 104011015840 1210 120621015840 756 (d 24) 1100 756 (d 21) 109831015840 1481 148041015840 1508 151751015840 694 (dd 84 24) 1160 713 (d 84) 116261015840 755 (dd 84 24) 1220 768 (dd 96 18) 12165-OH 12978-OH 107941015840-OH 99631015840-OCH3 389 (s) 560 385 (s) 56041015840-OCH3 388 (s) 560

synergistically with regard to the gastroprotective potentialpresented here possibly involving other mechanisms ofaction beyond those cited by Yoo et al [25ndash27] such asactivation of alpha-2 adrenergic receptors as proposed bySilva et al [13] when evaluating the hydroalcoholic extract ofC martii in an ethanol-induced ulcer model

There is a scarcity of studies that assess the phytochemicalprofile at the level of identifying the principal compoundsfor C martii Such work was pioneered by Silva et al[13 14] in identifying two sesquiterpene lactones directlyfrom a hydroalcoholic extract The present study docu-ments for the first time identification of two flavones (3101584041015840-dimethoxyluteolin and chrysoeriol) from the genus Chrestawith relevant evidence for them being the agents responsiblefor the gastroprotective effect seen inCmartii given that thisinvestigation occurred through direct monitoring betweengastroprotective pharmacological activity and processes ofextraction purification and identification of compoundsFinally the results presented here provide rational supportfor the purported use of the species in the treatment of gas-trointestinal disorders according to ethnopharmacologicalinformation

Conflict of Interests

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

References

[1] HD S Falcao J A Leite JM Barbosa-Filho et al ldquoGastric andduodenal antiulcer activity of alkaloids a reviewrdquoMolecules vol13 no 12 pp 3198ndash3223 2008

[2] C E P Araujo R F R Rodrigues and A F OliveiraldquoAnalise preliminar da atividade antiulcerogenica do extratohidroalcoolico de Solanum cernuum Vellrdquo Acta FarmaceuticaBonaerense vol 21 no 4 pp 283ndash286 2002

[3] R S Donatini T Ishikawa S B M Barros and E M BacchildquoAtividade antiulcera e antioxidante do extrato de folhas deSyzygium jambos (L) Alston (Myrtaceae)rdquo Revista Brasileira deFarmacognosia vol 19 no 1 pp 89ndash94 2009

[4] A B A De Almeida A Luiz-Ferreira M Cola et al ldquoAnti-ulcerogenic mechanisms of the sesquiterpene lactoneonopordopicrin-enriched fraction from Arctium lappa L(Asteraceae) role of somatostatin gastrin and endogenoussulfhydryls and nitric oxiderdquo Journal of Medicinal Food vol 15no 4 pp 378ndash383 2012

[5] L C Ddine C C Ddine C C R Rodrigues V R Kirsten andE Colpo ldquoFactors associated with chronic gastritis in patientswith presence and absence of Helicobacter pylorirdquo ArquivosBrasileiros de Cirgia Digestiva vol 25 no 2 pp 96ndash100 2012

[6] G Tobin D Giglio and O Lundgren ldquoMuscarinic receptorsubtypes in the alimentary tractrdquo Journal of Physiology andPharmacology vol 60 no 1 pp 3ndash21 2009

[7] A S T Carvalho ldquoUlcera pepticardquo Jornal de Pediatria vol 76no 2 pp 127ndash134 2000

[8] M W Mulholland and H T Debas ldquoRecent advances in thetreatment of duodenal ulcer disease A surgical perspectiverdquoTheWestern Journal of Medicine vol 147 no 3 pp 301ndash308 1987

Evidence-Based Complementary and Alternative Medicine 7

[9] P Malfertheiner F K Chan and K E McColl ldquoPeptic ulcerdiseaserdquoThe Lancet vol 374 no 9699 pp 1449ndash1461 2009

[10] Agencia Nacional de Vigilancia Sanitaria Formulario deFitoterapicos da Farmacopeia Brasileira ANVISA Rio deJaneiro Brzil 2011

[11] C F C B R de Almeida T C de Lima E Silva E L C deAmorim M B D S Maia and U P de Albuquerque ldquoLifestrategy and chemical composition as predictors of the selectionof medicinal plants from the caatinga (Northeast Brazil)rdquoJournal of Arid Environments vol 62 no 1 pp 127ndash142 2005

[12] C D F C B R Almeida E L C de Amorim U P de Albu -querque andM B S Maia ldquoMedicinal plants popularly used inthe Xingo regionmdasha semi-arid location in Northeastern BrazilrdquoJournal of Ethnobiology and Ethnomedicine vol 2 article 152006

[13] AA R SilvaMMarques BezerraHVasconcelosChaves et alldquoProtective effect of Chresta martii extract on ethanol-inducedgastropathy depends on alpha-2 adrenoceptors pathways butnot on nitric oxide prostaglandins or opioidsrdquo Journal ofEthnopharmacology vol 142 no 1 pp 206ndash212 2012

[14] A A R Silva M M Bezerra H V Chaves et al ldquoProtectiveeffect of Chresta martii extract against indomethacin-inducedgastric lesions inmicerdquo Journal of Natural Medicines vol 67 no1 pp 143ndash151 2013

[15] J Singh ldquoMaceration percolation and infusion techniques forthe extraction of medicinal and aromatic plantsrdquo in ExtractionTechnologies for Medicinal and Aromatic Plants p 259 UnitedNations Industrial Development Organization and the Interna-tional Centre for Science and High Technology Triseste Italy2008

[16] A F Costa Farmacognosia vol 2 Fundacao Calouste Gul-benkian Lisbon Portugal 4th edition 1994

[17] S Szabo J S Trier A Brown J Schnoor H D Homanand J C Bradford ldquoA quantitative method for assessing theextent of experimental gastric erosions and ulcersrdquo Journal ofPharmacological Methods vol 13 no 1 pp 59ndash66 1985

[18] Y Morimoto K Shimohara S Oshima and T SukamotoldquoEffects of the new anti-ulcer agent KB-5492 on experimentalgastric mucosal lesions and gastric mucosal defensive factorsas compared to those of teprenone and cimetidinerdquo JapaneseJournal of Pharmacology vol 57 no 4 pp 495ndash505 1991

[19] F Borrelli and A A Izzo ldquoThe plant kingdom as a source ofanti-ulcer remediesrdquo Phytotherapy Research vol 14 no 8 pp581ndash591 2000

[20] J R Vane ldquoInhibition of prostaglandin synthesis as a mecha-nism of action for aspirin-like drugsrdquo Nature New Biology vol231 no 25 pp 232ndash235 1971

[21] F M Birdane M Cemek Y O Birdane I Gulcin and EBuyukokuroglu ldquoBeneficial effects of Foeniculum vulgare onethanol-induced acute gastric mucosal injury in ratsrdquo WorldJournal of Gastroenterology vol 13 no 4 pp 607ndash611 2007

[22] E Marhuenda M J Martin and C A de La Lastra ldquoAntiul-cerogenic activity of aescine in different experimental modelsrdquoPhytotherapy Research vol 7 no 1 pp 13ndash16 1993

[23] C la-Casa I Villegas C Alarcon de la Lastra V Motilva andM J Martın Calero ldquoEvidence for protective and antioxidantproperties of rutin a natural flavone against ethanol inducedgastric lesionsrdquo Journal of Ethnopharmacology vol 71 no 1-2pp 45ndash53 2000

[24] S C F Van and P J Lea The Biochemistry of Plant PhenolicsProceedings of the Phytochemical Society of Europe OxfordUniversity Press Oxford UK 1985

[25] M Yoo M W Son I K Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 004541 1998

[26] M Yoo M W Son I Y Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo US Patent no 6025387 2000

[27] M Yoo M W Son I Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 0915864 B104092002

[28] C D Sadik H Sies and T Schewe ldquoInhibition of 15-lipoxygen-ases by flavonoids structure-activity relations and mode ofactionrdquo Biochemical Pharmacology vol 65 no 5 pp 773ndash7812003

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

coupling constants were reported inHertz (Hz) Tetramethyl-silane (TMS) was used as an internal reference standard Thecrystals were solubilized in DMSO-d

6

26 Study of the Gastroprotective Effect of the Organic Extractsand Semipurified Fraction of C martii

261 Gastric Lesions Induced by Indomethacin Gastric ulcer-ations were induced in Swiss Webster mice (119899 = 6 animalsper group) held under fasting (24 h) by the administrationof indomethacin (Sigma) (40mgkg sc) The animals werepretreated with extracts (ECCm EACm or EECm) eachin doses of 50 100 or 200mgkg (po) with a positivecontrol of omeprazole (30mgkg po) or a negative controlof saline (5mLkg po) 1 h before the administration ofindomethacinA group containingmice (119899 = 3)without ulcerinduction received only saline (5mLkg po) to be used asthe default physiological group The doses of extracts werechosen taking into consideration the prior determinationof LD

50(results not presented) and by comparison with

the dose used by Silva et al [13 14] for the same plantspecies and model of ulcer The animals were euthanizedsix hours after the ulcerogenic procedure Their stomachswere removed and opened along the greater curvature Theulcer index was evaluated using the quantitative method forgauging the extent of erosion and experimental gastric ulcersas described by Szabo et al [17] The percentage of inhibitionwas calculated in relation to the saline group according to thefollowing formula inhibition = UItUIs times 100 where UItand UIs correspond to the ulcer indices of the treated andsaline groups respectively

262 Ethanol-Induced Gastropathy The gastric damage wasinduced by the administration of 999 ethanol (02mLanimal) in Swiss Webster mice (119899 = 6 animals per group)kept under fasting (24 h) [18] The animals were treated anhour before induction with EACm (50 100 or 200mgkgpo) or semipurified fractions F1 F2 F3 and F4 (50mgkgpo) or only F1 (125 25 or 50mgkg po)The control groupswere treated with ranitidine (80mgkg po) as a positivecontrol or saline (5mLkg po) as a negative control A groupcontaining mice (119899 = 3) without ulcer induction receivedsaline (5mLkg po) to be used as a physiological parametergroup All the animals were euthanized in a CO

2chamber

30min after the injurious procedure Their stomachs wereremoved and opened along the greater curvature washed insaline fixed between Petri dishes and photographed (SonyCyber-Shot Dsc-h2) at 72 dpi resolution (2816 times 2112 pixels)Hemorrhagic or ulcerative lesions were measured and com-pared to the total area of each stomach through computerizedplanimetry using the Image J program (National Institutesof Health 9000 Rockville Pike Bethesda Maryland USA)The percentage of inhibition was calculated in relation to thesaline group according to the following formula inhibition= UItUIs times 100 where UIt and UIs correspond to the indicesof ulcers in the treated and saline groups respectively [18]

0

5

10

15

20

25

30

35

40

Ulc

er in

dex

Salin

e

Om

epra

zole

(30

mg

kg)

ECCm

(50

mg

kg)

ECCm

(100

mg

kg)

ECCm

(200

mg

kg)

EACm

(50

mg

kg)

EACm

(100

mg

kg)

EACm

(200

mg

kg)

EECm

(50

mg

kg)

EECm

(100

mg

kg)

EECm

(200

mg

kg)

aa a

a

b

c c

b

d dd

Figure 1 Gastroprotective effect of cyclohexane extract of C martii(ECCm) ethyl acetate extract of C martii (EACm) and ethanolicextract of C martii (EECm) at doses of (50 100 or 200mgkgvo) on gastric lesions induced by indomethacin in mice (119899 = 6)Different letters indicateminimal significance (119875 lt 005)Bonferronitest

27 Statistical Analysis All values were expressed as mean plusmnSD For the ulcerativehemorrhagic area ANOVA followedby the Bonferroni test formultiple comparisons was usedThedifferences were considered significant when 119875 lt 005

3 Results and Discussion

The gastroprotective effect of organic extracts (ECCmEACm and EECm) on gastric lesions induced by indometha-cin is as shown in Figure 1

As can be observed the EACmwas the extract which con-ferred the best gastroprotective activityThe activity observedin the group treated with EACm (50mgkg) was significantly(119875 lt 005) different from the control group (saline) andcomparable (119875 gt 005) to that observed in the grouptreated with omeprazole (30mgkg po) A previous studyusing a hydroalcoholic extract of aerial parts of C martii(100 or 400mgkg po) showed significant gastroprotectiveactivity in an ulcer model induced by indomethacin withinhibition indices of 5466 and 8130 respectively [14] Ourresults demonstrate superior gastroprotective activity withEACm (100 or 200mgkg po) (7989 and 8083 resp)taking into consideration the fact that the second dose ofEACm was 50 less than the highest dose used by Silva etal [14] The best performance observed in our work was dueto the extractive process and choice of solvent These twofactors favored the best extraction of substances present in Cmartii resulting in significant gastroprotective activity Theability of indomethacin to induce ulcers experimentally andin humans has been well documented in the literature [19]because it is a potent inhibitor of cyclooxygenases preventing

4 Evidence-Based Complementary and Alternative Medicine

0

10

20

30

40

50

60

70

80

d

ca

b b

Ulc

er in

dex

Salin

e

Rani

tidin

e (80

mg

kg)

EACm

(50

mg

kg)

EACm

(100

mg

kg)

EACm

(200

mg

kg)

Figure 2 Gastroprotective effect of an ethyl acetate extract of Cmartii (EACm) (50 100 or 200mgkg vo) in ethanol-inducedgastric lesions in mice (119899 = 6) Different letters indicate minimalsignificance (119875 lt 005) Bonferroni test

the biosynthesis of prostaglandins [20] The reduction inbiosynthesis of prostanoids would result in decreased resis-tance of the gastric mucosa to the action of HCl and pepsin asit induces an imbalance between the protective and aggressivefactors of the mucosa [1] taking into account the previousknowledge of the mechanism of action of indomethacin onthe gastric mucosa The likely mechanism of action assignedto EACm can be deduced to involve the biosynthesis ofprostaglandins or mimetic action the same as that whichoccurs with synthetic analogues of PGE1 (eg misoprostol)This mimetic effect can act through stimulation of secretionof mucus and bicarbonate andor increased blood flow to thestomach

Similar to what was verified with the model of gas-tric injury induced by indomethacin EACm (50mgkgpo) also presented a cytoprotective effect against ethanol-induced gastric lesions The gastroprotective effect was dose-dependent and in all doses tested superior to that verifiedwith ranitidine (Figure 2)

Ethanol-induced gastric lesions involve different mech-anisms such as a reduction in bicarbonate secretion areduction in mucus production damage to gastric bloodflow and direct injury to mucosal cells [21 22] Theselesions are associated with the excessive production of freeradicals which attack essential cellular constituents such asnucleic acids proteins and lipids [23] The increase of thecontent of lipid peroxides and oxygen-derived free radicalsresults in significant changes at the cellular level and causesdamage to membranes cell death exfoliation and epithelialerosion [21] Studies have demonstrated the presence offlavonoids in species belonging to the family Asteraceaeand this chemical class has been shown to have importantantioxidant properties This could be one of the forms of

action of the compounds present in EACm in providingsignificant cytoprotection given that free radicals are one ofthe harmful factors of the gastric mucosa induced by ethanol

Evaluation of the semipurified fractions (F1 F2 F3 andF4) (50mgkg po) against the ethanol-induced ulcer modelrevealed that animals treated with F1 and F2 showed lowerrates of ulcers when compared to the ranitidine controlHowever when compared with each other F1 presented asignificantly lower ulcer index (Figure 3(a)) Additionally thegastroprotective effect presented by F1 was dose-dependent(Figure 3(b))

After verification of the significant gastroprotective activ-ity of F1 it was refractioned giving rise to 23 subfractionsin which the presence of bands was determined throughTLC revealing anisaldehydesulfuric acid characteristics offlavonoids (yellow) steroids (lilac) and terpenoids (blue)(Figure 4) The existence of a larger number of bands withcolors ranging from orange to yellow can be seen Two puresubstances F11 (Rf 031) and F12 (Rf 048) were recrystal-lized showing a yellow amorphous physical appearance

The NMR (1H and 13C) presented peaks from which itwas possible to calculate the chemical shift (120575) in ppm andthe coupling constants in Hertz (Table 1)

From the interpretation of the spectra two flavones wereidentified chrysoeriol (F11) and 3101584041015840-dimethoxyluteolin(F12) (Figures 5(a) and 5(b)) The yield of 3101584041015840-dimethoxyluteolin was 058 and was higher than thatobserved for chrysoeriol at 043 It was verified in thisstudy that the principal compounds present in EACm areflavones

This study is a pioneer in the identification of two flavonesof Chresta martii According to Van and Lea [24] flavonespresent at least four important physiological mechanismsthey (1) bind to enzymes in the cell membrane (2) bindto heavy metal ions (3) participate in electron transfer ofenzymatic systems and (4) sequester free radicals

The flavone (3101584041015840-dimethoxyluteolin) among otherflavonoids was assessed as to its gastroprotective and anti-inflammatory potential (in different experimental modelsof gastric ulcers and inflammation using rodents) Themechanism of gastroprotective action proposed for thesecompounds has been reported to be the metabolic activationof arachidonic acid via cyclooxygenase activation leadingto biosynthesis of prostaglandins such as PGE2 and PGI2both with important protective function of the gastricmucosa The compounds were furthermore able to inhibitthe activation of 5-lipoxygenase (leukotrienes) which isan important inflammatory mediator besides acting in thesequestration of free radicals These results have given rise tosome patents by Yoo et al [25] (EPO 004541) Yoo et al [26](US 6025387) and Yoo et al [27] (EPO 0915864B1) SimilarlySadik et al [28] verified potent antioxidant and inhibitoryactivity of lipoxygenase in a culture of rabbit reticulocyteswhen different flavonoids were used including luteolinwhose structure is very similar to 3101584041015840-dimethoxyluteolin

However the fraction (F1) is a mixture (terpenesflavonoids and steroids) and these compounds can act

Evidence-Based Complementary and Alternative Medicine 5

0

10

20

30

40

50

60

70

80

a

b

cd

ef

Ulc

er in

dex

Salin

e (5

mL

kg)

Rani

tidin

e (80

mg

kg)

F1(50

mg

kg)

F2(50

mg

kg)

F3(50

mg

kg)

F4(50

mg

kg)

(a)

0

10

20

30

40

50

60

70

80

a

bc

d d

Ulc

er in

dex

Salin

e (5

mL

kg)

Rani

tidin

e (80

mg

kg)

F1(125

mg

kg)

F1(25

mg

kg)

F1(50

mg

kg)

(b)

Figure 3 Gastroprotective effect of fractions (F1 F2 F3 and F4) (50mgkg vo) (a) and the fraction (F1) in isolation (50 25 or 125mgkgvo) (b) obtained from the ethyl acetate extract of C martii on ethanol-induced gastric lesions in mice (119899 = 6) Different letters indicateminimal significance (119875 lt 005) Bonferroni test

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Figure 4 Chromatographic profile by TLC of the eluded fraction (F1) (chloroform ethyl acetate 8 2) revealed with anisaldehydesulfuricacid

HO O

O

OMe

OH

OH

(a)

O

O

HO

OMe

OMe

OH

(b)

Figure 5 Chemical structure of chrysoeriol (a) and 3101584041015840-dimethoxyluteolin (b) isolated from Chresta martii

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Data of 1H- and 13C-NMR of flavonoids (F11 and F12) isolated from the F1 of C martii (300MHz) in DMSO-d6

Flavonoid (F11) Flavonoid (F12)Positions Chrysoeriol 3101584041015840-Dimethoxyluteolin

1205751H (119869 Hz) 120575

13C 1205751H (119869 Hz) 120575

13C2 1637 16393 688 (s) 1041 697 (s) 10604 1818 18155 1620 16306 619 (d 18) 980 621 (d 21) 9827 1642 16478 652 (d 18) 940 653 (d 18) 9409 1574 157910 1040 104011015840 1210 120621015840 756 (d 24) 1100 756 (d 21) 109831015840 1481 148041015840 1508 151751015840 694 (dd 84 24) 1160 713 (d 84) 116261015840 755 (dd 84 24) 1220 768 (dd 96 18) 12165-OH 12978-OH 107941015840-OH 99631015840-OCH3 389 (s) 560 385 (s) 56041015840-OCH3 388 (s) 560

synergistically with regard to the gastroprotective potentialpresented here possibly involving other mechanisms ofaction beyond those cited by Yoo et al [25ndash27] such asactivation of alpha-2 adrenergic receptors as proposed bySilva et al [13] when evaluating the hydroalcoholic extract ofC martii in an ethanol-induced ulcer model

There is a scarcity of studies that assess the phytochemicalprofile at the level of identifying the principal compoundsfor C martii Such work was pioneered by Silva et al[13 14] in identifying two sesquiterpene lactones directlyfrom a hydroalcoholic extract The present study docu-ments for the first time identification of two flavones (3101584041015840-dimethoxyluteolin and chrysoeriol) from the genus Chrestawith relevant evidence for them being the agents responsiblefor the gastroprotective effect seen inCmartii given that thisinvestigation occurred through direct monitoring betweengastroprotective pharmacological activity and processes ofextraction purification and identification of compoundsFinally the results presented here provide rational supportfor the purported use of the species in the treatment of gas-trointestinal disorders according to ethnopharmacologicalinformation

Conflict of Interests

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

References

[1] HD S Falcao J A Leite JM Barbosa-Filho et al ldquoGastric andduodenal antiulcer activity of alkaloids a reviewrdquoMolecules vol13 no 12 pp 3198ndash3223 2008

[2] C E P Araujo R F R Rodrigues and A F OliveiraldquoAnalise preliminar da atividade antiulcerogenica do extratohidroalcoolico de Solanum cernuum Vellrdquo Acta FarmaceuticaBonaerense vol 21 no 4 pp 283ndash286 2002

[3] R S Donatini T Ishikawa S B M Barros and E M BacchildquoAtividade antiulcera e antioxidante do extrato de folhas deSyzygium jambos (L) Alston (Myrtaceae)rdquo Revista Brasileira deFarmacognosia vol 19 no 1 pp 89ndash94 2009

[4] A B A De Almeida A Luiz-Ferreira M Cola et al ldquoAnti-ulcerogenic mechanisms of the sesquiterpene lactoneonopordopicrin-enriched fraction from Arctium lappa L(Asteraceae) role of somatostatin gastrin and endogenoussulfhydryls and nitric oxiderdquo Journal of Medicinal Food vol 15no 4 pp 378ndash383 2012

[5] L C Ddine C C Ddine C C R Rodrigues V R Kirsten andE Colpo ldquoFactors associated with chronic gastritis in patientswith presence and absence of Helicobacter pylorirdquo ArquivosBrasileiros de Cirgia Digestiva vol 25 no 2 pp 96ndash100 2012

[6] G Tobin D Giglio and O Lundgren ldquoMuscarinic receptorsubtypes in the alimentary tractrdquo Journal of Physiology andPharmacology vol 60 no 1 pp 3ndash21 2009

[7] A S T Carvalho ldquoUlcera pepticardquo Jornal de Pediatria vol 76no 2 pp 127ndash134 2000

[8] M W Mulholland and H T Debas ldquoRecent advances in thetreatment of duodenal ulcer disease A surgical perspectiverdquoTheWestern Journal of Medicine vol 147 no 3 pp 301ndash308 1987

Evidence-Based Complementary and Alternative Medicine 7

[9] P Malfertheiner F K Chan and K E McColl ldquoPeptic ulcerdiseaserdquoThe Lancet vol 374 no 9699 pp 1449ndash1461 2009

[10] Agencia Nacional de Vigilancia Sanitaria Formulario deFitoterapicos da Farmacopeia Brasileira ANVISA Rio deJaneiro Brzil 2011

[11] C F C B R de Almeida T C de Lima E Silva E L C deAmorim M B D S Maia and U P de Albuquerque ldquoLifestrategy and chemical composition as predictors of the selectionof medicinal plants from the caatinga (Northeast Brazil)rdquoJournal of Arid Environments vol 62 no 1 pp 127ndash142 2005

[12] C D F C B R Almeida E L C de Amorim U P de Albu -querque andM B S Maia ldquoMedicinal plants popularly used inthe Xingo regionmdasha semi-arid location in Northeastern BrazilrdquoJournal of Ethnobiology and Ethnomedicine vol 2 article 152006

[13] AA R SilvaMMarques BezerraHVasconcelosChaves et alldquoProtective effect of Chresta martii extract on ethanol-inducedgastropathy depends on alpha-2 adrenoceptors pathways butnot on nitric oxide prostaglandins or opioidsrdquo Journal ofEthnopharmacology vol 142 no 1 pp 206ndash212 2012

[14] A A R Silva M M Bezerra H V Chaves et al ldquoProtectiveeffect of Chresta martii extract against indomethacin-inducedgastric lesions inmicerdquo Journal of Natural Medicines vol 67 no1 pp 143ndash151 2013

[15] J Singh ldquoMaceration percolation and infusion techniques forthe extraction of medicinal and aromatic plantsrdquo in ExtractionTechnologies for Medicinal and Aromatic Plants p 259 UnitedNations Industrial Development Organization and the Interna-tional Centre for Science and High Technology Triseste Italy2008

[16] A F Costa Farmacognosia vol 2 Fundacao Calouste Gul-benkian Lisbon Portugal 4th edition 1994

[17] S Szabo J S Trier A Brown J Schnoor H D Homanand J C Bradford ldquoA quantitative method for assessing theextent of experimental gastric erosions and ulcersrdquo Journal ofPharmacological Methods vol 13 no 1 pp 59ndash66 1985

[18] Y Morimoto K Shimohara S Oshima and T SukamotoldquoEffects of the new anti-ulcer agent KB-5492 on experimentalgastric mucosal lesions and gastric mucosal defensive factorsas compared to those of teprenone and cimetidinerdquo JapaneseJournal of Pharmacology vol 57 no 4 pp 495ndash505 1991

[19] F Borrelli and A A Izzo ldquoThe plant kingdom as a source ofanti-ulcer remediesrdquo Phytotherapy Research vol 14 no 8 pp581ndash591 2000

[20] J R Vane ldquoInhibition of prostaglandin synthesis as a mecha-nism of action for aspirin-like drugsrdquo Nature New Biology vol231 no 25 pp 232ndash235 1971

[21] F M Birdane M Cemek Y O Birdane I Gulcin and EBuyukokuroglu ldquoBeneficial effects of Foeniculum vulgare onethanol-induced acute gastric mucosal injury in ratsrdquo WorldJournal of Gastroenterology vol 13 no 4 pp 607ndash611 2007

[22] E Marhuenda M J Martin and C A de La Lastra ldquoAntiul-cerogenic activity of aescine in different experimental modelsrdquoPhytotherapy Research vol 7 no 1 pp 13ndash16 1993

[23] C la-Casa I Villegas C Alarcon de la Lastra V Motilva andM J Martın Calero ldquoEvidence for protective and antioxidantproperties of rutin a natural flavone against ethanol inducedgastric lesionsrdquo Journal of Ethnopharmacology vol 71 no 1-2pp 45ndash53 2000

[24] S C F Van and P J Lea The Biochemistry of Plant PhenolicsProceedings of the Phytochemical Society of Europe OxfordUniversity Press Oxford UK 1985

[25] M Yoo M W Son I K Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 004541 1998

[26] M Yoo M W Son I Y Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo US Patent no 6025387 2000

[27] M Yoo M W Son I Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 0915864 B104092002

[28] C D Sadik H Sies and T Schewe ldquoInhibition of 15-lipoxygen-ases by flavonoids structure-activity relations and mode ofactionrdquo Biochemical Pharmacology vol 65 no 5 pp 773ndash7812003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

0

10

20

30

40

50

60

70

80

d

ca

b b

Ulc

er in

dex

Salin

e

Rani

tidin

e (80

mg

kg)

EACm

(50

mg

kg)

EACm

(100

mg

kg)

EACm

(200

mg

kg)

Figure 2 Gastroprotective effect of an ethyl acetate extract of Cmartii (EACm) (50 100 or 200mgkg vo) in ethanol-inducedgastric lesions in mice (119899 = 6) Different letters indicate minimalsignificance (119875 lt 005) Bonferroni test

the biosynthesis of prostaglandins [20] The reduction inbiosynthesis of prostanoids would result in decreased resis-tance of the gastric mucosa to the action of HCl and pepsin asit induces an imbalance between the protective and aggressivefactors of the mucosa [1] taking into account the previousknowledge of the mechanism of action of indomethacin onthe gastric mucosa The likely mechanism of action assignedto EACm can be deduced to involve the biosynthesis ofprostaglandins or mimetic action the same as that whichoccurs with synthetic analogues of PGE1 (eg misoprostol)This mimetic effect can act through stimulation of secretionof mucus and bicarbonate andor increased blood flow to thestomach

Similar to what was verified with the model of gas-tric injury induced by indomethacin EACm (50mgkgpo) also presented a cytoprotective effect against ethanol-induced gastric lesions The gastroprotective effect was dose-dependent and in all doses tested superior to that verifiedwith ranitidine (Figure 2)

Ethanol-induced gastric lesions involve different mech-anisms such as a reduction in bicarbonate secretion areduction in mucus production damage to gastric bloodflow and direct injury to mucosal cells [21 22] Theselesions are associated with the excessive production of freeradicals which attack essential cellular constituents such asnucleic acids proteins and lipids [23] The increase of thecontent of lipid peroxides and oxygen-derived free radicalsresults in significant changes at the cellular level and causesdamage to membranes cell death exfoliation and epithelialerosion [21] Studies have demonstrated the presence offlavonoids in species belonging to the family Asteraceaeand this chemical class has been shown to have importantantioxidant properties This could be one of the forms of

action of the compounds present in EACm in providingsignificant cytoprotection given that free radicals are one ofthe harmful factors of the gastric mucosa induced by ethanol

Evaluation of the semipurified fractions (F1 F2 F3 andF4) (50mgkg po) against the ethanol-induced ulcer modelrevealed that animals treated with F1 and F2 showed lowerrates of ulcers when compared to the ranitidine controlHowever when compared with each other F1 presented asignificantly lower ulcer index (Figure 3(a)) Additionally thegastroprotective effect presented by F1 was dose-dependent(Figure 3(b))

After verification of the significant gastroprotective activ-ity of F1 it was refractioned giving rise to 23 subfractionsin which the presence of bands was determined throughTLC revealing anisaldehydesulfuric acid characteristics offlavonoids (yellow) steroids (lilac) and terpenoids (blue)(Figure 4) The existence of a larger number of bands withcolors ranging from orange to yellow can be seen Two puresubstances F11 (Rf 031) and F12 (Rf 048) were recrystal-lized showing a yellow amorphous physical appearance

The NMR (1H and 13C) presented peaks from which itwas possible to calculate the chemical shift (120575) in ppm andthe coupling constants in Hertz (Table 1)

From the interpretation of the spectra two flavones wereidentified chrysoeriol (F11) and 3101584041015840-dimethoxyluteolin(F12) (Figures 5(a) and 5(b)) The yield of 3101584041015840-dimethoxyluteolin was 058 and was higher than thatobserved for chrysoeriol at 043 It was verified in thisstudy that the principal compounds present in EACm areflavones

This study is a pioneer in the identification of two flavonesof Chresta martii According to Van and Lea [24] flavonespresent at least four important physiological mechanismsthey (1) bind to enzymes in the cell membrane (2) bindto heavy metal ions (3) participate in electron transfer ofenzymatic systems and (4) sequester free radicals

The flavone (3101584041015840-dimethoxyluteolin) among otherflavonoids was assessed as to its gastroprotective and anti-inflammatory potential (in different experimental modelsof gastric ulcers and inflammation using rodents) Themechanism of gastroprotective action proposed for thesecompounds has been reported to be the metabolic activationof arachidonic acid via cyclooxygenase activation leadingto biosynthesis of prostaglandins such as PGE2 and PGI2both with important protective function of the gastricmucosa The compounds were furthermore able to inhibitthe activation of 5-lipoxygenase (leukotrienes) which isan important inflammatory mediator besides acting in thesequestration of free radicals These results have given rise tosome patents by Yoo et al [25] (EPO 004541) Yoo et al [26](US 6025387) and Yoo et al [27] (EPO 0915864B1) SimilarlySadik et al [28] verified potent antioxidant and inhibitoryactivity of lipoxygenase in a culture of rabbit reticulocyteswhen different flavonoids were used including luteolinwhose structure is very similar to 3101584041015840-dimethoxyluteolin

However the fraction (F1) is a mixture (terpenesflavonoids and steroids) and these compounds can act

Evidence-Based Complementary and Alternative Medicine 5

0

10

20

30

40

50

60

70

80

a

b

cd

ef

Ulc

er in

dex

Salin

e (5

mL

kg)

Rani

tidin

e (80

mg

kg)

F1(50

mg

kg)

F2(50

mg

kg)

F3(50

mg

kg)

F4(50

mg

kg)

(a)

0

10

20

30

40

50

60

70

80

a

bc

d d

Ulc

er in

dex

Salin

e (5

mL

kg)

Rani

tidin

e (80

mg

kg)

F1(125

mg

kg)

F1(25

mg

kg)

F1(50

mg

kg)

(b)

Figure 3 Gastroprotective effect of fractions (F1 F2 F3 and F4) (50mgkg vo) (a) and the fraction (F1) in isolation (50 25 or 125mgkgvo) (b) obtained from the ethyl acetate extract of C martii on ethanol-induced gastric lesions in mice (119899 = 6) Different letters indicateminimal significance (119875 lt 005) Bonferroni test

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Figure 4 Chromatographic profile by TLC of the eluded fraction (F1) (chloroform ethyl acetate 8 2) revealed with anisaldehydesulfuricacid

HO O

O

OMe

OH

OH

(a)

O

O

HO

OMe

OMe

OH

(b)

Figure 5 Chemical structure of chrysoeriol (a) and 3101584041015840-dimethoxyluteolin (b) isolated from Chresta martii

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Data of 1H- and 13C-NMR of flavonoids (F11 and F12) isolated from the F1 of C martii (300MHz) in DMSO-d6

Flavonoid (F11) Flavonoid (F12)Positions Chrysoeriol 3101584041015840-Dimethoxyluteolin

1205751H (119869 Hz) 120575

13C 1205751H (119869 Hz) 120575

13C2 1637 16393 688 (s) 1041 697 (s) 10604 1818 18155 1620 16306 619 (d 18) 980 621 (d 21) 9827 1642 16478 652 (d 18) 940 653 (d 18) 9409 1574 157910 1040 104011015840 1210 120621015840 756 (d 24) 1100 756 (d 21) 109831015840 1481 148041015840 1508 151751015840 694 (dd 84 24) 1160 713 (d 84) 116261015840 755 (dd 84 24) 1220 768 (dd 96 18) 12165-OH 12978-OH 107941015840-OH 99631015840-OCH3 389 (s) 560 385 (s) 56041015840-OCH3 388 (s) 560

synergistically with regard to the gastroprotective potentialpresented here possibly involving other mechanisms ofaction beyond those cited by Yoo et al [25ndash27] such asactivation of alpha-2 adrenergic receptors as proposed bySilva et al [13] when evaluating the hydroalcoholic extract ofC martii in an ethanol-induced ulcer model

There is a scarcity of studies that assess the phytochemicalprofile at the level of identifying the principal compoundsfor C martii Such work was pioneered by Silva et al[13 14] in identifying two sesquiterpene lactones directlyfrom a hydroalcoholic extract The present study docu-ments for the first time identification of two flavones (3101584041015840-dimethoxyluteolin and chrysoeriol) from the genus Chrestawith relevant evidence for them being the agents responsiblefor the gastroprotective effect seen inCmartii given that thisinvestigation occurred through direct monitoring betweengastroprotective pharmacological activity and processes ofextraction purification and identification of compoundsFinally the results presented here provide rational supportfor the purported use of the species in the treatment of gas-trointestinal disorders according to ethnopharmacologicalinformation

Conflict of Interests

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

References

[1] HD S Falcao J A Leite JM Barbosa-Filho et al ldquoGastric andduodenal antiulcer activity of alkaloids a reviewrdquoMolecules vol13 no 12 pp 3198ndash3223 2008

[2] C E P Araujo R F R Rodrigues and A F OliveiraldquoAnalise preliminar da atividade antiulcerogenica do extratohidroalcoolico de Solanum cernuum Vellrdquo Acta FarmaceuticaBonaerense vol 21 no 4 pp 283ndash286 2002

[3] R S Donatini T Ishikawa S B M Barros and E M BacchildquoAtividade antiulcera e antioxidante do extrato de folhas deSyzygium jambos (L) Alston (Myrtaceae)rdquo Revista Brasileira deFarmacognosia vol 19 no 1 pp 89ndash94 2009

[4] A B A De Almeida A Luiz-Ferreira M Cola et al ldquoAnti-ulcerogenic mechanisms of the sesquiterpene lactoneonopordopicrin-enriched fraction from Arctium lappa L(Asteraceae) role of somatostatin gastrin and endogenoussulfhydryls and nitric oxiderdquo Journal of Medicinal Food vol 15no 4 pp 378ndash383 2012

[5] L C Ddine C C Ddine C C R Rodrigues V R Kirsten andE Colpo ldquoFactors associated with chronic gastritis in patientswith presence and absence of Helicobacter pylorirdquo ArquivosBrasileiros de Cirgia Digestiva vol 25 no 2 pp 96ndash100 2012

[6] G Tobin D Giglio and O Lundgren ldquoMuscarinic receptorsubtypes in the alimentary tractrdquo Journal of Physiology andPharmacology vol 60 no 1 pp 3ndash21 2009

[7] A S T Carvalho ldquoUlcera pepticardquo Jornal de Pediatria vol 76no 2 pp 127ndash134 2000

[8] M W Mulholland and H T Debas ldquoRecent advances in thetreatment of duodenal ulcer disease A surgical perspectiverdquoTheWestern Journal of Medicine vol 147 no 3 pp 301ndash308 1987

Evidence-Based Complementary and Alternative Medicine 7

[9] P Malfertheiner F K Chan and K E McColl ldquoPeptic ulcerdiseaserdquoThe Lancet vol 374 no 9699 pp 1449ndash1461 2009

[10] Agencia Nacional de Vigilancia Sanitaria Formulario deFitoterapicos da Farmacopeia Brasileira ANVISA Rio deJaneiro Brzil 2011

[11] C F C B R de Almeida T C de Lima E Silva E L C deAmorim M B D S Maia and U P de Albuquerque ldquoLifestrategy and chemical composition as predictors of the selectionof medicinal plants from the caatinga (Northeast Brazil)rdquoJournal of Arid Environments vol 62 no 1 pp 127ndash142 2005

[12] C D F C B R Almeida E L C de Amorim U P de Albu -querque andM B S Maia ldquoMedicinal plants popularly used inthe Xingo regionmdasha semi-arid location in Northeastern BrazilrdquoJournal of Ethnobiology and Ethnomedicine vol 2 article 152006

[13] AA R SilvaMMarques BezerraHVasconcelosChaves et alldquoProtective effect of Chresta martii extract on ethanol-inducedgastropathy depends on alpha-2 adrenoceptors pathways butnot on nitric oxide prostaglandins or opioidsrdquo Journal ofEthnopharmacology vol 142 no 1 pp 206ndash212 2012

[14] A A R Silva M M Bezerra H V Chaves et al ldquoProtectiveeffect of Chresta martii extract against indomethacin-inducedgastric lesions inmicerdquo Journal of Natural Medicines vol 67 no1 pp 143ndash151 2013

[15] J Singh ldquoMaceration percolation and infusion techniques forthe extraction of medicinal and aromatic plantsrdquo in ExtractionTechnologies for Medicinal and Aromatic Plants p 259 UnitedNations Industrial Development Organization and the Interna-tional Centre for Science and High Technology Triseste Italy2008

[16] A F Costa Farmacognosia vol 2 Fundacao Calouste Gul-benkian Lisbon Portugal 4th edition 1994

[17] S Szabo J S Trier A Brown J Schnoor H D Homanand J C Bradford ldquoA quantitative method for assessing theextent of experimental gastric erosions and ulcersrdquo Journal ofPharmacological Methods vol 13 no 1 pp 59ndash66 1985

[18] Y Morimoto K Shimohara S Oshima and T SukamotoldquoEffects of the new anti-ulcer agent KB-5492 on experimentalgastric mucosal lesions and gastric mucosal defensive factorsas compared to those of teprenone and cimetidinerdquo JapaneseJournal of Pharmacology vol 57 no 4 pp 495ndash505 1991

[19] F Borrelli and A A Izzo ldquoThe plant kingdom as a source ofanti-ulcer remediesrdquo Phytotherapy Research vol 14 no 8 pp581ndash591 2000

[20] J R Vane ldquoInhibition of prostaglandin synthesis as a mecha-nism of action for aspirin-like drugsrdquo Nature New Biology vol231 no 25 pp 232ndash235 1971

[21] F M Birdane M Cemek Y O Birdane I Gulcin and EBuyukokuroglu ldquoBeneficial effects of Foeniculum vulgare onethanol-induced acute gastric mucosal injury in ratsrdquo WorldJournal of Gastroenterology vol 13 no 4 pp 607ndash611 2007

[22] E Marhuenda M J Martin and C A de La Lastra ldquoAntiul-cerogenic activity of aescine in different experimental modelsrdquoPhytotherapy Research vol 7 no 1 pp 13ndash16 1993

[23] C la-Casa I Villegas C Alarcon de la Lastra V Motilva andM J Martın Calero ldquoEvidence for protective and antioxidantproperties of rutin a natural flavone against ethanol inducedgastric lesionsrdquo Journal of Ethnopharmacology vol 71 no 1-2pp 45ndash53 2000

[24] S C F Van and P J Lea The Biochemistry of Plant PhenolicsProceedings of the Phytochemical Society of Europe OxfordUniversity Press Oxford UK 1985

[25] M Yoo M W Son I K Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 004541 1998

[26] M Yoo M W Son I Y Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo US Patent no 6025387 2000

[27] M Yoo M W Son I Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 0915864 B104092002

[28] C D Sadik H Sies and T Schewe ldquoInhibition of 15-lipoxygen-ases by flavonoids structure-activity relations and mode ofactionrdquo Biochemical Pharmacology vol 65 no 5 pp 773ndash7812003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

0

10

20

30

40

50

60

70

80

a

b

cd

ef

Ulc

er in

dex

Salin

e (5

mL

kg)

Rani

tidin

e (80

mg

kg)

F1(50

mg

kg)

F2(50

mg

kg)

F3(50

mg

kg)

F4(50

mg

kg)

(a)

0

10

20

30

40

50

60

70

80

a

bc

d d

Ulc

er in

dex

Salin

e (5

mL

kg)

Rani

tidin

e (80

mg

kg)

F1(125

mg

kg)

F1(25

mg

kg)

F1(50

mg

kg)

(b)

Figure 3 Gastroprotective effect of fractions (F1 F2 F3 and F4) (50mgkg vo) (a) and the fraction (F1) in isolation (50 25 or 125mgkgvo) (b) obtained from the ethyl acetate extract of C martii on ethanol-induced gastric lesions in mice (119899 = 6) Different letters indicateminimal significance (119875 lt 005) Bonferroni test

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Figure 4 Chromatographic profile by TLC of the eluded fraction (F1) (chloroform ethyl acetate 8 2) revealed with anisaldehydesulfuricacid

HO O

O

OMe

OH

OH

(a)

O

O

HO

OMe

OMe

OH

(b)

Figure 5 Chemical structure of chrysoeriol (a) and 3101584041015840-dimethoxyluteolin (b) isolated from Chresta martii

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Data of 1H- and 13C-NMR of flavonoids (F11 and F12) isolated from the F1 of C martii (300MHz) in DMSO-d6

Flavonoid (F11) Flavonoid (F12)Positions Chrysoeriol 3101584041015840-Dimethoxyluteolin

1205751H (119869 Hz) 120575

13C 1205751H (119869 Hz) 120575

13C2 1637 16393 688 (s) 1041 697 (s) 10604 1818 18155 1620 16306 619 (d 18) 980 621 (d 21) 9827 1642 16478 652 (d 18) 940 653 (d 18) 9409 1574 157910 1040 104011015840 1210 120621015840 756 (d 24) 1100 756 (d 21) 109831015840 1481 148041015840 1508 151751015840 694 (dd 84 24) 1160 713 (d 84) 116261015840 755 (dd 84 24) 1220 768 (dd 96 18) 12165-OH 12978-OH 107941015840-OH 99631015840-OCH3 389 (s) 560 385 (s) 56041015840-OCH3 388 (s) 560

synergistically with regard to the gastroprotective potentialpresented here possibly involving other mechanisms ofaction beyond those cited by Yoo et al [25ndash27] such asactivation of alpha-2 adrenergic receptors as proposed bySilva et al [13] when evaluating the hydroalcoholic extract ofC martii in an ethanol-induced ulcer model

There is a scarcity of studies that assess the phytochemicalprofile at the level of identifying the principal compoundsfor C martii Such work was pioneered by Silva et al[13 14] in identifying two sesquiterpene lactones directlyfrom a hydroalcoholic extract The present study docu-ments for the first time identification of two flavones (3101584041015840-dimethoxyluteolin and chrysoeriol) from the genus Chrestawith relevant evidence for them being the agents responsiblefor the gastroprotective effect seen inCmartii given that thisinvestigation occurred through direct monitoring betweengastroprotective pharmacological activity and processes ofextraction purification and identification of compoundsFinally the results presented here provide rational supportfor the purported use of the species in the treatment of gas-trointestinal disorders according to ethnopharmacologicalinformation

Conflict of Interests

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

References

[1] HD S Falcao J A Leite JM Barbosa-Filho et al ldquoGastric andduodenal antiulcer activity of alkaloids a reviewrdquoMolecules vol13 no 12 pp 3198ndash3223 2008

[2] C E P Araujo R F R Rodrigues and A F OliveiraldquoAnalise preliminar da atividade antiulcerogenica do extratohidroalcoolico de Solanum cernuum Vellrdquo Acta FarmaceuticaBonaerense vol 21 no 4 pp 283ndash286 2002

[3] R S Donatini T Ishikawa S B M Barros and E M BacchildquoAtividade antiulcera e antioxidante do extrato de folhas deSyzygium jambos (L) Alston (Myrtaceae)rdquo Revista Brasileira deFarmacognosia vol 19 no 1 pp 89ndash94 2009

[4] A B A De Almeida A Luiz-Ferreira M Cola et al ldquoAnti-ulcerogenic mechanisms of the sesquiterpene lactoneonopordopicrin-enriched fraction from Arctium lappa L(Asteraceae) role of somatostatin gastrin and endogenoussulfhydryls and nitric oxiderdquo Journal of Medicinal Food vol 15no 4 pp 378ndash383 2012

[5] L C Ddine C C Ddine C C R Rodrigues V R Kirsten andE Colpo ldquoFactors associated with chronic gastritis in patientswith presence and absence of Helicobacter pylorirdquo ArquivosBrasileiros de Cirgia Digestiva vol 25 no 2 pp 96ndash100 2012

[6] G Tobin D Giglio and O Lundgren ldquoMuscarinic receptorsubtypes in the alimentary tractrdquo Journal of Physiology andPharmacology vol 60 no 1 pp 3ndash21 2009

[7] A S T Carvalho ldquoUlcera pepticardquo Jornal de Pediatria vol 76no 2 pp 127ndash134 2000

[8] M W Mulholland and H T Debas ldquoRecent advances in thetreatment of duodenal ulcer disease A surgical perspectiverdquoTheWestern Journal of Medicine vol 147 no 3 pp 301ndash308 1987

Evidence-Based Complementary and Alternative Medicine 7

[9] P Malfertheiner F K Chan and K E McColl ldquoPeptic ulcerdiseaserdquoThe Lancet vol 374 no 9699 pp 1449ndash1461 2009

[10] Agencia Nacional de Vigilancia Sanitaria Formulario deFitoterapicos da Farmacopeia Brasileira ANVISA Rio deJaneiro Brzil 2011

[11] C F C B R de Almeida T C de Lima E Silva E L C deAmorim M B D S Maia and U P de Albuquerque ldquoLifestrategy and chemical composition as predictors of the selectionof medicinal plants from the caatinga (Northeast Brazil)rdquoJournal of Arid Environments vol 62 no 1 pp 127ndash142 2005

[12] C D F C B R Almeida E L C de Amorim U P de Albu -querque andM B S Maia ldquoMedicinal plants popularly used inthe Xingo regionmdasha semi-arid location in Northeastern BrazilrdquoJournal of Ethnobiology and Ethnomedicine vol 2 article 152006

[13] AA R SilvaMMarques BezerraHVasconcelosChaves et alldquoProtective effect of Chresta martii extract on ethanol-inducedgastropathy depends on alpha-2 adrenoceptors pathways butnot on nitric oxide prostaglandins or opioidsrdquo Journal ofEthnopharmacology vol 142 no 1 pp 206ndash212 2012

[14] A A R Silva M M Bezerra H V Chaves et al ldquoProtectiveeffect of Chresta martii extract against indomethacin-inducedgastric lesions inmicerdquo Journal of Natural Medicines vol 67 no1 pp 143ndash151 2013

[15] J Singh ldquoMaceration percolation and infusion techniques forthe extraction of medicinal and aromatic plantsrdquo in ExtractionTechnologies for Medicinal and Aromatic Plants p 259 UnitedNations Industrial Development Organization and the Interna-tional Centre for Science and High Technology Triseste Italy2008

[16] A F Costa Farmacognosia vol 2 Fundacao Calouste Gul-benkian Lisbon Portugal 4th edition 1994

[17] S Szabo J S Trier A Brown J Schnoor H D Homanand J C Bradford ldquoA quantitative method for assessing theextent of experimental gastric erosions and ulcersrdquo Journal ofPharmacological Methods vol 13 no 1 pp 59ndash66 1985

[18] Y Morimoto K Shimohara S Oshima and T SukamotoldquoEffects of the new anti-ulcer agent KB-5492 on experimentalgastric mucosal lesions and gastric mucosal defensive factorsas compared to those of teprenone and cimetidinerdquo JapaneseJournal of Pharmacology vol 57 no 4 pp 495ndash505 1991

[19] F Borrelli and A A Izzo ldquoThe plant kingdom as a source ofanti-ulcer remediesrdquo Phytotherapy Research vol 14 no 8 pp581ndash591 2000

[20] J R Vane ldquoInhibition of prostaglandin synthesis as a mecha-nism of action for aspirin-like drugsrdquo Nature New Biology vol231 no 25 pp 232ndash235 1971

[21] F M Birdane M Cemek Y O Birdane I Gulcin and EBuyukokuroglu ldquoBeneficial effects of Foeniculum vulgare onethanol-induced acute gastric mucosal injury in ratsrdquo WorldJournal of Gastroenterology vol 13 no 4 pp 607ndash611 2007

[22] E Marhuenda M J Martin and C A de La Lastra ldquoAntiul-cerogenic activity of aescine in different experimental modelsrdquoPhytotherapy Research vol 7 no 1 pp 13ndash16 1993

[23] C la-Casa I Villegas C Alarcon de la Lastra V Motilva andM J Martın Calero ldquoEvidence for protective and antioxidantproperties of rutin a natural flavone against ethanol inducedgastric lesionsrdquo Journal of Ethnopharmacology vol 71 no 1-2pp 45ndash53 2000

[24] S C F Van and P J Lea The Biochemistry of Plant PhenolicsProceedings of the Phytochemical Society of Europe OxfordUniversity Press Oxford UK 1985

[25] M Yoo M W Son I K Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 004541 1998

[26] M Yoo M W Son I Y Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo US Patent no 6025387 2000

[27] M Yoo M W Son I Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 0915864 B104092002

[28] C D Sadik H Sies and T Schewe ldquoInhibition of 15-lipoxygen-ases by flavonoids structure-activity relations and mode ofactionrdquo Biochemical Pharmacology vol 65 no 5 pp 773ndash7812003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Data of 1H- and 13C-NMR of flavonoids (F11 and F12) isolated from the F1 of C martii (300MHz) in DMSO-d6

Flavonoid (F11) Flavonoid (F12)Positions Chrysoeriol 3101584041015840-Dimethoxyluteolin

1205751H (119869 Hz) 120575

13C 1205751H (119869 Hz) 120575

13C2 1637 16393 688 (s) 1041 697 (s) 10604 1818 18155 1620 16306 619 (d 18) 980 621 (d 21) 9827 1642 16478 652 (d 18) 940 653 (d 18) 9409 1574 157910 1040 104011015840 1210 120621015840 756 (d 24) 1100 756 (d 21) 109831015840 1481 148041015840 1508 151751015840 694 (dd 84 24) 1160 713 (d 84) 116261015840 755 (dd 84 24) 1220 768 (dd 96 18) 12165-OH 12978-OH 107941015840-OH 99631015840-OCH3 389 (s) 560 385 (s) 56041015840-OCH3 388 (s) 560

synergistically with regard to the gastroprotective potentialpresented here possibly involving other mechanisms ofaction beyond those cited by Yoo et al [25ndash27] such asactivation of alpha-2 adrenergic receptors as proposed bySilva et al [13] when evaluating the hydroalcoholic extract ofC martii in an ethanol-induced ulcer model

There is a scarcity of studies that assess the phytochemicalprofile at the level of identifying the principal compoundsfor C martii Such work was pioneered by Silva et al[13 14] in identifying two sesquiterpene lactones directlyfrom a hydroalcoholic extract The present study docu-ments for the first time identification of two flavones (3101584041015840-dimethoxyluteolin and chrysoeriol) from the genus Chrestawith relevant evidence for them being the agents responsiblefor the gastroprotective effect seen inCmartii given that thisinvestigation occurred through direct monitoring betweengastroprotective pharmacological activity and processes ofextraction purification and identification of compoundsFinally the results presented here provide rational supportfor the purported use of the species in the treatment of gas-trointestinal disorders according to ethnopharmacologicalinformation

Conflict of Interests

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

References

[1] HD S Falcao J A Leite JM Barbosa-Filho et al ldquoGastric andduodenal antiulcer activity of alkaloids a reviewrdquoMolecules vol13 no 12 pp 3198ndash3223 2008

[2] C E P Araujo R F R Rodrigues and A F OliveiraldquoAnalise preliminar da atividade antiulcerogenica do extratohidroalcoolico de Solanum cernuum Vellrdquo Acta FarmaceuticaBonaerense vol 21 no 4 pp 283ndash286 2002

[3] R S Donatini T Ishikawa S B M Barros and E M BacchildquoAtividade antiulcera e antioxidante do extrato de folhas deSyzygium jambos (L) Alston (Myrtaceae)rdquo Revista Brasileira deFarmacognosia vol 19 no 1 pp 89ndash94 2009

[4] A B A De Almeida A Luiz-Ferreira M Cola et al ldquoAnti-ulcerogenic mechanisms of the sesquiterpene lactoneonopordopicrin-enriched fraction from Arctium lappa L(Asteraceae) role of somatostatin gastrin and endogenoussulfhydryls and nitric oxiderdquo Journal of Medicinal Food vol 15no 4 pp 378ndash383 2012

[5] L C Ddine C C Ddine C C R Rodrigues V R Kirsten andE Colpo ldquoFactors associated with chronic gastritis in patientswith presence and absence of Helicobacter pylorirdquo ArquivosBrasileiros de Cirgia Digestiva vol 25 no 2 pp 96ndash100 2012

[6] G Tobin D Giglio and O Lundgren ldquoMuscarinic receptorsubtypes in the alimentary tractrdquo Journal of Physiology andPharmacology vol 60 no 1 pp 3ndash21 2009

[7] A S T Carvalho ldquoUlcera pepticardquo Jornal de Pediatria vol 76no 2 pp 127ndash134 2000

[8] M W Mulholland and H T Debas ldquoRecent advances in thetreatment of duodenal ulcer disease A surgical perspectiverdquoTheWestern Journal of Medicine vol 147 no 3 pp 301ndash308 1987

Evidence-Based Complementary and Alternative Medicine 7

[9] P Malfertheiner F K Chan and K E McColl ldquoPeptic ulcerdiseaserdquoThe Lancet vol 374 no 9699 pp 1449ndash1461 2009

[10] Agencia Nacional de Vigilancia Sanitaria Formulario deFitoterapicos da Farmacopeia Brasileira ANVISA Rio deJaneiro Brzil 2011

[11] C F C B R de Almeida T C de Lima E Silva E L C deAmorim M B D S Maia and U P de Albuquerque ldquoLifestrategy and chemical composition as predictors of the selectionof medicinal plants from the caatinga (Northeast Brazil)rdquoJournal of Arid Environments vol 62 no 1 pp 127ndash142 2005

[12] C D F C B R Almeida E L C de Amorim U P de Albu -querque andM B S Maia ldquoMedicinal plants popularly used inthe Xingo regionmdasha semi-arid location in Northeastern BrazilrdquoJournal of Ethnobiology and Ethnomedicine vol 2 article 152006

[13] AA R SilvaMMarques BezerraHVasconcelosChaves et alldquoProtective effect of Chresta martii extract on ethanol-inducedgastropathy depends on alpha-2 adrenoceptors pathways butnot on nitric oxide prostaglandins or opioidsrdquo Journal ofEthnopharmacology vol 142 no 1 pp 206ndash212 2012

[14] A A R Silva M M Bezerra H V Chaves et al ldquoProtectiveeffect of Chresta martii extract against indomethacin-inducedgastric lesions inmicerdquo Journal of Natural Medicines vol 67 no1 pp 143ndash151 2013

[15] J Singh ldquoMaceration percolation and infusion techniques forthe extraction of medicinal and aromatic plantsrdquo in ExtractionTechnologies for Medicinal and Aromatic Plants p 259 UnitedNations Industrial Development Organization and the Interna-tional Centre for Science and High Technology Triseste Italy2008

[16] A F Costa Farmacognosia vol 2 Fundacao Calouste Gul-benkian Lisbon Portugal 4th edition 1994

[17] S Szabo J S Trier A Brown J Schnoor H D Homanand J C Bradford ldquoA quantitative method for assessing theextent of experimental gastric erosions and ulcersrdquo Journal ofPharmacological Methods vol 13 no 1 pp 59ndash66 1985

[18] Y Morimoto K Shimohara S Oshima and T SukamotoldquoEffects of the new anti-ulcer agent KB-5492 on experimentalgastric mucosal lesions and gastric mucosal defensive factorsas compared to those of teprenone and cimetidinerdquo JapaneseJournal of Pharmacology vol 57 no 4 pp 495ndash505 1991

[19] F Borrelli and A A Izzo ldquoThe plant kingdom as a source ofanti-ulcer remediesrdquo Phytotherapy Research vol 14 no 8 pp581ndash591 2000

[20] J R Vane ldquoInhibition of prostaglandin synthesis as a mecha-nism of action for aspirin-like drugsrdquo Nature New Biology vol231 no 25 pp 232ndash235 1971

[21] F M Birdane M Cemek Y O Birdane I Gulcin and EBuyukokuroglu ldquoBeneficial effects of Foeniculum vulgare onethanol-induced acute gastric mucosal injury in ratsrdquo WorldJournal of Gastroenterology vol 13 no 4 pp 607ndash611 2007

[22] E Marhuenda M J Martin and C A de La Lastra ldquoAntiul-cerogenic activity of aescine in different experimental modelsrdquoPhytotherapy Research vol 7 no 1 pp 13ndash16 1993

[23] C la-Casa I Villegas C Alarcon de la Lastra V Motilva andM J Martın Calero ldquoEvidence for protective and antioxidantproperties of rutin a natural flavone against ethanol inducedgastric lesionsrdquo Journal of Ethnopharmacology vol 71 no 1-2pp 45ndash53 2000

[24] S C F Van and P J Lea The Biochemistry of Plant PhenolicsProceedings of the Phytochemical Society of Europe OxfordUniversity Press Oxford UK 1985

[25] M Yoo M W Son I K Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 004541 1998

[26] M Yoo M W Son I Y Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo US Patent no 6025387 2000

[27] M Yoo M W Son I Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 0915864 B104092002

[28] C D Sadik H Sies and T Schewe ldquoInhibition of 15-lipoxygen-ases by flavonoids structure-activity relations and mode ofactionrdquo Biochemical Pharmacology vol 65 no 5 pp 773ndash7812003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

[9] P Malfertheiner F K Chan and K E McColl ldquoPeptic ulcerdiseaserdquoThe Lancet vol 374 no 9699 pp 1449ndash1461 2009

[10] Agencia Nacional de Vigilancia Sanitaria Formulario deFitoterapicos da Farmacopeia Brasileira ANVISA Rio deJaneiro Brzil 2011

[11] C F C B R de Almeida T C de Lima E Silva E L C deAmorim M B D S Maia and U P de Albuquerque ldquoLifestrategy and chemical composition as predictors of the selectionof medicinal plants from the caatinga (Northeast Brazil)rdquoJournal of Arid Environments vol 62 no 1 pp 127ndash142 2005

[12] C D F C B R Almeida E L C de Amorim U P de Albu -querque andM B S Maia ldquoMedicinal plants popularly used inthe Xingo regionmdasha semi-arid location in Northeastern BrazilrdquoJournal of Ethnobiology and Ethnomedicine vol 2 article 152006

[13] AA R SilvaMMarques BezerraHVasconcelosChaves et alldquoProtective effect of Chresta martii extract on ethanol-inducedgastropathy depends on alpha-2 adrenoceptors pathways butnot on nitric oxide prostaglandins or opioidsrdquo Journal ofEthnopharmacology vol 142 no 1 pp 206ndash212 2012

[14] A A R Silva M M Bezerra H V Chaves et al ldquoProtectiveeffect of Chresta martii extract against indomethacin-inducedgastric lesions inmicerdquo Journal of Natural Medicines vol 67 no1 pp 143ndash151 2013

[15] J Singh ldquoMaceration percolation and infusion techniques forthe extraction of medicinal and aromatic plantsrdquo in ExtractionTechnologies for Medicinal and Aromatic Plants p 259 UnitedNations Industrial Development Organization and the Interna-tional Centre for Science and High Technology Triseste Italy2008

[16] A F Costa Farmacognosia vol 2 Fundacao Calouste Gul-benkian Lisbon Portugal 4th edition 1994

[17] S Szabo J S Trier A Brown J Schnoor H D Homanand J C Bradford ldquoA quantitative method for assessing theextent of experimental gastric erosions and ulcersrdquo Journal ofPharmacological Methods vol 13 no 1 pp 59ndash66 1985

[18] Y Morimoto K Shimohara S Oshima and T SukamotoldquoEffects of the new anti-ulcer agent KB-5492 on experimentalgastric mucosal lesions and gastric mucosal defensive factorsas compared to those of teprenone and cimetidinerdquo JapaneseJournal of Pharmacology vol 57 no 4 pp 495ndash505 1991

[19] F Borrelli and A A Izzo ldquoThe plant kingdom as a source ofanti-ulcer remediesrdquo Phytotherapy Research vol 14 no 8 pp581ndash591 2000

[20] J R Vane ldquoInhibition of prostaglandin synthesis as a mecha-nism of action for aspirin-like drugsrdquo Nature New Biology vol231 no 25 pp 232ndash235 1971

[21] F M Birdane M Cemek Y O Birdane I Gulcin and EBuyukokuroglu ldquoBeneficial effects of Foeniculum vulgare onethanol-induced acute gastric mucosal injury in ratsrdquo WorldJournal of Gastroenterology vol 13 no 4 pp 607ndash611 2007

[22] E Marhuenda M J Martin and C A de La Lastra ldquoAntiul-cerogenic activity of aescine in different experimental modelsrdquoPhytotherapy Research vol 7 no 1 pp 13ndash16 1993

[23] C la-Casa I Villegas C Alarcon de la Lastra V Motilva andM J Martın Calero ldquoEvidence for protective and antioxidantproperties of rutin a natural flavone against ethanol inducedgastric lesionsrdquo Journal of Ethnopharmacology vol 71 no 1-2pp 45ndash53 2000

[24] S C F Van and P J Lea The Biochemistry of Plant PhenolicsProceedings of the Phytochemical Society of Europe OxfordUniversity Press Oxford UK 1985

[25] M Yoo M W Son I K Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 004541 1998

[26] M Yoo M W Son I Y Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo US Patent no 6025387 2000

[27] M Yoo M W Son I Kim et al ldquoGastroprotective flavoneflavanone compounds with therapeutic effect on inflammatorybowel diseaserdquo European Patent Office (EPO) 0915864 B104092002

[28] C D Sadik H Sies and T Schewe ldquoInhibition of 15-lipoxygen-ases by flavonoids structure-activity relations and mode ofactionrdquo Biochemical Pharmacology vol 65 no 5 pp 773ndash7812003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom