Antiulcerogenic Activity of the Hydroalcoholic Extract of Leaves of Croton campestris A. St.-Hill in...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 579346 10 pageshttpdxdoiorg1011552013579346

Research ArticleAntiulcerogenic Activity of the Hydroalcoholic Extract of Leavesof Croton campestris A St-Hill in Rodents

Francisco E B Juacutenior Dayanne R de Oliveira Elizacircngela B Bento Laura H I LeiteDaniele O Souza Ana Luiza A Siebra Renata S Sampaio Anita O P B MartinsAndreza G B Ramos Saulo R Tintino Luiz J Lacerda-Neto Patricia R L FigueiredoLarissa R Oliveira Cristina K S Rodrigues Valterluacutecio S SalesFrancisco R S D N Figueiredo Emmily P Nascimento Alefe B MonteiroEacuterika N Amaro Joseacute G M Costa Henrique Douglas Melo CoutinhoIrwin R A de Menezes and Marta R Kerntopf

Department of Biological Chemistry Regional University of Cariri Crato CE Brazil

Correspondence should be addressed to Henrique Douglas Melo Coutinho hdmcoutinhogmailcom

Received 20 February 2013 Revised 24 May 2013 Accepted 27 May 2013

Academic Editor Ulysses Paulino de Albuquerque

Copyright copy 2013 Francisco E B Junior et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Croton campestris A St-Hill popularly known as ldquovelame do campordquo is a species native to the savannah area of Northeast Brazilwhich is used by traditional communities in folk medicine for variety of health problems especially detoxification inflammationand gastritisThe hydroalcoholic extract of C campestris leaves (HELCC) was assessed for its antiulcerogenic effect in gastric lesionmodels and effect on intestinal motility in mice and possible mechanisms of action were examined HELCC showed significantgastroprotective action in all models of gastric ulcer evaluated the results suggest that this action probably involves the nitric oxidepathway HELCC did not show alteration of intestinal motility in mice It was also found that C campestris represents a promisingnatural source with important biological potential justifying some of its uses in folk medicine

1 Introduction

Peptic ulcer is a term used to describe a group of ulcerativedisorders that occur in areas of the upper gastrointestinaltract which are exposed to acidic secretions and pepsin Itrepresents a chronic health problem Approximately 10 ofthe population have or will develop peptic ulcer Its incidenceis slightly higher in men than women (13 1) and althoughit occurs at any age duodenal ulcer occurs most often in therange of 30ndash55 years whereas gastric ulcer occurs in the rangeof 50ndash70 years [1]

In Brazil despite the absence of epidemiological recordsof ulcer cases it is known that there are numerous cases in-volving this disease which means a significant public healthproblem prompting the search for new substances with anti-ulcerogenic activity Although there is a large arsenal of drugswith antiulcerogenic activity on the market none produces

100 remission of ulcers with reduced side effects andwithout compromising the patientrsquos wellbeing which usuallyresults in chronic use of these drugs Published studies havereported the widespread identification of new drugs derivedfrom natural antiulcerogenic sources [2]

Studies have shown that lipid peroxidation and oxidativereactions are implicated in the pathogenesis of lesionsinduced by ethanol by attacking biological molecules andprostaglandinsThus research in animalmodels has attractedthe interest of many investigators as a promising source forthe discovery of new drugs [3ndash5]

The genusCroton whose namemeans ldquotickrdquo is the secondlargest in the family Euphorbiaceae and belongs to the sub-family Crotonoidae Crotoneae and tribe [6] It is subdividedinto 40 sections and has more than 1300 species of mainlypantropical distribution of which 350 species in 29 sections

2 Evidence-Based Complementary and Alternative Medicine

occur in Brazil [7] Of these a total of 52 species in 18 sectionsare referred to Northeast Brazil [8]

Several species of Croton have long played an importantrole in traditional uses of medicinal plants in Africa Asiaand South America including the treatment of cancer con-stipation diarrhea and other digestive problems diabetesexternal wounds fever hypercholesterolemia hypertensioninflammation intestinal worms malaria pain ulcers andobesity [9]

Studies involving several species of the genusCroton haveshown different pharmacological activities such as anti-inflammatory and antioxidant activities of C celtidifolius[10] antileishmanial effect of C cajucara [11] antinociceptiveeffect ofC cajucara [12] antiulcerogenic and cytotoxic activi-ties ofC cajucara [13] hypoglycemic and hypolipidemic pro-perties of C cajucara [14] antimicrobial and the antimalarialeffects C kongensis [15] antinociceptive and anti-inflam-matory properties of C malambo [16] antimicrobial effect ofC sonderianus [17] purgative effect of C campestris [18] andhypotensive and narcotic activities of C eluteria [19]

A study of C campestris in the traditional community ofthe bioregion of Chapada doAraripe revealed the popular useof the leaves and roots of this plant for gastric hematologicaland inflammatory disturbances wounds and respiratoryproblems For their medicinal uses the local populationutilizes infusion in alcoholic beverages teas and macerateswhich justifies the use of the hydroalcoholic extract for theverification of the antiulcer activity

The hydroalcoholic extract of leaves of C campestris ASt-Hill (HELCC) was assessed for its antiulcerogenic effectin models of gastric lesions using absolute ethanol acidifiedethanol and indomethacin and mechanisms of action wereexamined The effect of HELCC on gastric motility was alsoruled out

2 Materials and Methods

21 Plant Material The leaves of the plant species Crotoncampestris A St-Hill were collected in the municipality ofCrato Ceara Brazil using a GPS device (7∘ 221015840 2810158401015840 S 39∘ 28101584042410158401015840 W altitude 892 meters) The procedures for specimenpreparation followed the recommendations described byMing 1996 apud Di Stasi 1996 The material was depositedin the Herbarium of the Federal University of Rio Grande doNorte (UFRN) and registered under no 7095 The researchwas reviewed by the Ethics Committee for Animal ResearchCEPA Faculty of Medicine of Juazeiro do Norte (FMJ) andwas approved (case no 2009 0432 FR 271610)

22 Preparation of Hydroalcoholic Extract of the Leaves of Cro-tonCampestris A St-Hill (HELCC) Theplantmaterial (freshleaves 852 g C campestris) was washed under running watercrushed and macerated and then submitted to cold extrac-tionwith 999ethanol andwater in a 1 1 proportion (87 L ofsolvent) The solvent was removed using a rotary evaporatorunder reduced pressure and temperature of 40ndash50∘C and ayield of 7089 g crude extract was obtained using a lyophilizer

23 Phytochemical Screening The material was submitted tophytochemical screening for the presence of tanninsflavonoids saponins steroids triterpenes coumarins quin-ones organic acids and alkaloids Accordingly the hydro-alcoholic extract (03 g) was diluted in distilled water (9mL)and 70 ethanol (21mL) and after dilution distributed intosix flasks in which the tests for different compounds wereperformed Phenols and tannins 3 drops of ferric chloridewere added to the flask and development of a blue colordenoted the presence of pyrogallic tannins which could befurther confirmed with the addition of gelatin resulting in aprecipitate with the sample Anthocyanins anthocyanidinsand flavonoids the sample was acidified (pH 3) by the addi-tion of 18 drops of HCl and no change in color of the flaskdemonstrated the presence of flavones flavonols xanthonesand flavanonols

The test for leucoanthocyanidins catechins and flavon-ones was performed by alkalinization to pH 85 with 1sodium hydroxide in the first flask acidification (pH 3) bymeans of 18 drops of HCl and heating for 2min in the secondflask and alkalinization (pH 11) with 18 drops of 1 NaOHand also heating for 2min in the third flask Thus flask 2did not show a change in color demonstrating the presenceof flavonones and still the orange red color of flask 3 alsorevealed the presence of flavonones The test for chalconesand aurones by alkalinization (pH 11) with 18 drops of 1NaOH gave a purple red color in the flask indicating thepresence of these compounds

The test for alkaloids was done by diluting the extract(03 g) in 30mL of 5 acetic acid and 15mL of ammonia foralkalinization The preparation was heated until boiling and10mL of 10 ammonium hydroxide was then added alongwith 15mL of chloroform After homogenization and allow-ing to stand in a separatory funnel the chloroform phase wascollected in a beaker and the solvent was evaporated Theresidue was resuspended in 1 HCl and homogenized andsolution was placed on a slide along with a drop of Dragen-dorff reagent where the formation of a precipitate was indi-cative of the presence of alkaloids

The test for steroids and triterpenoids was carried out bydilution of the extract (03 g) in 6mL of chloroform and filtra-tion using cotton covered with sodium sulfate collecting thefiltrate in a test tube After the addition of 01mL of anhydrousacetic acid and three drops of sulfuric acid the developmentof a green color indicated the presence of steroids [20]

24 Assays

241 Assessment of Oral Acute Toxicity Swissmice were usedthroughout to evaluate HELCC for toxicity and effect ongastric lesions The animals were fasted for 3 h prior to theexperiment and were given a single dose of extract dissolvedin 2wv Tween 80 at 175 55 175 550 2000 and 5000mgkgand observed for mortality for up to 48 h (short-term toxi-city) Based on the short-term toxicity the dose of the nextanimal was determined as per OECD guideline 425 Allanimals were also observed for long-term toxicity (14 days)The LD50 of the test extracts was calculated using ldquoAOT 425rdquo

Evidence-Based Complementary and Alternative Medicine 3

software provided by the Environmental Protection AgencyUSA [21]

25 Gastric Lesions Induced by Ethanol [22] The mice weredivided into groups (119899 = 6) fasted for a period of 15 h andtreated with HELCC (50 75 125 250 500 and 750mgkgpo) omeprazole (30mgkg po) or vehicle (saline 01mL10 g po) 1 h before administration of absolute ethanol(02mLanimal po) After 30min the animals were sacri-ficed by cervical dislocation Their stomach was removedopened along the greater curvature and rinsed with salineand digitized the ulcerated area was expressed as a percent-age relative to the total area of the gastric body using ImageJsoftware

26 Gastric Lesions Induced by Acidified Ethanol [23] Themice were treated with HELCC (50 75 125 250 500 and750mgkg po) omeprazole (30mgkg po) or vehicle(saline 01mL10 g po for the control lesion group) Onehour after treatment the animals received 02mL of 03Mhydrochloric acid (HCl) in 60 ethanol and were sacrificed1 h later The percentage of stomach ulceration was deter-mined as described above

27 Gastric Lesions Induced by Indomethacin [24] The micewere pretreated with HELCC (250 500 and 750mgkg po)omeprazole (30mgkg po) or vehicle (saline 01mL10 gpo for the control lesion group) Six hours after administra-tion of the ulcerogenic agent (indomethacin 10mgkg sc)the animals were sacrificed The percentage of stomachulceration was determined as described above

28 Evaluation of the Mechanism of Action of GastroprotectiveHELCC To determine the mechanism of action experi-mentswere performed to separately examine the involvementof 120572-2 receptors prostaglandins [23] nitric oxide [25] andATP-dependent K+ channel activation [26] in the gastropro-tective effect of HELCC (75mgkg po) These experi-ments used the appropriate antagonists including yohim-bine (2mgkg ip) indomethacin (10mgkg po) L-NAME(10mgkg ip) and glibenclamidee (5mgkg po) or ago-nists including L-arginine (600mgkg po) as a positive con-trol for L-NAME and misoprostol (0016mgkg po) as acontrol for indomethacin before the oral administration of02mL of 96 ethanol

In each case the animals were pretreated with the specificantagonist or agonist for 30min before the administrationof HELCC Finally 02mL of 96 ethanol was orally admin-istered one hour after HELCC The animals were then sacri-ficed and their stomach was removed opened along thegreater curvature washed in saline and compressed betweenglass slides for better viewing The slides were scanned at1200 dpi The percentage area of gastric lesions (glandularportion) was determined with the aid of ImageJ softwareTheinjured area is expressed as a relative percentage of the totalarea of the gastric body [23 26]

29 Effect of HELCC on Intestinal Motility [27] The ani-mals were treated with HELCC (75mgkg po) vehicle

(saline 01mL10 g po) or atropine (001 gkg po) and 10activated charcoal (01mL10 g po) HELCC atropine orvehicle was administered first followed by activated char-coal 1 h later Thirty minutes after charcoal administrationthe animals were sacrificed and their small intestine wasremoved The total length of the intestine (the pyloric regionto the ileocecal junction) was then measured the distancetraveled by the charcoal was determined based on the dis-tance from the pylorus to the last portion of the intestine thatcontained at least 1 cmof continuous charcoalThirtyminutesafter administration of the charcoal the animals were sacri-ficed and analyzed as described above

210 Statistical Analysis Data are presented as the mean plusmnstandard deviation The level of significance was 005 Dif-ferences between the means were determined by analysis ofvariance (ANOVA) and the Newman-Keuls test was used todetermine statistical significance Analyses were performedusing GraphPad Prism software version 50

3 Results

Phytochemical prospecting [20] ofHELCC identified the pre-sence of tannins flavonols flavones flobabenics flavanoneflavonals xanthones terpenes and alkaloids (Table 1) possi-bly involved in the bioactive properties evaluated

31 Toxicological Data HELCC did not show evidence ofacute toxicity The daily monitoring of weight of animals didnot indicate a significant variation compared to the controlgroup (saline)There were no signs of morbidity or death justafter extract administration or during the period of obser-vation and weighing (14 days after treatment) in the grouptreated orally The median lethal dose (LD50) for the oraladministration of HELCC was greater than or equal to5000mgkg with a 95 confidence interval

The animals showed decreased activity andmild dyspneashowing recovery within four hours

32 Gastroprotection Tests The effects of HELCC on gastriclesions induced by absolute ethanol (02mLanimal po) areshown in Figure 1 The animals that received only vehiclecombined with oral administration of absolute ethanolshowed an extensive area of gastric lesion (1857 plusmn 187)HELCCproduced amarked reduction in the area damaged byabsolute ethanol at all doses tested 134 plusmn 066 at 50mgkg(9278 reduction) 111plusmn067 at 75mgkg (9402) 093plusmn043 at 125mgkg (9499) 087 plusmn 054 at 250mgkg(9531) 082 plusmn 031 at 500mgkg (9558) and 053 plusmn016 at 750mgkg (9714) when compared to the control(119875 lt 0001 in all cases)The animals that received omeprazole(30mgkg po) also showed a significant decrease in gastriclesion area and a substantial reduction percentage in ulcerarea that is 078plusmn034 and 9579 respectively comparedto control (119875 lt 0001)

The effects of HELCC on gastric lesions induced by acidi-fied ethanol (02mLanimal po) are shown in Figure 2


Table 1 Prospecting photochemistry of hydroalcoholic extract oflyophilized of leaves of Croton campestris A St-Hill

Metabolites (+) presence(minus) absence

Phenols minus

Pyrogallic tannins minus

Flobabenic tannins +Anthocyanins minus

Anthocyanidins minus

Flavones +Flavonols +Xanthones +Chalcones minus

Aurones minus

Flavanonols +Leucoanthocyanidin minus

Catechin minus

Flavonone +Alkaloids +Terpenes +Steroids minus

HEFCCEthanol absolute (01 mL10 g vo)

(mgkg)

Gas

tric

ulc

erat

ed ar

ea (

)

CL305075

125250500750

0

5

10

15

20

25

lowastlowastlowast

CL 30 50 75 125 250 500 750

lowastlowastlowast lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

Omeprazole

Figure 1 Effect of oral administration of the HEFCC in gastriclesions induced by ethanol abs in mice Significance lowastlowastlowast119875 lt 0001when compared with control group

The animals that received only vehicle combined with oraladministration of acidified ethanol showed an extensive areaof gastric lesion (2319 plusmn 309) HELCC showed substantialreduction in the areas damaged by acidified ethanol at alldoses tested 304plusmn080 at 50mgkg (reduction of 8689)194 plusmn 070 at 75mgkg (9163) 167 plusmn 075 at 125mgkg(9279) 240 plusmn 099 at 250mgkg (8965) 052 plusmn 030at 500mgkg (9775) and 059 plusmn 026 at 750mgkg

Gas

tric

ulc

erat

ed ar

ea (

)

HEFCC

Ethanol-HCL (01 mL10 g vo)

(mgkg)

CL305075

125250500750

0

10

20

30

lowastlowastlowast

CL 30 50 75 125 250 500 750

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast


Omeprazole

Figure 2 Effect of oral administration of the HEFCC in gastriclesions induced by acid ethanol in mice Significance lowastlowastlowast119875 lt 0001when compared with control group

(9745) respectively when compared to the control (119875 lt0001 in all cases) The animals that received omeprazole(30mgkg po) also showed a significant reduction in theinjured areas and a high percentage reduction in ulcer areathat is 358 plusmn 056 and 8456 respectively compared tocontrol (119875 lt 0001)

The effects of HELCC on gastric lesions induced by indo-methacin (10mgkg sc) are shown in Figure 3 The animalsthat received only vehicle combined with sc administrationof indomethacin showed a large area of gastric lesion (1261plusmn327) HELCC produced a significant reduction in the areasdamaged by indomethacin at all doses tested 063plusmn011 at250mgkg (reduction of 9500) 033 plusmn 008 at 500mgkg(9738) and 047 plusmn 017 at 750mgkg (9738) respec-tively when compared to the control (119875 lt 0001 in all cases)The animals that received omeprazole (30mgkg po) alsoshowed significant reduction in gastric lesion area and a sub-stantial percentage reduction in ulcer area that is 040 plusmn024 and 9682 respectively compared to control (119875 lt0001)

In the evaluation of the role of nitric oxide (NO) in theprotective effect of HELCC against gastric lesions induced byabsolute ethanol inmice it was observed that the animals thatreceived only vehicle combined with oral administration ofabsolute ethanol showed gastric lesion (2052 plusmn 229) Theanimals that received L-NAME (10mgkg ip) an inhibitorof nitric oxide synthase (NOS) along with absolute ethanol(02mLanimal po) also showed a large percentage ofulcerated area (2873 plusmn 383) However the animals thatreceived L-arginine (600mgkg po) in combination withabsolute ethanol exhibited reductions in injured area (147 plusmn


HEFCC

(mgkg)

Gas

tric

ulc

erat

ed ar

ea (

)

CL 30 250 500 7500

5

10

15

20

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

CL30250

500750

Omeprazole

Indomethacin (10 mgkg sc)

Figure 3 Effect of oral administration of the HEFCC in gastriclesions induced by indomethacin inmice Significance lowastlowastlowast119875 lt 0001when compared with control group

038) with percentage of ulcer reduction of 9283HELCC(75mgkg po) produced a significant (119875 lt 0001) reductionin ulcerated area to 111plusmn 067 or a percentage reduction of9459 compared to control However HELCC (75mgkgpo) when given with L-NAME (10mgkg ip) had its effectblocked resulting in a percentage of ulcerated area of 3065plusmn655 (Figure 4)

In the investigation of the role of prostaglandins in theprotective effect of HELCC against gastric lesions induced byabsolute ethanol in mice it was seen that the animals thatreceived only vehicle in combination with oral administra-tion of absolute ethanol showed a gastric lesion area of 1857plusmn187 The animals that received indomethacin (10mgkgsc) together with absolute ethanol (02mLanimal po)displayed a percentage of ulcerated area of 1309 plusmn 192However the animals that receivedmisoprostol (0016mgkgpo) combined with absolute ethanol showed reductions ininjured area (057 plusmn 025) resulting in a percentage reduc-tion of 9695 HELCC (75mgkg po) produced a signi-ficant (119875 lt 0001) reduction in ulcerated area to 111plusmn 067or a percentage reduction of 9402 compared to the controlHELCC (75mgkg po) when combined with indomethacin(10mgkg sc) resulted in a percentage of ulcerated area of395 plusmn 115 and a percentage reduction in ulcer area of7872 (Figure 5)

In the investigation of the role of the alpha-2 noradrener-gic receptor in the protective effect of HELCC against gastriclesions induced by absolute ethanol in mice it was seen thatthe animals that received only vehicle combined with oraladministration of absolute ethanol showed a gastric lesionarea of 2322 plusmn 208 The animals that received yohimbine

HEFCC

L-NAME L-NAME

(mgkg)

HEFCC

abc abc

Gas

tric

ulc

erat

ed ar

ea (

)

CL 10 600 10 750

10

20

30

40

75CL10600

10

75

Ethanol absolute (01 mL10 g vo)

L-arginine

(mgkg)

Figure 4The role of nitric oxide (NO) in the gastroprotector effectof the HEFCC in gastric lesions model induced by ethanol absolutein mice Significance a

119875 lt 0001 versus CL b119875 lt 0001 versus L-

NAME (10mgKg vo) and c119875 lt 0001 versus L-NAME (10mgkg

vo) + EHFCC (75mgkg vo)

CL 10 0016 10 750

5

10

15

20

25

HEFCC75

HEFCC


ac

ac

ac

CL100016

1075

Gas

tric

ulc

erat

ed ar

ea (

)

(mgkg)

MisoprostolIndomethacinIndometacine

(mgkg)

Figure 5 The role of prostaglandins in gastroprotector effect of theHEFCC in gastric lesionmodel induced by ethanol absolute inmiceSignificance a

119875 lt 0001 versus CL b119875 lt 001 versus CL c

119875 lt 0001

versus indomethacin (10mgKg sc)


(2mgkg ip) togetherwith absolute ethanol (02mLanimalpo) exhibited an extensive ulcerated area of 3034 plusmn 496HELCC (75mgkg po) produced a significant (119875 lt 0001)reduction in ulcerated area to 091 plusmn 063 or a percentagereduction of 9634 compared to the control Also HELCC(75mgkg po) given togetherwith yohimbine (2mgkg ip)produced a significant (119875 lt 001) gastroprotective effect witha percentage of ulcerated area of 930plusmn059 or a percentagereduction in ulcer area of 5994 (Figure 6)

In the evaluation of the involvement of ATP-dependentK+ channels in the protective effect of HELCC against gastriclesions induced by absolute ethanol in mice it was observedthat the animals that received only vehicle in combinationwith oral administration of absolute ethanol displayed a gas-tric lesion area of (1717 plusmn 244) The animals that receivedglibenclamidee (5mgkg ip) along with absolute ethanol(02mLanimal po) showed an ulcerated area of 1330 plusmn225) HELCC (75mgkg po) produced a significant (119875 lt0001) reduction in ulcerated area to 111 plusmn 067 or per-centage reduction of 9165 compared to the control AlsoHELCC (75mgkg po) when given with glibenclamidee(5mgkg ip) still exerted a significant gastroprotective effect(119875 lt 0001)with a percentage of ulcerated area of 123plusmn053or percentage reduction in ulcer area of 9075 (Figure 7)

33 Evaluation of Intestinal Motility The percentage of thedistance traveled by the marker (activated charcoal) in thesmall intestine of mice was (8277 plusmn 429) The animals thatreceived HELCC (75mgkg) orally had a distance traveledby the marker (8420 plusmn 394) that was similar to thatobtained by vehicle group demonstrating that HELCC didnot significantly affect gastrointestinal motility However inthe group treated with atropine (001 gkg po) a muscarinicantagonist there was a decrease in intestinal motility with adistance traveled of 6321 plusmn 205 compared to the vehiclecontrol group (saline po) showing a significant effect (119875 lt001) as seen in the data presented in Figure 8

4 Discussion

Several species of the genus Croton have played an importantrole in traditional uses of medicinal plants in Africa Asiaand South America Such uses include the treatment ofcancer constipation diarrhea and other digestive problemsexternal wounds diabetes dyslipidemia hypertension feverinflammation intestinal worms malaria pain ulcers andobesity [9] In the toxicological evaluation HELCC did notshow signs of significant acute toxicity in the animals testedand at all doses administered corroborating a previous studythat demonstrated the low acute toxicity of Croton campestrisA St-Hill in two guinea pigs [28]

As seen in the evaluation of the gastroprotective activityof HELCC experimental animals play an important role inthe search for new drugs with protective properties Consid-ering that the etiology of ulcer is multifactorial lesions inthe gastric mucosa can be induced in different experimentalmodels through various mechanisms [29] Some of the most

CL 2 2 750

10

20

30

40

HEFCC


HEFCC75

(mgkg)

(mgkg)

ab

bc

CL2

275

Gas

tric

ulc

erat

ed ar

ea (

)

Yohimbine Yohimbine

Figure 6 The role of noradrenergic receptor alpha2

in the gas-troprotector effect of HEFCC in gastric lesion model induced byethanol abs in mice Significance a

119875 lt 0001 versus CL b119875 lt 0001

versus glibenclamide (5mgkg ip)

used acute models for the evaluation of antiulcerogenic sub-stances in animals are the models of gastric lesion induced byabsolute ethanol or indomethacin [30] In this work besidesthe use of these methodologies the effects of HELCC werealso evaluated with the gastric ulceration model using acidi-fied ethanol

Ethanol-induced gastric ulcer occurs predominantly inthe glandular portion of the stomach as a result of a directnecrotizing action and moreover the lack of defense factorssuch as secretion of bicarbonate and mucus and increasedoxidative stress [31] HELCC at all doses tested by oral admin-istration prevented in a dose-dependent manner gastriclesion induced by the administration of absolute ethanolThedoses decreased the percentage of gastric ulcer area in thesame manner as with omeprazole a well-known protonpump inhibitor when compared to the vehicle-treated con-trol lesion group It was also demonstrated that the gastro-protective action of HELCC at all orally administered dosesagainst gastric ulcer induced by acidified ethanol resultedin a percentage reduction in ulcer area similar to that withomeprazole in comparison with the vehicle-treated controllesion group

Indomethacin like the majority of nonsteroidal anti-inflammatory drugs (NSAIDs) acts as an inhibitor of pros-taglandin synthesis and consequently diminishes the defensemechanisms of gastric mucus [32ndash34] It is the primarymechanism by which this class of drugs cause damage to thegastrointestinal tract [35]

This research demonstrates the ability of the HELCC toinhibit the gastric damage caused by NSAIDs where we


CL 5 5 750

5

10

15

20

25

HEFCC


HEFCC

75

(mgkg)

ab ab

CL5

575

Gas

tric

ulc

erat

ed ar

ea (

)

Glibenclamida Glibenclamida

(mgkg)

Figure 7The role of ATP-dependent K+ channels in the gastropro-tector effect of HEFCC in gastric lesion model induced by ethanolabsolute in mice Significance a

119875 and a119875 lt 0001 versus CL b

119875 lt

0001 versus glibenclamide (5mgkg ip)

used a classic model of induction of gastric lesion by scindomethacin Orally administered HELCC at the dosestested was able to prevent the appearance of gastric lesions inanimals subjected to the treatment with this NSAID showingsignificant results when compared to the gastric lesion con-trol

Among the various factors involved in maintaining theintegrity of the gastric mucus and protection against injurycaused by ulcerogenic agents we examined the contributionof endogenous nitric oxide prostaglandins ATP-sensitivepotassium channels (KATP) and alpha-2 noradrenergicreceptor [36 37] to the action of HELCC on the gastricmucosa

The discovery of nitric oxide (NO) as an agent of cellsignaling was one of the most important events in humanphysiology of the last 80ndash90 years NO is a free radical gaswhich has drawn attention because of its role in signal trans-duction related to various physiological processes such assmooth muscle relaxation and vasodilation neurotransmis-sion platelet aggregationmechanisms regulation of pro- andantiapoptoticmechanisms and control of blood pressure andblood flow [38 39]

Related to the modulation of the physiological compo-nents of gastrointestinal tract NO plays an important role inthe control of gastrointestinalmotility [40] gastric blood flow[41] recruitment of neutrophils [42] and secretion of mucus[43]

In the present research the assessment of the role of NOin the gastroprotective effect of HELCC in models of gastriclesions induced by ethanol in mice showed that the ani-mals that received L-NAME (a nonspecific inhibitor of NO

HEFCC (75 mgkg vo)

Activate charcoal 10 (01 mL10 g vo)

Atropine (001 gKg vo)

Dist

ance

trav

elle

d pe

lo b

y ch

arco

al (

)

C 001 750

20

40

60

80

100

lowastlowast

C00175

Figure 8 The effect of HEFCC on intestinal motility in miceSignificance lowastlowast119875 lt 001 versus C (vehicle control)

synthase) showed a large ulcerated gastric area after admin-istration of absolute ethanol as in the control lesion groupHELCC managed to inhibit the appearance of gastric lesionsin the gastroprotection model using L-NAMEThe extract inthis situation showed a non-significant percentage reductionin ulcer area Similarly it was seen that the L-arginine (a sub-strate of NO synthase) significantly reduced the percentageof ulcerated gastric area when compared with the vehicle-treated control lesion group This suggests that the com-pounds present in the extract that exert a gastroprotectiveeffect involving the cytoprotective effect of NO

Considering the traditional use of the leaves of the speciesCroton campestris A St-Hill for gastric disturbances in theform of teas and infusion and in view of the presence of suchsecondarymetabolites as tannins flavonoids and alkaloids inthe extract it is possible to correlate the antiulcerogenic effectdemonstrated by the plant with these compounds Also it ispossible to correlate studies that demonstrate an associationbetween plant species that contain tannins flavonoids andalkaloids and gastroprotective effects [44 45] as probablyoccur with Croton campestris A St-Hill

The protective action of prostaglandins (PGs) on thegastric mucosa is mediated by an increase in mucus produc-tion and secretion of bicarbonate modulation of gastric acidsecretion inhibition of the release of inflammatorymediatorsbymast cells and themaintenance of blood flowduring expo-sure to irritants It is known that prostaglandin E2 (PGE2)has a gastroprotective action against lesions caused by ethanoland that this protection stems from an increase in intracellu-lar guanosine-31015840 51015840-cyclic monophosphate (cGMP) which ismediated by an increase in intracellular free calcium concen-tration and nitric oxide production [46]


In the evaluation of a possible involvement of prostaglan-dins in the gastroprotective effect of HELCC we used miso-prostol an analogue of prostaglandin E1 (PGE1) as well asindomethacin and an inhibitor of PG synthesis Misoprostolsignificantly inhibited the development of gastric lesionswhen comparedwith the vehicle-treated control lesion groupPretreatment with indomethacin did not reverse the cytopro-tective effect of HELCC demonstrating just a trend in thisdirection On the basis of this result we cannot state thatprostaglandins play a role in the relaxant effect of HELCC

Themodulation of 1205722 receptors located in the intramuralperipheral parasympathetic ganglia decreases the dischargeof vagal acetylcholine which reduces gastric secretion andmotility and increases blood flow The 1205722-noradrenergicreceptors and the 1205722 presynaptic are involved in the regula-tion of gastric acid secretion and are effective in protectionagainst chemical agents such as NSAIDs and ethanol wheretheir effectsmay bemediated central and peripheral receptors[47]

In our study the administration of yohimbine (10mgkgip) an indole alkaloid that promotes the release of neuro-transmitters by blocking the presynaptic 1205722 receptors [48]did not reverse the HELCC effect This indicates that thegastroprotective effect of HELCC does not act by modulationof 1205722 receptor activity

KATP channels are regulated by ligands and their mecha-nism is defined based on their sensitivity to intracellular ATPwhich inhibits its activity It has been postulated that KATPchannels are involved in a variety of pathophysiological func-tions in the stomach such as regulation of blood flow gastricacid secretion and gastric muscle contractility [49] In thevascular system these channels are related to the relaxationof vascular smoothmuscle having an important role in bloodpressure control This vasodilation can be blocked by gly-buride a sulfonylurea that blocks KATP channels [50] Ourstudies showed that HELCC when combined with gliben-clamidee significantly retained its gastroprotective effectindicating that the mechanism of action of the active ingredi-ents of the extract may not involve the stimulation of KATPchannels

Another way in which HELCC could promote protectionof the gastric mucosa would be through increased gastro-intestinal motility so increased intestinal transit would acce-lerate gastric emptying thereby decreasing the effect ofaggressor acid in the stomach and duodenum Cholinergicinnervation of the circular muscle layer of TGI acting onmuscarinic receptors M1 andM3 is primarily responsible forgastrointestinal motility

In this sense HELCC did not significantly alter the intes-tinal transit of mice when compared to the vehicle group(10 activated charcoal) and the group that received atropinewhich is a drug blocking the muscarinic action of acetyl-choline

5 Conclusion

In short we demonstrate here for the first time that HELCCan extract of leaves of Croton campestris A St-Hill has

a protective effect against acute gastric lesion induced byabsolute ethanol acidified ethanol or indomethacin in micethus indicating a cytoprotective action of HELCC whichoccurs through the potentiation of the NOcGMP pathwayThe leaves of C campestris contain compounds (metabolites)that possibly act in synergy in the activation of defense factorsand in reducing aggressor factors of gastric mucosa whichmakes this extract promising for the development of newtherapies to combat gastropathy associated with NSAID-induced peptic ulcer disease

References

[1] E Rubin and J P Palazzo ldquoTrato gastrointestinalrdquo in PatologiaBases Clinicopatologicas E Rubin Ed chapter 13 pp 673ndash750Guanabara Koogan Rio de Janeiro Brazil 2006

[2] A Jamal K Javed M Aslam andM A Jafri ldquoGastroprotectiveeffect of cardamom Elettaria cardamomum Maton fruits inratsrdquo Journal of Ethnopharmacology vol 103 no 2 pp 149ndash1532006

[3] J E CarvalhoAtividade antiulcerogenica e anticancer de produ-tos naturais e de sıntese Divisao de Farmacologia e ToxicologiaUniversidade de Campinas 2006

[4] A H Gilani andAtta-ur-Rahman ldquoTrends in ethnopharmacol-ogyrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 43ndash492005

[5] D J Newman G M Cragg and K M Snader ldquoNatural prod-ucts as sources of new drugs over the period 1981ndash2002rdquo Jour-nal of Natural Products vol 66 no 7 pp 1022ndash1037 2003

[6] R Braga Plantas do Nordeste especialmente do Ceara EscolaSuperior de Agricultura de Mossoro Fortaleza Brazil 3rd edi-tion 1976

[7] P Berry Croton Research Network University of WisconsinBoard of Regents Madison Wis USA 2006

[8] D S Carneiro-Torres I Cordeiro and F A Franca ldquoA famıliaEuphorbiaceae na flora de inselbergs da regiao de MilagresBahia Brasilrdquo Boletim de Botanica da Universidade de SaoPaulo vol 20 pp 31ndash47 2002

[9] A Salatino M L F Salatino and G Negri ldquoTraditional useschemistry and pharmacology of croton species (Euphor-biaceae)rdquo Journal of the Brazilian Chemical Society vol 18 no1 pp 11ndash33 2007

[10] G M Nardi R Felippi S DalBo et al ldquoAnti-inflammatory andantioxidant effects of Croton celtidifolius Barkrdquo Phytomedicinevol 10 no 2-3 pp 176ndash184 2003

[11] M D S S Rosa R RMendonca-Filho H R Bizzo et al ldquoAnti-leishmanial activity of a linalool-rich essential oil from Crotoncajucarardquo Antimicrobial Agents and Chemotherapy vol 47 no6 pp 1895ndash1901 2003

[12] A R Campos F A A Albuquerque V S N Rao M A MMaciel and A C Pinto ldquoInvestigations on the antinociceptiveactivity of crude extracts from Croton cajucara leaves in micerdquoFitoterapia vol 73 no 2 pp 116ndash120 2002

[13] A A B Almeida P S Melo C A Hiruma-Lima et al ldquoAnti-ulcerogenic effect and cytotoxic activity of semi-synthetic cro-tonin obtained from Croton cajucara Benthrdquo European Journalof Pharmacology vol 472 no 3 pp 205ndash212 2003

[14] R M Silva F A Santos V S N Rao M A Maciel and A CPinto ldquoBlood glucose- and triglyceride-lowering effect of trans-dehydrocrotonin a diterpene from Croton cajucara Benth in


ratsrdquo Diabetes Obesity and Metabolism vol 3 no 6 pp 452ndash456 2001

[15] J Thongtan P Kittakoop N Ruangrungsi J Saenboonruengand Y Thebtaranonth ldquoNew antimycobacterial and antimalar-ial 89-secokaurane diterpenes from Croton kongensisrdquo Journalof Natural Products vol 66 no 6 pp 868ndash870 2003

[16] A I Suarez R S Compagnone M M Salazar-Bookaman etal ldquoAntinociceptive and anti-inflammatory effects of Crotonmalambo Bark aqueous extractrdquo Journal of Ethnopharmacologyvol 88 no 1 pp 11ndash14 2003

[17] J D Mcchesney A M Clark and E R Silveira ldquoAntimicrobialditerpenes of Croton sonderianus II ent-Beyer-15-en-18-oicacidrdquoPharmaceutical Research vol 8 no 10 pp 1243ndash1247 1991

[18] F E Babili C Moulis M Bon M-J Respaud and I FourasteldquoThree furano-diterpenes from the Bark of Croton campestrisrdquoPhytochemistry vol 48 no 1 pp 165ndash169 1998

[19] C Vigor N Fabre I Fouraste and C Moulis ldquoThree clerodanediterpenoids from Croton eluteria Bennettrdquo Phytochemistryvol 57 no 8 pp 1209ndash1212 2001

[20] F J A Matos Introducao a fitoquımica experimental EdicoesUFC Fortaleza Brazil 1997

[21] OECDmdashGuidlines for the testing of chemicals Acute OralToxicity-Up-and-Down-Procedure (UDP) 2008

[22] A Robert J E Nezamis C Lancaster and A J Hanchar ldquoCyto-protection by prostaglandins in rats Prevention of gastricnecrosis produced by alcohol HCl NaOH hypertonic NaCland thermal injuryrdquoGastroenterology vol 77 no 3 pp 433ndash4431979

[23] TMizui N Shimono andMDoteuchi ldquoA possiblemechanismof protection by polyamines against gastric damage inducedby acidified ethanol in rats polyamine protection may dependon its antiperoxidative propertiesrdquo The Japanese Journal ofPharmacology vol 44 no 1 pp 43ndash50 1987

[24] B Djahanguiri ldquoThe production of acute gastric ulceration byindomethacin in the ratrdquo Scandinavian Journal of Gastroenterol-ogy vol 4 no 3 pp 265ndash267 1969

[25] H Matsuda Y Li and M Yoshikawa ldquoRoles of capsaicin-sen-sitive sensory nerves endogenous nitric oxide sulfhydryls andprostaglandins in gastroprotection by momordin Ic an oleano-lic acid oligoglycoside on ethanol-induced gastric mucosallesions in ratsrdquo Life Sciences vol 65 no 2 pp PL27ndashPL32 1999

[26] M Rahgozar H Pazokitoroudi A Bakhtiarian and B Dja-hanguiri ldquoDiazoxide a K

119860119879119875

opener accelerates restitutionof ethanol or indomethacin-induced gastric ulceration in ratsindependent of polyaminesrdquo Journal of Gastroenterology andHepatology vol 16 no 3 pp 290ndash296 2001

[27] A J Lapa C Souccar M T R Lima-Landman M A SCastro and T C M Lima Metodos de avaliacao da atividadefarmacologica de plantas medicinais Sociedade Brasileira dePlantas Medicinais Sao Paulo Brazil 2008

[28] EM Ribeiro PrataM Q Paulo and A RM Souza Brito ldquoIso-lamento do princıpio ativo de Croton campestris St Hill(Euphorbiaceae)rdquo Revista Brasileira de Farmacologia vol 74no 2 pp 36ndash41 1993

[29] G E Samonina G N Kopylova G V Lukjanzeva et al ldquoAnti-ulcer effects of amylin a reviewrdquo Pathophysiology vol 11 no 1pp 1ndash6 2004

[30] R S Pandian C V Anuradha and P Viswanathan ldquoGastropro-tective effect of fenugreek seeds (Trigonella foenum graecum) onexperimental gastric ulcer in ratsrdquo Journal of Ethnopharmacol-ogy vol 81 no 3 pp 393ndash397 2002

[31] C Rujjanawate D Amornlerdpison S Pojanagaroon and DKanjanapothi ldquoAnti-gastric ulcer effect of Kaempferia parvi-florardquo Journal of Ethnopharmacology vol 102 no 1 pp 120ndash1222005

[32] D A Lewis and P J Hanson ldquoAnti-ulcer drugs of plant originrdquoin Progress Medicinal Chemistry G P Ellis and G BWest Edsvol 28 pp 201ndash231 Elsevier Science Publishers AmsterdamThe Netherlands 1991

[33] Y Morimoto K Shimohara S Oshima and T SukamotoldquoEffects of the new anti-ulcer agent KB-5492 on experimentalgastricmucosal lesions and gastricmucosal defensive factors ascompared to those of teprenone and cimetidinerdquo The JapaneseJournal of Pharmacology vol 57 no 4 pp 495ndash505 1991

[34] F Evans ldquoThe gastro-intestinal Tractrdquo in Selection Preparationand Pharmacological Evaluation of Plant Material pp 25ndash451996

[35] J L Wallace ldquoNonsteroidal anti-inflammatory drugs and gas-troenteropathy the second hundred yearsrdquo Gastroenterologyvol 112 no 3 pp 1000ndash1016 1997

[36] O S Zayachkivska S J Konturek D Drozdowicz T Brzo-zowski and M R Gzhegotsky ldquoInfluence of plant-originatedgastroproteciive and antiulcer substances on gastric mucosalrepairrdquo Fiziologicheskiı Zhurnal vol 50 no 6 pp 118ndash127 2004

[37] T Brzozowski P C Konturek D Drozdowicz et al ldquoGrape-fruit-seed extract attenuates ethanol-and stress-induced gastriclesions via activation of prostaglandin nitric oxide and sensorynerve pathwaysrdquo World Journal of Gastroenterology vol 11 no41 pp 6450ndash6458 2005

[38] M W Radomski and S Moncada ldquoRegulation of vascularhomeostasis by nitric oxiderdquo Thrombosis and Haemostasis vol70 no 1 pp 36ndash41 1993

[39] P K M Kim R Zamora P Petrosko and T R Billiar ldquoTheregulatory role of nitric oxide in apoptosisrdquo International Im-munopharmacology vol 1 no 8 pp 1421ndash1441 2001

[40] S Ueki K Takeuchi and S Okabe ldquoGastric motility is animportant factor in the pathogenesis of indomethacin-inducedgastric mucosal lesions in ratsrdquo Digestive Diseases and Sciencesvol 33 no 2 pp 209ndash216 1988

[41] B J RWhittle G L Kauffman and SMoncada ldquoVasoconstric-tion with thromboxane A2 induces ulceration of the gastricmucosardquo Nature vol 292 no 5822 pp 472ndash474 1981

[42] P Kubes M Suzuki and D N Granger ldquoNitric oxide an endo-genous modulator of leukocyte adhesionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 11 pp 4651ndash4655 1991

[43] J LWallace andM J SMiller ldquoNitric oxide inmucosal defensea little goes a long wayrdquoGastroenterology vol 119 no 2 pp 512ndash520 2000

[44] F F G Rodrigues B S Cabral H D M Coutinho et al ldquoAnti-ulcer and antimicrobial activities of Stryphnodendron rotundi-folium Martrdquo Pharmacognosy Magazine vol 4 no 15 pp 193ndash196 2008

[45] J A S Zuanazzi and J A Montanha ldquoFlavonoidesrdquo in Farma-cognosia da planta ao medicamento pp 577ndash614 Editora daUFRGSEditora da UFSC Porto Alegre Brazil 2004

[46] H Sakai E Kumano A Ikari and N Takeguchi ldquoA gastrichousekeeping Cl-channel activated via prostaglandin EP3receptor-mediated Ca2+nitric oxidecGMP pathwayrdquoThe Jour-nal of Biological Chemistry vol 270 no 32 pp 18781ndash187851995


[47] B Yelken T Dorman S Erkasap E Dundar and B TanriverdildquoClonidine pretreatment inhibits stress-induced gastric ulcer inratsrdquo Anesthesia amp Analgesia vol 89 no 1 pp 159ndash162 1999

[48] B B Hoffman ldquoFarmacos antagonistas dos adrenorreceptoresrdquoin Farmacologia basica e clınica B G Katzung Ed pp 127ndash141McGraw-Hill Sao Paulo Brazil 10th edition 2007

[49] N B Standen J M Quayle N W Davies J E BraydenY Huang and M T Nelson ldquoHyperpolarizing vasodilatorsactivate ATP-sensitive K+ channels in arterial smooth musclerdquoScience vol 245 no 4914 pp 177ndash180 1989

[50] M T Nelson and J M Quayle ldquoPhysiological roles and pro-perties of potassium channels in arterial smooth musclerdquo TheAmerican Journal of Physiology vol 268 no 4 part 1 pp C799ndashC822 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch


OphthalmologyJournal of


ISRN Allergy


BioMed Research International


ObesityJournal of


ISRN Addiction



Computational and Mathematical Methods in Medicine

ISRN AIDS


Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

Diabetes ResearchJournal of


Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom


Gastroenterology Research and Practice


ISRN Biomarkers


MEDIATORSINFLAMMATION

of


occur in Brazil [7] Of these a total of 52 species in 18 sectionsare referred to Northeast Brazil [8]

Several species of Croton have long played an importantrole in traditional uses of medicinal plants in Africa Asiaand South America including the treatment of cancer con-stipation diarrhea and other digestive problems diabetesexternal wounds fever hypercholesterolemia hypertensioninflammation intestinal worms malaria pain ulcers andobesity [9]

Studies involving several species of the genusCroton haveshown different pharmacological activities such as anti-inflammatory and antioxidant activities of C celtidifolius[10] antileishmanial effect of C cajucara [11] antinociceptiveeffect ofC cajucara [12] antiulcerogenic and cytotoxic activi-ties ofC cajucara [13] hypoglycemic and hypolipidemic pro-perties of C cajucara [14] antimicrobial and the antimalarialeffects C kongensis [15] antinociceptive and anti-inflam-matory properties of C malambo [16] antimicrobial effect ofC sonderianus [17] purgative effect of C campestris [18] andhypotensive and narcotic activities of C eluteria [19]

A study of C campestris in the traditional community ofthe bioregion of Chapada doAraripe revealed the popular useof the leaves and roots of this plant for gastric hematologicaland inflammatory disturbances wounds and respiratoryproblems For their medicinal uses the local populationutilizes infusion in alcoholic beverages teas and macerateswhich justifies the use of the hydroalcoholic extract for theverification of the antiulcer activity

The hydroalcoholic extract of leaves of C campestris ASt-Hill (HELCC) was assessed for its antiulcerogenic effectin models of gastric lesions using absolute ethanol acidifiedethanol and indomethacin and mechanisms of action wereexamined The effect of HELCC on gastric motility was alsoruled out

2 Materials and Methods

21 Plant Material The leaves of the plant species Crotoncampestris A St-Hill were collected in the municipality ofCrato Ceara Brazil using a GPS device (7∘ 221015840 2810158401015840 S 39∘ 28101584042410158401015840 W altitude 892 meters) The procedures for specimenpreparation followed the recommendations described byMing 1996 apud Di Stasi 1996 The material was depositedin the Herbarium of the Federal University of Rio Grande doNorte (UFRN) and registered under no 7095 The researchwas reviewed by the Ethics Committee for Animal ResearchCEPA Faculty of Medicine of Juazeiro do Norte (FMJ) andwas approved (case no 2009 0432 FR 271610)

22 Preparation of Hydroalcoholic Extract of the Leaves of Cro-tonCampestris A St-Hill (HELCC) Theplantmaterial (freshleaves 852 g C campestris) was washed under running watercrushed and macerated and then submitted to cold extrac-tionwith 999ethanol andwater in a 1 1 proportion (87 L ofsolvent) The solvent was removed using a rotary evaporatorunder reduced pressure and temperature of 40ndash50∘C and ayield of 7089 g crude extract was obtained using a lyophilizer

23 Phytochemical Screening The material was submitted tophytochemical screening for the presence of tanninsflavonoids saponins steroids triterpenes coumarins quin-ones organic acids and alkaloids Accordingly the hydro-alcoholic extract (03 g) was diluted in distilled water (9mL)and 70 ethanol (21mL) and after dilution distributed intosix flasks in which the tests for different compounds wereperformed Phenols and tannins 3 drops of ferric chloridewere added to the flask and development of a blue colordenoted the presence of pyrogallic tannins which could befurther confirmed with the addition of gelatin resulting in aprecipitate with the sample Anthocyanins anthocyanidinsand flavonoids the sample was acidified (pH 3) by the addi-tion of 18 drops of HCl and no change in color of the flaskdemonstrated the presence of flavones flavonols xanthonesand flavanonols

The test for leucoanthocyanidins catechins and flavon-ones was performed by alkalinization to pH 85 with 1sodium hydroxide in the first flask acidification (pH 3) bymeans of 18 drops of HCl and heating for 2min in the secondflask and alkalinization (pH 11) with 18 drops of 1 NaOHand also heating for 2min in the third flask Thus flask 2did not show a change in color demonstrating the presenceof flavonones and still the orange red color of flask 3 alsorevealed the presence of flavonones The test for chalconesand aurones by alkalinization (pH 11) with 18 drops of 1NaOH gave a purple red color in the flask indicating thepresence of these compounds

The test for alkaloids was done by diluting the extract(03 g) in 30mL of 5 acetic acid and 15mL of ammonia foralkalinization The preparation was heated until boiling and10mL of 10 ammonium hydroxide was then added alongwith 15mL of chloroform After homogenization and allow-ing to stand in a separatory funnel the chloroform phase wascollected in a beaker and the solvent was evaporated Theresidue was resuspended in 1 HCl and homogenized andsolution was placed on a slide along with a drop of Dragen-dorff reagent where the formation of a precipitate was indi-cative of the presence of alkaloids

The test for steroids and triterpenoids was carried out bydilution of the extract (03 g) in 6mL of chloroform and filtra-tion using cotton covered with sodium sulfate collecting thefiltrate in a test tube After the addition of 01mL of anhydrousacetic acid and three drops of sulfuric acid the developmentof a green color indicated the presence of steroids [20]

24 Assays

241 Assessment of Oral Acute Toxicity Swissmice were usedthroughout to evaluate HELCC for toxicity and effect ongastric lesions The animals were fasted for 3 h prior to theexperiment and were given a single dose of extract dissolvedin 2wv Tween 80 at 175 55 175 550 2000 and 5000mgkgand observed for mortality for up to 48 h (short-term toxi-city) Based on the short-term toxicity the dose of the nextanimal was determined as per OECD guideline 425 Allanimals were also observed for long-term toxicity (14 days)The LD50 of the test extracts was calculated using ldquoAOT 425rdquo











3 Results









Phenols minus





Aurones minus


Catechin minus



(mgkg)

Gas

tric

ulc

erat

ed ar

ea (

)

CL305075

125250500750

0

5

10

15

20

25

lowastlowastlowast

CL 30 50 75 125 250 500 750


lowastlowastlowast

Omeprazole



Gas

tric

ulc

erat

ed ar

ea (

)

HEFCC


(mgkg)

CL305075

125250500750

0

10

20

30

lowastlowastlowast

CL 30 50 75 125 250 500 750

lowastlowastlowast


lowastlowastlowast


Omeprazole






HEFCC

(mgkg)

Gas

tric

ulc

erat

ed ar

ea (

)

CL 30 250 500 7500

5

10

15

20


lowastlowastlowast

CL30250

500750

Omeprazole






HEFCC

L-NAME L-NAME

(mgkg)

HEFCC

abc abc

Gas

tric

ulc

erat

ed ar

ea (

)

CL 10 600 10 750

10

20

30

40

75CL10600

10

75


L-arginine

(mgkg)





CL 10 0016 10 750

5

10

15

20

25

HEFCC75

HEFCC


ac

ac

ac

CL100016

1075

Gas

tric

ulc

erat

ed ar

ea (

)

(mgkg)


(mgkg)



119875 lt 0001






4 Discussion



CL 2 2 750

10

20

30

40

HEFCC


HEFCC75

(mgkg)

(mgkg)

ab

bc

CL2

275

Gas

tric

ulc

erat

ed ar

ea (

)

Yohimbine Yohimbine



119875 lt 0001 versus CL b119875 lt 0001







CL 5 5 750

5

10

15

20

25

HEFCC


HEFCC

75

(mgkg)

ab ab

CL5

575

Gas

tric

ulc

erat

ed ar

ea (

)


(mgkg)



119875 lt







HEFCC (75 mgkg vo)



Dist

ance

trav

elle

d pe

lo b

y ch

arco

al (

)

C 001 750

20

40

60

80

100

lowastlowast

C00175












5 Conclusion



References





























119860119879119875


































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of











3 Results









Phenols minus





Aurones minus


Catechin minus



(mgkg)

Gas

tric

ulc

erat

ed ar

ea (

)

CL305075

125250500750

0

5

10

15

20

25

lowastlowastlowast

CL 30 50 75 125 250 500 750


lowastlowastlowast

Omeprazole



Gas

tric

ulc

erat

ed ar

ea (

)

HEFCC


(mgkg)

CL305075

125250500750

0

10

20

30

lowastlowastlowast

CL 30 50 75 125 250 500 750

lowastlowastlowast


lowastlowastlowast


Omeprazole






HEFCC

(mgkg)

Gas

tric

ulc

erat

ed ar

ea (

)

CL 30 250 500 7500

5

10

15

20


lowastlowastlowast

CL30250

500750

Omeprazole






HEFCC

L-NAME L-NAME

(mgkg)

HEFCC

abc abc

Gas

tric

ulc

erat

ed ar

ea (

)

CL 10 600 10 750

10

20

30

40

75CL10600

10

75


L-arginine

(mgkg)





CL 10 0016 10 750

5

10

15

20

25

HEFCC75

HEFCC


ac

ac

ac

CL100016

1075

Gas

tric

ulc

erat

ed ar

ea (

)

(mgkg)


(mgkg)



119875 lt 0001






4 Discussion



CL 2 2 750

10

20

30

40

HEFCC


HEFCC75

(mgkg)

(mgkg)

ab

bc

CL2

275

Gas

tric

ulc

erat

ed ar

ea (

)

Yohimbine Yohimbine



119875 lt 0001 versus CL b119875 lt 0001







CL 5 5 750

5

10

15

20

25

HEFCC


HEFCC

75

(mgkg)

ab ab

CL5

575

Gas

tric

ulc

erat

ed ar

ea (

)


(mgkg)



119875 lt







HEFCC (75 mgkg vo)



Dist

ance

trav

elle

d pe

lo b

y ch

arco

al (

)

C 001 750

20

40

60

80

100

lowastlowast

C00175












5 Conclusion



References





























119860119879119875


































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of




Phenols minus





Aurones minus


Catechin minus



(mgkg)

Gas

tric

ulc

erat

ed ar

ea (

)

CL305075

125250500750

0

5

10

15

20

25

lowastlowastlowast

CL 30 50 75 125 250 500 750


lowastlowastlowast

Omeprazole



Gas

tric

ulc

erat

ed ar

ea (

)

HEFCC


(mgkg)

CL305075

125250500750

0

10

20

30

lowastlowastlowast

CL 30 50 75 125 250 500 750

lowastlowastlowast


lowastlowastlowast


Omeprazole






HEFCC

(mgkg)

Gas

tric

ulc

erat

ed ar

ea (

)

CL 30 250 500 7500

5

10

15

20


lowastlowastlowast

CL30250

500750

Omeprazole






HEFCC

L-NAME L-NAME

(mgkg)

HEFCC

abc abc

Gas

tric

ulc

erat

ed ar

ea (

)

CL 10 600 10 750

10

20

30

40

75CL10600

10

75


L-arginine

(mgkg)





CL 10 0016 10 750

5

10

15

20

25

HEFCC75

HEFCC


ac

ac

ac

CL100016

1075

Gas

tric

ulc

erat

ed ar

ea (

)

(mgkg)


(mgkg)



119875 lt 0001






4 Discussion



CL 2 2 750

10

20

30

40

HEFCC


HEFCC75

(mgkg)

(mgkg)

ab

bc

CL2

275

Gas

tric

ulc

erat

ed ar

ea (

)

Yohimbine Yohimbine



119875 lt 0001 versus CL b119875 lt 0001







CL 5 5 750

5

10

15

20

25

HEFCC


HEFCC

75

(mgkg)

ab ab

CL5

575

Gas

tric

ulc

erat

ed ar

ea (

)


(mgkg)



119875 lt







HEFCC (75 mgkg vo)



Dist

ance

trav

elle

d pe

lo b

y ch

arco

al (

)

C 001 750

20

40

60

80

100

lowastlowast

C00175












5 Conclusion



References





























119860119879119875


































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of


HEFCC

(mgkg)

Gas

tric

ulc

erat

ed ar

ea (

)

CL 30 250 500 7500

5

10

15

20


lowastlowastlowast

CL30250

500750

Omeprazole






HEFCC

L-NAME L-NAME

(mgkg)

HEFCC

abc abc

Gas

tric

ulc

erat

ed ar

ea (

)

CL 10 600 10 750

10

20

30

40

75CL10600

10

75


L-arginine

(mgkg)





CL 10 0016 10 750

5

10

15

20

25

HEFCC75

HEFCC


ac

ac

ac

CL100016

1075

Gas

tric

ulc

erat

ed ar

ea (

)

(mgkg)


(mgkg)



119875 lt 0001






4 Discussion



CL 2 2 750

10

20

30

40

HEFCC


HEFCC75

(mgkg)

(mgkg)

ab

bc

CL2

275

Gas

tric

ulc

erat

ed ar

ea (

)

Yohimbine Yohimbine



119875 lt 0001 versus CL b119875 lt 0001







CL 5 5 750

5

10

15

20

25

HEFCC


HEFCC

75

(mgkg)

ab ab

CL5

575

Gas

tric

ulc

erat

ed ar

ea (

)


(mgkg)



119875 lt







HEFCC (75 mgkg vo)



Dist

ance

trav

elle

d pe

lo b

y ch

arco

al (

)

C 001 750

20

40

60

80

100

lowastlowast

C00175












5 Conclusion



References





























119860119879119875


































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of





4 Discussion



CL 2 2 750

10

20

30

40

HEFCC


HEFCC75

(mgkg)

(mgkg)

ab

bc

CL2

275

Gas

tric

ulc

erat

ed ar

ea (

)

Yohimbine Yohimbine



119875 lt 0001 versus CL b119875 lt 0001







CL 5 5 750

5

10

15

20

25

HEFCC


HEFCC

75

(mgkg)

ab ab

CL5

575

Gas

tric

ulc

erat

ed ar

ea (

)


(mgkg)



119875 lt







HEFCC (75 mgkg vo)



Dist

ance

trav

elle

d pe

lo b

y ch

arco

al (

)

C 001 750

20

40

60

80

100

lowastlowast

C00175












5 Conclusion



References





























119860119879119875


































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of


CL 5 5 750

5

10

15

20

25

HEFCC


HEFCC

75

(mgkg)

ab ab

CL5

575

Gas

tric

ulc

erat

ed ar

ea (

)


(mgkg)



119875 lt







HEFCC (75 mgkg vo)



Dist

ance

trav

elle

d pe

lo b

y ch

arco

al (

)

C 001 750

20

40

60

80

100

lowastlowast

C00175












5 Conclusion



References





























119860119879119875


































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of








5 Conclusion



References





























119860119879119875


































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of















119860119879119875


































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of













Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of

Antiulcerogenic Activity of the Hydroalcoholic Extract of Leaves of Croton campestris A. St.-Hill in...

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