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Peptides 31 (2010) 1426–1433

Contents lists available at ScienceDirect

Peptides

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dentification of E. dysenterica laxative peptide: A novel strategy in the treatmentf chronic constipation and irritable bowel syndrome

.B. Limaa, O.N. Silvaa,b, J.T.A. Oliveirac, I.M. Vasconcelosc, F.B. Scalabrina, T.L. Rochad,.F. Grossi-de-Sáa,d, L.P. Silvad, R.V. Guadagnine, B.F. Quirinoa, C.F.S. Castro f,

. Leonardecz f,∗∗, O.L. Francoa,b,∗

Center for Proteomic and Biochemical Analyses, Post-Graduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasilia, SGAN Quadra 916, Modulo B, Av.5, 70.790-160 Brasília, DF, Brazil

Post-Graduate Program in Biological Sciences (Immunology/Genetics and biotechnology), Federal University of Juiz de Fora, Martelos, 36036-900 Juiz de Fora, MG, BrazilDepartment of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, CE, BrazilEmbrapa Genetic Resources and Biotechnology, Laboratory of Plant–Pest Molecular Interaction, PqEB, Av. W5 Norte, 70770-900 Brasília, DF, BrazilCatholic University of Brasilia, SGAN Quadra 916, Modulo B, Av. W5, 70.790-160 Brasília, DF, BrazilUniversity of Brasília, Campus Universitário de Planaltina, Vila Nossa Senhora de Fátima, 73300-000 Brasília, DF, Brazil

r t i c l e i n f o

rticle history:eceived 19 February 2010eceived in revised form 10 May 2010ccepted 10 May 2010vailable online 16 May 2010

eywords:. dysentericaaxative

a b s t r a c t

Plants have contributed over the years to the discovery of various pharmacological products. Amongst theenormous diversity of herbs with remarkable medicinal use and further pharmacological potential, herein this report we evaluated pulp extracts from Eugenia dysenterica fruits and further identified the activeprinciple involved in such laxative activity in rats. For protein isolation, fruits were macerated with anextraction solution following precipitation with (NH4)2SO4 (100%). After dialysis, the peptide was appliedonto a reversed-phase semi-preparative HPLC column, and the major fraction was eluted with 26% and66% acetonitrile. The evaluation of molecular masses by MALDI-TOF and Tris/Tricine SDS-PAGE of HPLCfractions showed the presence of a major peptide with approximately 7 kDa. The N-terminal amino acid

emolytic activeioactive peptides

peptide sequence was determined and showed no similarity to other proteins deposited in the DataBank. Peptide from E. dysenterica was able to enhance rats’ intestinal motility by approximately 20.8%,probably being responsible for laxative activity. Moreover, these proteins were non-toxic to mammals,as observed in histopathology and hemolytic analyses. In conclusion, results here reported indicate that,in the near future, proteins synthesized by E. dysenterica fruits could be utilized in the development

l phatrea

of novel biotechnologicairritable bowel syndrome

. Introduction

Chronic constipation is a highly prevalent gastrointestinal dis-rder characterized by altered bowel habits, abdominal discomfort

Abbreviations: TFA, trifluoroacetic acid; HPLC, high performance liquidhromatography; PRF, protein-rich fraction; PBS, phosphate buffered saline; MALDI-oF, Matrix-Assisted Laser Desorption Ionization Time-of-Flight Analyses; 5-HT4,-hydroxytryptamine (serotonin) receptor 4; ALT, alanine transaminase; AST, aspar-ate transaminase; DMF, dimethylformamide; FGF23, fibroblast growth factor 23;

equiv./L, milliequivalents per liter; PTH, parathyroid hormone; T0, no admin-stration; T1, one dose administration; T2, consecutive doses for 7 days; T3,epeated-doses for 14 days.∗ Corresponding author at: Octávio Luiz Franco, SGAN Quadra 916, Av. W5 Norte,ódulo C 70.790-160, Brasília, DF, Brazil.

el.: +55 61 3448 7167; fax: +55 61 3347 4797.∗∗ Corresponding author at: University of Brasília, Campus Universitário delanaltina, Vila Nossa Senhora de Fátima, 73300-000 Brasília, DF, Brazil.

E-mail addresses: leonardecz@unb.br (E. Leonardecz), ocfranco@pos.ucb.brO.L. Franco).

196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.peptides.2010.05.003

rmaceutics with laxative properties for use in chronic constipation andtment.

© 2010 Elsevier Inc. All rights reserved.

and/or difficult defecation [39]. It is estimated to affect between2% and 27% of the population and to have a significant impact onpatients’ quality of life, as well as on the world economy [32]. Thishealth problem could cost a minimum of $19 million year, basedon 76,854 constipation-related consultations in the California Med-icaid program [38]. This is an overall cost since constipation is amultifactorial disorder involving endocrine or metabolic deregula-tions, neurologic disorders (Parkinson’s disease, multiple sclerosis,spinal lesions and autonomic neuropathy, for example), psychiatricdisorders (depression and eating disorders), pharmacologic agents(opiates, anticholinergics and antidepressants), structural lesions(tumors and anal fissures) and lifestyle factors (dietary, repressedurge to defecate, and immobility) [32].

Traditionally, the pharmacologic treatment of constipation pri-marily uses bulking agents and/or laxatives, which act in variousways, causing water retention due to osmotic effects [26], stim-ulating intestinal secretion [22], or directly acting in intestinalmotility [14]. However, these therapies often do not provide the

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esired improvement and have a short-lived efficacy. Furthermore,he chronic use of these compounds, mainly of anthranoid laxa-ives, has been associated with colorectal cancer and loss of colonic

otility [12,27]. For these reasons, new treatment options areeeded.

In this field, several new pharmacologic classes have beeneveloped for chronic idiopathic constipation and irritable bowelyndrome including 5-HT4 receptor agonists, chloride channelctivators and guanylate cyclase activators [40]. Among these phar-acological agents it is important to emphasize the activity of a

nique peptide with laxative action named Linaclotide. This pep-ide is a guanylate cyclase agonist that presents a 14-amino acideptide as its active principle with the ability to stimulate intestinalecretion and transit [19].

In view of promising developments in the pharmacologic treat-ent of chronic constipation using peptides and phytotherapies

s a model for the production of laxative drugs, this study aims tonvestigate the laxative activity of Eugenia dysenterica DC. known inrazil as cagaita, this is a fruit-bearing tree of the Myrtaceae family,ative to the Brazilian upland savannah or “Cerrado”. In general,

ts leaves are used as an antidiarrheic in Brazilian folk medicine,hile its fruit has laxative properties, according to popular use

31]. For these reasons, our analyses were conducted with pulpn natura, and the peptide fraction was used in the detection of

otility alteration and serum electrolyte levels, showing excep-ional activity of these peptides in promoting increased intestinal

otility. Hemolytic activity in vitro and histopathological effectsere also evaluated.

. Materials and methods

.1. Plant extraction and peptide purification

E. dysenterica fruits were collected in the Brazilian savannahCerrado) near Brasilia, Federal District, Brazil. In natura pulp wasxtracted from the fruit, and a further protein-rich fraction (PRF)as obtained by using an extraction solution containing 0.6 M NaCl

nd 0.1% HCl in a proportion of 1:3 (w/v). The suspension was cen-rifuged at 4500 × g for 90 min, at 4 ◦C, and the supernatant wasrecipitated with ammonium sulfate at 100% saturation, at 25 ◦C.fter centrifugation under similar conditions to those describedbove, the precipitated material was dialyzed (3.0 kDa cut-off)gainst distilled water. This fraction was filtered and centrifuged at500 × g for 20 min, at 4 ◦C. Supernatant was separated, lyophilizednd diluted with 0.1% trifluoroacetic acid (TFA), which was useds ion pairing. Rich fractions were applied onto a reversed-phasePLC (Vydac C-18 TP510 semi-preparative column) equilibratedith 0.1% TFA. Proteins were eluted at a flow rate of 1.0 mL min−1

ith a linear acetonitrile gradient (0–100%). Eluted proteins andeptides were detected at 216 nm.

.2. Molecular mass analyses

Molecular mass and purity degree of peptide eluted fromPLC were determined by two different methods, Tris/Tricinend MALDI-TOF. Tris–tricine–sodium dodecyl sulphate (TSDS)olyacrylamide gel electrophoresis was conducted according tostablished procedures [36]. Electrophoretic separations were car-ied out in discontinuous buffer system (cathode buffer: 0.1 Mris, 0.1 M Tricine, 1% SDS at pH 8.25 and anode buffer: 0.2 M

ris–HCl at pH. 8.9), using a 10% separation gel and 4% stackingel. An equal amount of 10 �l peptide was denatured into theuffer solution of 20% glycerol, 10% �-Mercaptoethanol, 4.6% SDS,.01% bromophenol blue and 124 mM Tris at pH 6.8 for 5 min at5 ◦C, and then loaded to gels. Electrophoresis separations were

(2010) 1426–1433 1427

conducted at 10 mA/gel until the dye front reached the bottomof the gel and then maintained for 4 h with 20 mA/gel stable cur-rent. Separation gels were stained with 0.1% Coomassie Blue R-250(Sigma) for 3 h. Molecular mass marker FERMENTAS-UnstainedProtein Molecular Weight Marker #SM0431 (MW 14.4–116.0 kDa)was used to estimate the molecular mass. Protein-rich fractionwas submitted to mass spectrometry analyses. Freeze-dried sam-ples were prepared for Matrix-Assisted Laser Desorption IonizationTime-of-Flight Analyses (MALDI-ToF) in Ultraflex II. Samples weredissolved in sinapinic acid matrix solution (1:3, v:v), spotted onto aMALDI target plate and dried at room temperature for 15 min. Themonoisotopic (600–6000 Da) or average (4000–40,000 Da) com-pound masses were obtained in a positive reflector mode withexternal calibration, using the Peptide Calibration Standard forMass Spectrometry calibration mixture (up to 4000 Da mass range,Bruker Daltonics).

2.3. Amino acid sequencing and in silico analysis

The N-terminal amino acid sequence of purified peptidewas determined on a Shimadzu PPSQ-23A Automated ProteinSequencer performing Edman degradation [13]. PHT-amino acidswere detected at 269 nm after separation on a reversed phase C18column (4.6 mm × 2.5 mm) under isocratic conditions, according tothe manufacturer’s instructions. Amino acid sequence was com-pared to SWISSPROT Data Bank, using BLAST2 Program.

2.4. Animals

Male rats (Rattus novergicus Wistar) (280–300 g) were acquiredat the Biotery of the Catholic University of Brasilia. All animals wereacclimatized for 1 week (temperature 23 ± 2 ◦C; humidity 60%) andhad free access to water and food. Thirty-two male adult Wistar ratswere separated into four groups of eight animals. The study wasapproved by the Animal Use Committee (CEUA) at the Institute ofBiological Sciences, University of Brasilia. Project number 0177-07was approved on 03.27.07 under the name of “Activity of drugs inthe presence or absence of nanocapsules and/or magnetic fluids inthe treatment of several diseases in experimental animal models”.The experiments reported here comply with ethical procedureswith investigated animals [15].

2.5. Evaluation of intestinal motility

Evaluation of laxative effects of E. dysenterica was conductedaccording to the method described by Abdullahi et al. [1]. Evalu-ated animals were starved for 12 h prior to the experiment, butconsumed water ad libitum. Positive and negative control groupsorally received castor oil and water at the dose of 10 mL kg−1. Testgroups received the pulp at 10 mL kg−1 and peptide at 60 mg kg−1

re-suspended in 1 mL of deionized water. Ten minutes after admin-istration of treatments, animals of each group were fed on 1 mLof a charcoal meal (3% suspension of deactivated charcoal in 0.5%aqueous methylcellulose).

Thirty minutes after administration of charcoal meal, the ani-mals of each group were anesthetized using CO2. The abdomenwas opened and the entire small intestine starting from the pyloricsphincter to caecum was removed. The length of intestine (pyloricsphincter to caecum) and distance transited by charcoal were mea-

sured as previously described by Nwafor et al. [29]. Peristalsismovements were calculated according to the distance covered bythe charcoal divided by the total length of the small intestine. Themean values for each group were calculated and results obtainedin the control and test groups were compared.

1428 T.B. Lima et al. / Peptides 31 (2010) 1426–1433

by Tr

2

tcdodpscuB

2

pmRtct5Ti4sasohr

2

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Fig. 1. Analyses of molecular mass from the protein-rich fraction

.6. Evaluation of serum levels

The method described by Shibayama [37] was adapted forhis study. Blood utilized for the evaluation of serum levels wasollected from animals used in the previous experiment. Afterifferent drug administration periods (no administration – T0,ne dose – T1, repeated-doses for 7 days – T2 and repeated-oses for 14 days – T3) 1 mL of blood was collected by heartuncture, without anti-coagulant substances, in order to obtainerum samples, and further analyzed according to several bio-hemical variables such as chloride, magnesium, phosphorus andsing commercial kits (Labtest Diagnostic S.A., Lagoa Santa, MG,razil).

.7. Hemolytic activity

Hemolytic assay with bovine erythrocytes was performed asreviously described by Aparicio and collaborators [4] with minorodifications. Bovine blood was obtained from Embrapa Genetic

esources and Biotechnology, Cenargen Unit. Blood samples drawno obtain erythrocytes for subsequent use as target cells wereollected in anticoagulant (Heparine). Erythrocytes were washedhree times and resuspended in PBS containing 22 mM Na2HPO4,.6 mM KH2PO4, and 123.3 mM NaCl in distilled water (pH 7.4).he hemolytic activity of peptide fraction of pulp was evaluatedn the concentration range 100–400 �g mL−1 (100, 200, 300 and00 �g mL−1) in association with aliquots of 25 �l of erythrocyteuspension. Erythrocytes were incubated with samples for 30 mint 37 ◦C and then centrifuged for 5 min at 5000 × g. The hemoly-is percentages were determined by measuring the optical densityf the supernatant at 540 nm. Zero hemolysis (blank) and 100%emolysis were determined in PBS buffer and 0.2% Triton X-100,espectively.

.8. Histopathological analysis

All animals that survived after 22 days of treatment were sub-itted to laparotomy for removal of the small intestine and liver.

hese tissues were fixed in 10% neutral buffered formalin. The fixedamples were dehydrated in ascending series of ethanol, clearedn methyl benzoate and embedded in paraffin wax. Sections of

�m thickness were prepared using a microtome, stained withematoxylin and eosin (HE) according to Luna [24]. The photomi-rographs were taken by digital camera (AxioCam MRc 5, Carleiss) driven by Axio Vision 4.6.3 software (Carl Zeiss) coupled inn optical microscope (Carl Zeiss). The tissue lesion in intestine

is/Tricine SDS-PAGE (A) and mass spectrometry (MALDI-ToF) (B).

was classified by occurrence of villi alterations and ulceration orinflammatory process. Tissue lesion of the liver was determined bypresence of congestion, hydropic and fatty degeneration, inflam-matory infiltrate and necrosis.

2.9. Statistical analysis

Results were expressed as means ± SD of four separate experi-ments. Data from all assays were subjected to a Shapiro-Wilk testof normality, followed by post hoc Dunnett testing. Analyses wereperformed using BioEstat software (v. 5.0). Values were held to besignificant if P < 0.05.

3. Results

3.1. Purification and characterization of laxative peptides

E. dysenterica (“cagaita”) is a typical “Cerrado” fruit known inLatin America due to its activity as a laxative. In order to identifyproteins with laxative activity in the “cagaita” fruit, the analy-ses were performed with the pulp in natura and peptides isolatedfrom the fruit pulp. Samples were submitted to precipitation withammonium sulfate, through salting out at 100% of saturation. Afterdialysis, using cut off of 3.5 kDa, the extraction yield was assessedby a previously described method of protein quantification [8].

The evaluation of protein-rich molecular masses from E. dysen-terica by Tris/Tricine SDS-PAGE showed greater intensity of stainingof proteins with molecular weight of about 7 kDa (Fig. 1A). Addi-tionally, the analysis by mass spectrometry (MALDI-ToF) revealed amass range of 6 to 13 kDa, corresponding to the molecular massesobserved in the gel (Fig. 1B). In order to isolate the peptides, theprotein-rich fraction from E. dysenterica was then submitted toseparation by reversed-phase high performance liquid chromatog-raphy (HPLC), revealing the presence of major fractions eluted with26% and 66% of acetonitrile (Fig. 2A). Via MS analysis, the presenceof a major peptide with 7274.0 Da eluted with 26% of acetonitrile,

fraction I (Fig. 2b), was observed. In addition, the N-terminal aminoacid sequence of this peptide was determined, obtaining fifteenacid-amino residues (DPMPAEDIVDLAYES). The sequence was thencompared to Expasy Data Bank (http://www.expasy.ch/), using theBLAST 2 Program, demonstrating no similarity with other protein.

T.B. Lima et al. / Peptides 31 (2010) 1426–1433 1429

F n. (A) Chromatogram of the protein-rich fraction applied to reverse-phase HPLC (VydacC 0%); (B) MALDI-ToF spectrum of purified peptide from E. dysenterica.

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Fig. 4. Analyses of chloride ions in experimental groups (pulp and peptide-rich frac-tion of E. dysenterica fruits) and negative (water) and positive control (castor oil).

ig. 2. Isolation and mass spectrum of the major peptide from protein-rich fractio-18 TP510 semi-preparative column), using a linear gradient of acetonitrile (0–10

.2. Effects of E. dysenterica on gastrointestinal transit ofharcoal meal

Experiments carried out on intestinal transit after charcoal mealdministrations revealed that the E. dysenterica pulp in naturand peptide at doses of 10 mL kg−1 and 6 mg kg−1 significantlyncreased intestinal transit in rats by 14.8% and 19%, respectivelyFig. 3). Nevertheless, it is important to observe that pulp in naturaid not present a significant difference to the positive control (cas-or oil), which increased intestinal motility by 15%, evidencing thathese samples clearly show a laxative activity.

.3. Ion effects

Increased fluid secretion causes luminal distension, secondaryeristalsis and laxation. For this reason, we analyzed the absorptionf chloride, magnesium and phosphorus in different periods (oneose, repeated-doses for 7 and 14 days) and its correlation with

axative activity. Animal studies have demonstrated that pulp inatura from E. dysenterica significantly increases serum chloride

evels by 16% after one dose (T1), by 20% after consecutive doses

or 7 days (T2) and by 24% after administration for 14 days (T3), asemonstrated in Fig. 4.

Furthermore, analyses of serum phosphorus concentrationere also carried out. In general, serum phosphorus significantly

ig. 3. Analysis of intestinal motility after administration of pulp and peptide-richractions. The expressed value is related to average distance covered by the activeoal utilized to mark motility. The negative control was water and positive controlas castor oil. *Statistically different from control (P ≤ 0.05) by Dunnett test.

These tests involve interference in sample after one dose (T1), consecutive doses forseven (T2) and 14 days (T3). SI conversation factors (mg/dL): T0 multiply by 0.368; T1multiply by 0.357; T2 multiply by 0.413; T3 multiply by 0.379. *Statistically differentfrom control (P ≤ 0.05) by Dunnett test.

increased (P < 0.05) by 75% (in test groups) and by 100% (positivecontrol) after prolonged use for 14 days (T3), as shown in Fig. 5.Serum magnesium concentration increased significantly (P < 0.05)in experimental groups (pulp and peptides) and in the positive con-trol (castor oil) (Fig. 6). This increase of 50% in magnesium levels inanimals treated for 14 days with consecutive doses (T3) occurredin all groups when compared with the negative control, as shownin Fig. 6.

3.4. Histopathological evaluation and in vitro hemolytic activity

Based on potential activity of protein-rich fraction from E.dysenterica, which was able to accelerate intestinal transit, andin order to evaluate the toxic effects, the histopathologic analy-ses were conducted after administration of prolonged treatment.Histopathology examination of the liver showed that animals

treated with castor oil and pulp in natura presented congestion inthe centrolobular vein and sinusoids (Fig. 7b and c). In addition,histopathological examination of the liver of animals treated withpeptide-rich fraction of E. dysenterica showed no alteration (Fig. 7d).

1430 T.B. Lima et al. / Peptides 31 (2010) 1426–1433

Fig. 5. Analyses of serum phosphorus levels in experimental groups (pulp andpeptide-rich fraction of E. dysenterica fruits) and control groups (negative was waterand positive was castor oil). These tests involve interference in sample after onedose (T1), consecutive doses for seven (T2) and 14 days (T3). SI conversation factors(mg/dL): T0 multiply by 18, 52; T1 multiply by 20, 08; T2 multiply by 21, 83; T3multiply by 20, 08. *Statistically different from control (P ≤ 0.05) by Dunnett test.

Fig. 6. Evaluation of magnesium ions in experimental groups (pulp and peptide-richfraction of E. dysenterica fruits) and control groups (negative was water and posi-tive was castor oil). These tests involve interference in sample after one dose (T1),consecutive doses for seven (T2) and 14 days (T3). SI conversation factors (mg/dL):T0 multiply by 8, 81; T1 multiply by 13, 33; T2 multiply by 9, 76; T3 multiply by 10,31. *Statistically different from control (P ≤ 0.05) by Dunnett test.

Fig. 7. Liver of rats treated with water (a), castor oil (b), pulp in natura (c), peptide-rich fraction (d). The arrows indicate congestion in the centrolobular vein; the tips ofarrows indicate congestion in the sinusoid capillary (scale bar = 100 �m).

T.B. Lima et al. / Peptides 31 (2010) 1426–1433 1431

F r oil (i orph

ipan

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4

soua

ig. 8. Small intestine of rats treated with water (a), protein-rich fraction (b), castonfiltrate in the mucosa and submucosa; the tips of arrows indicate changes in the m

Histopathological analysis of the small intestine showed seriousnjury in intestinal villi in animals treated with castor oil, used asositive control (Fig. 8c). The lesions in the epithelium observed innimals treated with these samples, including hemorrhage (dataot shown), are indicative of several possible mucosal injuries.

Additionally, it was also observed that animals treated withastor oil for a long period (22 days) presented infiltration withononuclear and polymorphonuclear cells in the intestinal mucosa

nd epithelial alterations.In addition, a histopathological examination of the small intes-

ine of animals treated with pulp in natura and peptide of E.ysenterica did not show any alterations (Fig. 8b and d). In ordero assess the cytotoxicity of the peptide-rich fraction of E. dysen-erica against mammalian cells, the hemolysis of cow erythrocytesas measured at various peptide-rich fraction concentrations. Theeptide-rich fraction of E. dysenterica did not show any hemolyticctivity (data not shown).

. Discussion

The disorders of bowel motility, namely incontinence and con-tipation, are common and cause a major impact on the life-stylef sufferers. Incontinence affects 2% of the general population andp to 10% of the healthy elderly [28]. In the present study, laxativectivity by increase of intestinal motility and alterations in blood

c), pulp in natura (d). The arrows indicate intense mononuclear cell inflammatoryology of the villi (scale bar = 100 �m).

ion levels after administration of pulp in natura and peptide isolatedfrom E. dysenterica are made available.

Increase in intestinal motility was demonstrated after admin-istration of pulp in natura and peptide. The result showed thatpeptides are capable of stimulating intestinal transit to a greaterdegree than castor oil, used as positive control (Fig. 3). Various plantextracts are described as having laxative activity by acceleratingintestinal transit, by presence of rhein and other anthraquinoniccompounds such as Croton tiglium [35] and Allamanda cathartica L.[2]. In general, most remedies for constipation, such as the seroton-ergic enterokinetic agents phenylmethanes and anthraquinones,focus on modulating the motility of the gastrointestinal (GI) tract[39]. In recent years, such agents have appeared to promise greatertherapeutic efficacy by increasing intestinal motility, targeting 5-Hydroxytryptamine receptor agonists such as 5-HT4 and 5-HT3[19]. Among these, plants are described that have in their com-position molecules with the ability to act in 5-HT receptor agonist,such as Rikkunshito (or TJ-43), a traditional herbal drug containingacyl ghrelin mimetics, which stimulates the release of acyl ghrelinby blocking the serotonin mechanism [11].

In addition, some medicines have a similar action mechanism,such as cisapride, a 5-HT3 receptor antagonist and 5-HT4 receptoragonist; prucalopride, a 5-HT4 agonist; and renzapride, a mixed5-HT4 receptor agonist and 5-HT3 receptor antagonist [34]. Otherpromotility agents have been described by Johnston et al. [19], such

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s Tegaserod, TD-5108, ATI-7505 and PF-00885706, all of which actia 5-HT4 receptor agonists, and are being used to develop remediesor chronic idiopathic constipation and irritable bowel syndromeith constipation.

It is known that some laxatives can cause electrolyte distur-ance due to increased fluid secretion that can be occasioned byn increase in intestinal motility [33]. These analyses showed thatulp in natura led to an increase in chloride levels (Fig. 4), andnimals treated with pulp, peptide and castor oil exhibited higherhosphorus concentration (Fig. 5) and magnesium concentrationFig. 6). Similar effects have been observed in Chinese medicinallants [9] categorized as laxatives, including Rheum palmatum, C.iglium and Cannabis sativa. Currently, medications with activityn chloride channels that can stimulate intestinal fluid secretion

ithout increasing serum electrolyte levels are being introducedor treatment of constipation [9]. Among these drugs, is importanto emphasize Lubiprostone, a fatty acid derivative of a metabo-ite of prostaglandin E1, acting as a selective chloride channel-2ctivator, which increases chloride ion transport into the intestinalumen, gut motility, frequency of stool passage, and which allevi-tes abdominal discomfort/pain [30].

In addition, another new pharmacological class that is able toause electrolyte transport alterations in chloride levels consists ofuanylate Cyclase C Activators [32]. In general, the activation of

his receptor results in an increase in cyclic guanosine monophos-hate, which leads to increased chloride and bicarbonate secretion

nto the intestinal lumen. These action mechanisms are describedor Linaclotide, a 14-amino acid peptide that acts in the intestinehrough activation of the luminal receptor guanylate cyclase C onnterocytes [40].

In relation to the hyperphosphatemy encoded in animals fromhe experimental and positive control group, this corroboratesaxative activity because it occurs by an increase in intestinalbsorption, cellular release or rapid intracellular to extracellularhifts, or decreased renal activity [25]. A similar result was obtainedsing cathartic laxative, which showed an increase in serum levelsf phosphorus and chloride, associated with a decrease in serumevels of calcium and potassium [7].

In addition, other information that corroborates the activityescribed was the increase in serum levels of magnesium, which

n general is associated with malabsorption, dehydration causedy diarrhea, and alterations in vitamin D and parathyroid hormoneetabolism [21,44]. These effects also encoded in laxatives that

ontain magnesium, and the same herbal laxatives, such as Cassiacutifolia and Cassia angustifolia and Rhamnus purshiana, can leado disruption, erosion of the mucosal lining and general irritationf the intestine, modifying intestinal electrolytes and water trans-ort, thus producing a systemic electrolytic imbalance [20,23,32].

t has been suggested that this action mechanism involves the par-icipation of muscarinic receptors located in the intestinal mucosa14,43]. A new generation of anticholinergic drugs, represented byhe muscarinic receptor antagonists zamifenacin and darifenacin,as been claimed to be effective in patients with irritable bowelyndrome because these drugs control ion and fluid transport [5].his information corroborates the laxative activity of the peptiderom E. dysenterica by electrolyte disturbance, which probably leadso an increase in intestinal motility.

In addition, toxicity tests were conducted and results showedhat peptides do not present toxicity in the liver and intestine. How-ver, histopathologic analyses of the liver demonstrate that pulp inatura and castor oil presented congestion in the centrolobular vein

nd sinusoids (Fig. 7b and c). Similar results have been describedy other authors [6] with the use of venom from Crotalus durissuserrificus. In addition, a cytotoxic effect on the liver due to the usef plant extracts or their derivates has been described; for examplean [18] and Van Gorkom et al. [41] demonstrated alterations such

(2010) 1426–1433

as hyperplasia of the Kupffer cells, intense ballooning degenerationof hepatocytes and portal tracts and centrolobular vennus conges-tion in rats treated with an aqueous extract obtained from leavesof Capraria biflora [42]. It is important to emphasize that the liveris a major site for the biotransformation, accumulation and excre-tion of exogenous chemicals and damage in nearby tissue indicatespossible toxicity.

Histopathologic analyses of the intestine revealed that castoroil presents toxicity in nearby tissue, and that epithelial alterationsand serious injury were exhibited in intestinal villi, including hem-orrhage (Fig. 8c). Epithelial erosion and degeneration may causeleakage of electrolytes such as sodium, potassium and hydrogenions into the muscle coat layer of the intestine, leading to amplifica-tion of the electrolytic activity of the mammalian small intestine [3].Some authors have suggested that intestinal hemorrhage is asso-ciated with a significant localized inflammatory response in themucosal and serosal, which they attributed to intestinal endotox-emy [23,28].

It is known that after administration castor oil is hydrolyzedin the small intestine to release glycerol and the active compo-nent, ricinoleic acid, which induces laxation by altering intestinalabsorption [16,17]. Cline et al. [10] studied the effects of sodiumricinoleate on hamsters’ small intestinal function and structure,observing that ricinoleate-induced intestinal secretion was alsoaccompanied by increased mucosal cell exfoliation. This was mea-sured by the appearance of DNA in the perfusate and by apparentinjury to epithelial cell membranes, as judged by measurement ofsucrase activity and phospholipids in cell-free aliquots of luminalfluid.

5. Conclusion

It can be concluded that the present study supports the claimsby practitioners of traditional medicine that E. dysenterica causeslaxative effects. The present study provides evidence that a pep-tide of E. dysenterica causes increase of intestinal motility, whichmay contribute to the clinical efficacy of this herbal drug in nor-malizing motility changes. The analyses demonstrate that the fruitof E. dysenterica has the ability to stimulate motility without caus-ing diarrhea. In addition, the laxative peptide it contains has beenseen to be different to all those sequenced in the Data Bank, withdifferent activity from other commercial laxatives and no toxic-ity in the proposed model. For these reasons, the laxative peptidesfrom E. dysenterica constitute a strong candidate for developing anovel pharmacological product in a promising market, representinga potentially multimillion-dollar business.

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