the effect of the diet in intestinal health and its relation with … · 2020-01-24 · the...

IES LAS MUSAS

TRABAJO DE INVESTIGACIÓN

The effect of the diet in intestinal health and

its relation with Diabetes.

Author: Bárbara Rodríguez Sarrión and Lorena Rodríguez Bravo

Tutors: Dr Mª Elvira López-Oliva Muñoz (Fac. Farmacia UCM)

and J.Carlos Ortega Lázaro

INDEX

ABSTRACT.............................................................................................................................2

INTRODUCTION.....................................................................................................................3

Type2DiabetesMellitus...........................................................................................................3

COLON:STRUCTUREANDFUNCTION.....................................................................................8

COLONANDMICROBIOTA...................................................................................................12

COLONANDDIETARYFIBRE.................................................................................................16

DIABETESANDGASTROINTESTINALHEALTH........................................................................18

CAROBFRUIT.......................................................................................................................21

Healthbenefitsofcarob.........................................................................................................23

OBJETIVE.............................................................................................................................26

MATERIALSANDMETHODS.................................................................................................27

Carobfruitpulpextract(CFE)..................................................................................................27

Diet..........................................................................................................................................27

Experimentaldesign................................................................................................................28

Analyticalmethods.................................................................................................................29

Preparationofparaffinsections..........................................................................................29

HaematoxylinandEosin(H&E)staining............................................................................30

PeriodicAcid-Schiffstaining(PAS)......................................................................................31

Imagesanalysis...................................................................................................................32

Statisticalanalysis...............................................................................................................32

RESULTS..............................................................................................................................34

DISCUSION..........................................................................................................................39

CONCLUSION.......................................................................................................................42

ACKNOWLEDGEMENTS

BIBLIOGRAPHY....................................................................................................................43

Trabajodeinvestigación BárbaraRodríguezSarrión

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ABSTRACT

Gastrointestinal health is the result of the triple interaction between the colonic

epithelial barrier, the microbiota and the nutrients. The balance between these

components is interrupted in some diseases such as diabetes mellitus (DM). DM-

inducedgastrointestinaldysfunction ispredominantlydueto the long-termoxidative

stresscausedbyhyperglycemia.Theintestinalbarrierpreventspathogeninvasionand

maintains host-microbiota homeostasis. The colonic mucosa exhibits numerous

morphologicalandfunctionalalterationsduringDM.Polyphenols,amainendproduct

ofmicrobialfermentationofdietaryfibersinthecolon,playanimportantrolein

the maintenance of intestinal oxidative status and homeostasis. Carob fruit pulp

extract (Ceratonia siliqua L.) (CFE) is a condensed source of polyphenols with

hipoglucemicproperties.ThepresentstudyevaluatedthebeneficialeffectsofCFEon

coloniccelldifferentiationandbarrierintegrityinthecolonicmucosaindiabeticrats.A

high-fat hypercholesterolemic diet combined with low-dose streptozotocin plus

nicotinamidewasusedtoinducetype2diabetes(DM).Morphometricandhistological

analysis of colon mucosa was done by standard method (haematoxylin-eosin). The

expressionofmucinwasevaluatedusingthehistochemicaltechniquesofPeriodicAcid

Schiff(PAS).Streptozotocin-inducedDMgeneratedpronounceddecreasesinthecolon

weight,crypt-lengthandincreasedthewallthicknessandcrypt-widthindistalsegment

butnotintheproximal.PASpositivecellsshowedalossofgobletcellsalongthecrypt

length in DM rats. CFE supplementation led to a notable restoration of intestinal

crypt/mucosa structure enhancing the morphometric parameters reaching those of

the non-diabetic group. CFE increased the colonic goblet cell density improving the

protective function of mucosal barrier. In summary, these data indicate that CFE

improved gut barrier integrity and colonic cell differentiation in DM rats. Dietary

supplementation of CFE could be advantageous for normalization of metabolic

alterationsasdiabetesviabeneficialmodulationofgastrointestinalhealth.


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INTRODUCTION

TYPE2DIABETESMELLITUS

Type2Diabetesmellitus (DM)-morecommonlyreferredtoas"diabetes"-achronic

diseaseassociatedwithabnormallyhighlevelsofthesugarglucoseintheblood.The

terms“Diabetes”and“Mellitus”arederivedfromGreeklanguage.“Diabetes”denotes

“a passer through, a siphon” whereas the “Mellitus” means “sweet”

(http://www.diabetesatlas.org/resources/2015-atlas.html).

The World Health Organization defines diabetes as the 7th leading death cause.

Recently, itwas recorded thatonly in2012at least 1.5milliondeaths induced from

diabetes(WHO,2016).Theglobalprevalenceofdiabetesamongadultsolderthan18

hasrisenfrom4.7%in1980to8.5%in2014,morerapidlyinmiddle-andlow-income

countries. Ithasbeenstimatedthatabout630millionswouldhavediabetes in2045.

Diabeteshasaconsiderableeconomicimpact.Infact12%ofglobalhealthexpenditure

is spent on these disease prevention and care, such as intensive blood glucose and

bloodpressurecontrol,theuseoflipidloweringagents,screeningforandtreatmentof

diabeticretinopathyandactivecareofthediabeticfeet.Duetoitshighcosts,diabetes

diseaseisanimportchallengeforthehealthcaresystem(Figure1)(Wildetal.,2004).

Figure1:Globalprevalenceandhealthexpenditureofdiabetes(Wildetal.,2004).


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DMisachronicdiseaseresultingfromthebody’sineffectiveuseofinsulin.Asaresult

ofthisdysfunction,glucagonandhepaticglucoselevelsthatriseduringfastingarenot

suppressed with a meal. Diabetes is due to one of two mechanisms: Inadequate

production of insulin (which ismade by the pancreas and lowers blood glucose), or

inadequate sensitivityof cells to theactionof insulin.Thus, initially theβ-pancreatic

cells produce and release insulin; but the hormone has a no-effect on the targets

organsdue to insulin resistanceof theorganism, leading to aprogressiveβ-cell loss

andinsulinsecretoryfailurethat inturn induceshyperglycemia(Olokobaetal2012).

Diabetesmaybediagnosedbasedonplasmaglucosecriteria,throughfastingplasma

glucose(FPG)testor2-hplasmaglucosevalueaftera75goralglucosetolerancetest

(OGTT). The cutpointof FPG test is ≥126mg/dL (7.0mmol/L); the cutpointof the

OGTTis≥200mg/dL.Accordingtofastingglucoselevel,peopleareclassifiedasnormal

(FPG = 100 mg/dL), prediabetes (FPG = 100–125 mg/dL), or diabetes (FPG ≥ 126

mg/dL).However,accordingtoOGTTresultspeoplearealsogroupedashavingnormal

glucose tolerance (2-hPG=140mg/dL),prediabetes (impaired fastingglucose [IFG]:

FPG=100–125mg/dLandimpairedglucosetolerance[IGT]:2-hplasmaglucose=140–

199mg/dL), anddiabetes (2-hplasmaglucose≥200mg/dL). FPGmeasure it carries

out,afterthepersonhasfastedovernight(atleast8hours),asinglesampleofbloodis

drawnand sent to the laboratory for analysis. This can alsobedoneaccurately in a

doctor'sofficeusingaglucosemeter. Impairedglucosetolerance(IGT)andimpaired

fastingglycaemia(IFG)areintermediateconditionsinthetransitionbetweennormality

anddiabetes.Peoplewith IGTor IFGareathighriskofprogressing toDM,although

this is not inevitable.When fasting blood glucose stays above 100mg/dl, but in the

rangeof100-126mg/dl,thisisknownasimpairedfastingglucose(IFG).Whilepatients

with IFGorprediabetesdonothave thediagnosisofdiabetes, this conditioncarries

withititsownrisksandconcerns,andisaddressedelsewhere(Table1)(ADA,2018).


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Table1:Diabetesdiagnosiscriteria

Somesymptomsofdiabetes include: increasedurineoutput,excessive thirst,weight

loss,hunger,fatigue,skinproblems,slowhealingwounds,yeastinfectionsandtingling

ornumbnessinthefeetortoes.TypeIIdiabetesclinicaldiagnosisisbasedonseveral

symptoms,suchasfrequenturination,excessivethirst,weightlossandblurredvision

which are lessmarked than those of type 1 diabetes. Thus, this formof diabetes is

frequentlyundiagnosedformanyyearsinthecourseofwhichthediseasegetsworse

and the organism gets damaged by hyperglycemia (International Expert Committee

2009). Patients with DM can present complications such as retinopathy, renal

dysfunction and diabetic foot. DM also is an important factor in accelerating the

hardeningandnarrowingofthearteries(atherosclerosis),leadingtostrokes,coronary

heart disease, and other large blood vessel diseases. Thus, this is referred as

macrovasculardisease(Figure2)(Sudesnaetal.2017).

Figure2:Diabetescomplications(Wuet1.,2014)

Normal Impaired

Fasting

Glycemia(IFG)

ImpairedGlucose

Tolerance(IGT)

Diabetes

Fastingplasmaglucose(FPG) <100mg/dL100–125mg/dL ≥126mg/dL

2-horalglucosetolerancetest

(OGTT)

<140mg/dL 140–200mg/dL ≥200mg/dL


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The exact cause of DM is uncertain until now. Nevertheless, scientists believe that

genes,environmental factorsandotherpathological conditions suchasautoimmune

eradication of the pancreatic β-cells which provoke insulin deficiency and other

abnormalities which cause resistance to insulin action. Some of the risk factors for

getting diabetes include being overweight or obese, leading a sedentary lifestyle, a

family historyof diabetes, hypertension (highbloodpressure), and low levels of the

"good"cholesterol (HDL)andelevated levelsof triglycerides intheblood.Theriskof

developingDMincreaseswithage,obesity,andlackofphysicalactivity.Itoccursmore

frequentlyinwomenwithpriorgestationaldiabetesmellitus(GDM)andinindividuals

withhypertensionordyslipidaemia.Itsfrequencyvariesinethnicsubgroups.Itisoften

associated with a strong genetic predisposition that exerts its effect following

exposure to an obesogenic environment characterised by sedentary behaviour and

excessivesugarandsaturatedfatconsumption.However,thegeneticsofthisformof

diabetesarecomplexandnotclearlydefined (AmericanDiabetesAssociation, 2012,

2014).

DMtreatmentisbasedonincreasingofinsulinsensitivitybyweightreduction,

increasedphysicalactivityandpharmacologictherapyofhyperglycemia(Table2),butis

notrestoredtonormal.

IDFDiabetesAtlas.6thed.Brussels:IDF;2013


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Furthermore, the prevention plays an important role in prediabetes, IGT or IFG,

especially in presence of BMI > 35 kg/m2, age < 60 years and women previously

diagnosedofGMD.Type2DMprevention is foundedto improvingdietandphysical

program,targetingweightloss(7%ofbodyweight)andincreasedphysicalactivity(≥

150min/weekofmoderateactivity) (Wuetal., 2014). Type2DMpreventiondiet is

settled according to International Diabetes Federation (IDF 2013). This kind of diet

aims tomaintainingoptimal glycemic value for preventingor delayingDM:Avoiding

sugaringdrinks;Eatingatleast3portionsofvegetables;Eatingfreshfruit,driedfruitor

yogurtassnack;Limitingalcoholintake;Preferringleanmeat,chickenorfishinsteadof

fatty meat or restructured (processed) meat; Eating whole bread, pasta or rice;

Choosingunsaturatedfatinsteadofsaturatedfat.Insummary,theinfluencingfactors

andmechanismofDMareshowninFigure3.

Figure3.TheinfluencingfactorsandmechanismofType2Diabetes(Wuetal.,2014)


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COLON:STRUCTUREANDFUNCTION

Thelargeintestineisapartofthedigestivesystem.Itisconnectedtotheiliumofthe

small intestinevia the ileocecalsphincter. Its length isabout1.5metersand7cm in

diameterinthelivingbody.Thevitaltaskthatthelargeintestineperformsisabsorbing

water and vitamins and metabolites produced by gut bacteria while converting

digestedfoodintofaeces(https://mejorconsalud.com/fisiologia-del-intestino-grueso).

Itisdividedinthreedifferentparts:cecum,colonandrectum.Thececumistheregion

whichislocatedbetweentheiliumandtheascendingcolon.Itslengthisabout5and7

cm.Thececumterminatesinthevermiformappendixwhichfunctionisproducingand

protectingthebacteriathatappearintheintestinealthoughitisbelievedthatitdoes

nothaveany important function.The followingregion iscalledcolon. It isdivided in

ascending,transverseanddescendingcolon;theydeterminethetransitionofthecud

throughthecolon.It isabout1.5meterslonganditsfunctionisobtainingwaterand

salts from the solid residues before they are eliminated. The colon has lumen and

mucus. There is a layer of bacteria located there calledmicrobiota. The descending

colonleadsintotherectumandafterwardsintheanus,ithassphinctersthatallowthe

expulsion of the faeces to the exterior

(http://www.innerbody.com/anatomy/digestive/large-intestine).

The functions of the rectum are the formation of the faeces and the absorption of

watertoavoiditsloss.Itproducesthefaecesbecauseofthestandstillofthecudthat

is caused by the peristaltic and anti-peristaltic movements that are contraction

motionsofthetubedigestive(Chengetal.2010).Liketherestofthegastrointestinal

canal,thelargeintestineismadeoffourtissuelayers:

- The innermost layer, known as the mucosa, is made of simple columnar

epithelialtissue.Manymucousglandssecretemucusintothehollowlumenof

thelargeintestinetolubricateitssurfaceandprotectitfromthebacteria.

- Surrounding the mucosa is a layer of blood vessels, nerves and connective

tissueknownas the submucosa,which supports theother layersof the large

intestine.

- The muscularis layer surrounds the submucosa and contains many layers of

visceralmusclecellsthatcontractandmovethelargeintestine.


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- Finally, the serosa forms the outermost layer. The serosa is a thin layer of

simplesquamousepithelialtissuethatsecreteswateryserousfluidtolubricate

the surface of the large intestine, protecting it from friction between

abdominalorgansandthesurroundingmusclesandbonesofthelowertorso.

In comparison to the small intestine, the large intestine does not have the same

intestinalvilli;itonlyhasglandularepitheliumformscylindricalstructurescalledCrypts

of Lieberkuhn, oriented perpendicular to the major axis of the colon. The large

intestine needs to absorbwater and not thatmany nutrients as the small intestine

(HowellandWells2011)(Figure4A).

The colonic intestinal epithelium is a complex environment composed of active cell

proliferation and differentiation of many cell types (Figure 4B). Intestinal epithelial

cells(IECs)provideaphysicalandbiochemicalbarrierthatsegregateshosttissueand

commensal bacteria to maintain intestinal homeostasis. Secretory IECs support this

function through the secretion ofmucins and antimicrobial peptides (Petersson and

artis,2014). Intestinalstemcells residenear thebottomofepithelialcryptsandgive

rise to progenitor cells, which rapidly divide and differentiate into subtypes of cells

thatcomprisetheintestinalepithelium.GobletThegobbetcellsareresponsibleforthe

production and maintenance of the protective mucus blanket by synthesizing and

secreting high-molecular-weight glycoproteins known as mucins. They create the

mucosa layer, with that; they avoid the entrance of the bacteria of themicrobiota.

Gobletcells,whicharemoreabundant in thecolon than thesmall intestine, secrete

mucins and other proteins that are used for lubrication and as a barrier defence

againstpathogens.Panethcellssecretelysozymetopreventbacterialinfection.Paneth

cellsalsoplayakeyrole inprovidinganiche for thestemcells in thecrypts.Finally,

enteroendocrine cells comprise approximately 1% of large intestinal epithelium, are

scattered throughout the mucosa as individual cells, and produce and secrete

hormones.

It is also known that the large intestinehas also adigestive function. If thebacteria

which are in the microbiota metabolize the fibre that is forming the faeces, the

enterocytewillabsorbdifferentacids.Butmostofthisfibreisaccumulatedinthelayer

mucosa.IECsalsoconveymicrobialsignalstomucosalimmunecellsandpromotethe


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coordination of appropriate immune responses against commensal bacteria and

enteric pathogens. Interactions between microbiota and IECs regulate immune cell

responsesthatactontheintestinalbarrier.

A)

B) C)

Figure4.AnatomyandHistologyoflargeintestinal(A)Anatomyoflargeintestine.(B)Incross-

section;bothsmallandlargeintestinecontainouterlayersofserosaandbothlongitudinaland

circularmusculature.Thelargeintestinehaslargemuscularribbons,calledtaeniacoli,toaidin

contraction and peristalsis. Middle layers include submucosa and muscularis mucosa. The

innermostlayersarethelaminapropriaandepithelium.Villiareabsentinthelargeintestine.

(C) Intestinal crypts contain resident stem cells capable of generating all cell types of the

mature epithelium: enterocytes (capable of absorption of nutrients and water) and mucin

secreting goblet cell. The hormone secreting enteroendocrine cells, and the aforementioned

Panethcellsarelargelyabsentinthelargeintestine(HowellandWells,2011).

The intestinal mucosal barrier is a heterogeneous entity composed of physical,

biochemical,and immuneelementselaboratedby the intestinalmucosa.Thecentral

component is the intestinal epithelial layer, which provides physical separation

betweenthelumenandthebody.Thesecretionofvariousmoleculesintothelumen


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reinforcesthebarrierfunctionontheextra-epithelialside,whileavarietyofimmune

cells provide additional protection below the epithelial layer. A disrupted intestinal

mucosal barrier can allow passage of microbes, microbial products, and foreign

antigens into the mucosa and the body proper. This can result in activation of the

immunesystemandsecretionofinflammatorymediators.Certainimmuneresponses

might, in turn might, cause cellular damage that could result in further barrier

dysfunction. Defects in intestinal mucosal barrier function with the accompanying

translocation of microbes and their products have been linked with a variety of

conditions,someofwhicharethoughttoadditionallyrequireageneticpredisposition.

Intestinalbarrierdysfunctionisthoughttobepreconditionforandexacerbatingfactor

of numerous autoimmune and inflammatory conditions, including food allergies,

inflammatoryboweldiseases,celiacdiseaseandDM(Turner,2009).

Figure5:Theintestinalmucosalbarrier(modifiedfromMaynardetal.,2012)


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COLONANDMICROBIOTA

The colon is colonisedby anentirepopulationofmicroorganisms, includingnot just

bacteria,butalsomicrobessuchasfungi,archaea,virusesandprotozoans.Majorityof

thegutbacteriaarenon-pathogenicand,cohabitwiththeenterocytes inasymbiotic

relationship. Gut microbiota (GM) has beneficial functions on health. The gut

commensalspredominantlyaid innutrientmetabolism,drugmetabolism,prevention

of colonization of pathogenic microorganisms and in intestinal barrier function. In

addition,healthymicrobiotacollaborateswiththeimmunesystemfightingoffinvasive

pathogenicmicroorganisms. The gutmicrobiota is formedby onehundredbillion of

bacteria,allofthemweightaround1.5and2kg,someofthemarepermanentandthe

othershaveatransitorilyrole.Thecolonicmicrobiotahasapproximately800to1,000

speciesper individual, but62%of themwereunknownand80%of thebacteria are

identifiedbymetagenomics.Thetechniqueusedtocultivatemicroorganismssuchas

theculturesinPetriplatesdonotallowustoaccesstoallofthemicroorganismthat

exist. The whole group of bacteria comprise 3 million of gens, a huge number in

comparisontothehumangenome,approximately150timesbigger.

Thegutmicrobiotawasdenominated«intestinal flora»because the importanceof it

was not known. Thanks to the new studies and developments in this area, a lot of

scientists have considered the gutmicrobiota as anorganbecauseof the important

influence that it has in different aspects, for example, in the maintenance of our

health.The functionof thegutmicrobiota isessential forourorganism; thebacteria

protectusfrompathogens.

The gut microbiota changes depending on the diet of the individual, it has been

observed by using mice that after a day of an occidental diet, the gut microbiota

changed.Afterweeksofconsumingthistypeofdiet,themicrobiotadeveloped,inthis

case, a better adiposity. Furthermore, they present an increase of Firmicutes and a

decrease ofBacteroidetes; therefore, we can conclude that any change on the diet

supposesanalterationofthecompositionofthegutmicrobiota.

Thegutmicrobiotaisformedafterthebirthofthebaby;wesupposethatduringthe

pregnancyperiodthebabyisinasterileenvironment.Thefirst100daysofthebaby´s

lifearecrucialforthetrainingoftheimmunitysystemofthebaby,andthatiswhythe


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adequate bacteria are needed during that period. In the first two years, the gut

microbiotaisformedbybifidobacterias.Lateron,thecompositiondiversifiesandnew

speciesofbacteriaappearsuchasFirmicutes.Afteranalysingthemicrobiotaofsome

families, it has been observed that they are similarities between them after been

comparedthemtopeoplenorelated.Themicrobiotawillhaveseveralchangesuntil

theageofthreeandafterthat,itwillbesimilartotheadults.Asofacertainage,this

layer of bacteria will start to be unstable, that will make the person be prone to

contractingillnessesbecauseitwillstartdisappearing(Figure5)(Jašarevićetal2016).

Figure5Timelineshowingthatcriticalshiftsinmaturationofthegut,hormonesandthebrain

occur in parallel, and that sex-specificity in these systems emerges at similar points in

development.(Jašarevićetal2016).


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The most important functions of the microbiota are: nutrition, helping keeping the

supplyofenergy,systemicinflammationandprotectingusfromvirusandbacteriathat

cause illnesses. Thebenefitsof thegutmicrobiotaare related tomolecules that are

producedwhenthebacteriafermentthealimentaryfibre.Adietwithoutalotoffibre

could provoke a diminution of the gut bacteria. In this sense, a dynamic interaction

between dietary fibre and metabolism of the microbiota composed of commensal

bacteria influencing the status of the colonic mucus layer and susceptibility to

pathogens that traverse gut barrier have been found. Using a gnotobiotic mouse

model,Desaietal.,(2016)providesamechanismbywhichadietdeficientincomplex

plantfibretriggersasyntheticgutmicrobiotatofeedonthecolonicmucuslayerthat

actsasaprimarybarrieragainstinvadingpathogens(Figure6).Regularconsumptionof

dietary fibre helps prevent erosion of the intestinal mucus barrier by the gut

microbiome.

Figure6.ThebalancebetweenfibredegradationandmucusdegradationinFibrerich(FR)diet-

fedmice;whereasanfibrefree(FF)dietleadstoproliferationofmucus-degradingbacteriaand

microbiota-mediateddegradationofthecolonicmucuslayer(ModifiedfromDesaietal.,2016)


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Entirecolonexhibitsconsiderablemorphologicalandphysiologicalheterogeneity(e.g.,

pH, redoxpotential andhydrogenconcentration)along its lengthdue to thedistinct

origins of the colonic segments. In proximal colon, bacterial growth is fast due to a

large availability of carbohydrates, and thus large amounts of beneficial SCFAs are

produced through fermentation, lowering the pH of its luminal content. However,

from the caecum toward the transverse colon, the mucosal oxygen concentration

increasesandthenitdecreasestowardthedistalcolonandrectum.Thethicknessand

compositionofthemucusbarrier,whichhasdirectcontactwiththeintestinallumen,

differsalongthegastrointestinaltract.Besidesaslowtransitandthemostdehydrated

luminalcontent,ahigherpHoccursinthedistalcoloncomparedtotheproximalcolon

asaresultofproteinfermentation,resultingintheproductionofammonia,branched-

chainfattyacids,andphenoliccompounds.Asthedistalcolonisoftenassociatedwith

several chronic disorders, the persistence of carbohydrate fermentation toward this

partofthehumancolonisoneofthedesirableeffectsofprebioticstakenupthrough

diet. Indeed, carbohydrates, such as inulin and arabinoxylans, may stimulate

bifidobacteriaselectivelyandincreaseSCFAproductioninthedistalcolon.

SeveralinvestigationssuchasJašarević´sincludeotheraspectsinwhichthemicrobiota

carriesoutafundamentalrole.Firstofall,thisinvestigatorexplainsthatafterstudies

madewithanimals,ithasbeenverifiedthatfactorssuchasthestressthatthemother

suffers during the pregnancy affect the cerebral growth of the litters, especially the

males. This factor also alters themicroorganisms that reside in the gut, altering its

development. The study was made by using mice that were exposed to factors of

stress; the results obtained were male litters with its cerebral health altered.

Consequently these litters had unusual responses in their adult ages. In addition,

during the experiment, faecal samples were taken and, after analysing them, the

conclusion obtained was that the stress interrupted the development of the gut

microbiota(Jašarević,etal.2015).AnotherstudyledbyProf.Ghannoumhasidentified

newspecific interkingdombacteriaand fungi interactions thatmaybekeyplayers in

Crohn’sdisease.Thisdiseaseconsistsinaninflammationusuallyplacedintheunionof

theileumandthecolon,butitcanalsoaffecttheeyes,skinetc.Thatmeansthatthe

microbiotaalsoaffectsseveraldiseases(Mukherjeeetal2015). Recentstudieshave

also provided new proof of the role of the gut bacteria in Parkinson´s disease


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revealing, by using mice, that the gut bacteria of people that suffer Parkinson got

worstmotorsymptoms(Keshavarzianetal.,2015).

COLONANDDIETARYFIBRE

Dietary fibers are heterogeneous, and thus different classifications are utilized to

describethem,includingfermentability,solubility,andviscosity,andtheseproperties

influence not only fermentation, but also the therapeutic effects of consumption.

Fermentationofnon-digestiblecarbohydratesinthehumancolonisconsideredtoplay

an important role in the maintenance of human health and well-being. Natural

products have adapted, during the course of evolution, optimum chemical scaffolds

against awide variety of diseases, including cancer and diabetes. Advances in high-

throughput screening assays, assisted by the continuous development on the

instrumentation’s capabilities and omics, have resulted in charting a large chemical

andbiologicalspaceofdrug-likecompounds,originatingfromnaturalsources.Thefate

offiberinthecolonlargelydependsonthecolonicmicrobiotaandthephysio-chemical

characteristicsof thefiber itself.Thus,dietary fiber iscommonlydivided intosoluble

andinsolublefibers(Mehtaetal.2013)(Figure7).Insolublefiberconsistsofcellulose,

partofhemicelluloseandligninandsolublefiberconsistsofpentosans,pectins,gums

andmucilages. Insoluble fibers are generally poorly fermentedby gutmicrobes, but

theirpresence in thediet increasesgut transit rateand thus reduces theamountof

timeavailable for colonicbacterial fermentationofnon-digested foodstuff. Theyare

partially fermented in the distal colon where transit time is slower, and bacterial

densitiesarehigher(Mehtaetal.2013).


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Figure7:Dietaryfibersclassification(Mehtaetal.2013).

Ontheotherhand,thetypeofdietaryfiberaffectsthemicrobialcompositionofthe

gut lumen. For example, inulin, a polymer of fructosemonomers present in onions,

garlicandasparagus,stimulatesthegrowthofBifidobacteria;whereas,itrestrictsthe

growthofpotentialpathogenicbacteriasuchasE.coli,Salmonella,andListeria(Zeng

etal.2014).Thus,dietary fiberwithpolyphenolsnotabsorbed in thesmall intestine

reachesthecolonandcanbefermented,modifyingtheproductionofcertainmicrobial

metabolitesandSCFA,whichcansignificantlyincreasethebiomass,reducetheluminal

pH,changethecompositionoffloraandmodifythegastrointestinalepithelialkinetic

pattern which inhibits the potentially harmful bacteria and promote the growth of

favorablelacticacidmicroflora.

Polyphenolsaresecondarymetabolitesandconstituteoneofthemostcommonand

widespread groups of substances in plants. Several thousand plant polyphenols are

known,encompassingawidevarietyofmoleculesconsistingofoneormorearomatic

rings with variable degrees of hydroxylation, methoxylation and glycosylation

(Manganaris, 2014). They comprise multiple simple phenols or phenolic acids, with

differingnumbersofphenolicringsandsubstitutinggroupsandareclassifiedintotwo

groups:theflavonoidsandthenon-flavonoids.Only5-10%ofthetotalconsumptionof


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polyphenols in the diet is absorbed directly in small intestines. The most ingested

polyphenols are metabolized by colon microorganisms before absorption. Certain

polyphenols present absorption facility. Absorbed polyphenolic metabolites can

interactwithadiposetissue,pancreas,muscleandliverexertinganti-diabeticeffects.

Thefibersourcepersemayhaveindependenteffectsoncolonichealth.First,dietary

fiber increases viscosity and fecal bulking (diluting potential carcinogens), and it

therefore shortens the time forproteolytic fermentation (andproductionofharmful

substances) and it also decreases the contact between potential carcinogens and

mucosal cells. In addition, dietary fiber could bind/excrete potential luminal

carcinogens(e.g.,secondarybileacids)andlowerfecalpHinthecolon.Second,dietary

fiber is not only a substrate for fermentation, but it is also a source of vitamins,

mineralsandslowlydigestibleenergy;forexample,branfractionsarerichinminerals,

vitamin B6, thiamine, folate and vitamin E. Third, dietary fiber is associated with

phytochemicals such as phenolics, carotenoids, lignans, beta-glucan and inulin. For

example, arabinoxylan, a constituent of hemicelluloses, is an important source of

phenolic compounds that may be released in the colon during fermentation of

complexedfibers.ThesebioactivesubstancesmayprotecttheGItractfromoxidative

damage,althoughthispossibilityiscontroversialduetotheanaerobicenvironmentin

thecolonandthefactthatthefiber-associatedphytochemicals(e.g.,carotenoids)do

notseemtobeabsorbedthroughtheGItractintotherestofthebody,eventhough

thecolonistheprimarysiteforfiberfermentationandthereleaseofthesechemicals.

However,sincetheconcentrationsofbioactivesubstancesderivedfromdietaryfiber

sources can bemuch higher in the colonic lumen than in plasma and other tissue,

thesephytochemicalsmaydelaytheonsetofcoloncancer(Zengetal.,2014)

DIABETESANDGASTROINTESTINALHEALTH

Intestinal morphology changes with nutritional variations, stress, aging, and (or)

diseaseandaffectsthephysiologyoftheintestine,specificallynutrientabsorptionand

metabolism. Because the absorptive functions of the intestine are related to its


19

morphology, alterations in morphology may predispose the intestine to functional

disorders.

DM patients often suffer from gastrointestinal (GI) disorders which are recently

recognized as one of themost common complications in this disease. ThewholeGI

tractcanbeaffectedintheDMandcommoncomplaintsincludediarrhea,constipation

and fecal incontinence. The symptoms areusually non-specific but occasionally they

maybesevereenoughtodecreasethequalityoflife.Thesymptomsarecloselyrelated

tohistomorphologicalandbiomechanicalchangesintheintestineandcolon.Disorders

ofintestineandcoloninDMareassociatedwithstructuralchangesintheconnective

tissuematrixandinthemusclesinthewallofintestineandcolonandfurthercauses

biomechanicalremodeling.Manymechanophysiologicalchangesoccurinthediabetic

intestineandcolonsuchaschangeddimensionsandchangedpassiveandactivetissue

properties.Remodelingalsooccursinthenervestructureandfunction.StudiesonSTZ-

inducedDMrats,carriedoutbyZhaoetal.(2015)haveshowntheopeningangleand

residualstrainbecamesmallerintheduodenumandjejunumandlargerinileumand

colon. Inaddition, ithasdemonstratedthestiffnessof thesmall intestinalandcolon

wallincreasedasfunctionoftimeofDMdevelopmentandthestressofintestinalwall

relaxedless.ThisGIremodellinghasbeenshownwasassociatedwithincreasingblood

glucose level and the consequent overproduction and accumulation of advanced

glycationendofproduct(AGE).Indeed,AGEsaltercellularstructureinducingchanges

in the extracellularmatrix architecture through the formation of protein cross-links.

Moreover, it has demonstratedAGEs damageperipheral nerves through a receptor-

dependentpathway,leadingadysfunctionofmechanic-sensitiveafferentsactivity.The

binding betweenAGEs and their receptormodifymajor axonal cytoskeletal proteins

suchastubulin,neurofilamentandactincontributingtothedevelopmentofatrophy

and degeneration of nerve fibres. Hyperglycemia markedly interfered with

homeostatic epithelial integrity, leading to abnormal influx of immune-stimulatory

microbialproductsandapropensityforsystemicspreadofentericpathogens (Thaiss

etal.,2018).

TheDM-induced intestinalhistomorphological changes suchasmucosadamagemay

also be related to GMmodification and function. The leaky epithelium presumably

alleviates the penetration of bacteria through the intestinal epithelium, initiating a


20

pathologic cascade and disturbing the intestinal immunology, which is a critical

element in thedevelopmentof type1DM.On theotherhand, theGMchangesmay

alsoaffectthe integrityof intestinalmucosaandsmoothmusclefunctions(Qinetal.

2012). In some recent studies, gut metagenome was shown to be a factor for the

development of DM. Different kinds of gut bacteria may play different roles in

maintaining or interacting with their environment. Two-stage metagenome-wide

association study (MGWAS) suggested thatDMpatients showamoderatedegreeof

gut microbial dysbiosis, with various butyrate-producing bacteria being decreased

(Clostridiales sp. SS3/4, Roseburia intestinalis, Roseburia inulinivorans, Eubacterium

rectale and Faecalibacterium prausnitzii) and some opportunistic pathogens being

increased (Bacteroides caccae, Clostridium hathewayi, Clostridium ramosum,

Clostridiumsymbiosum,EggerthellalentaandEscherichiacoli)(Mussoetal.,2011).

Studies together support the crucial role ofmicrobiota inmaintaining the intestinal

barrierintegrity,sustaininganormalmetabolichomeostasis,protectingthehostfrom

infection by pathogens, enhancing host defence system and even influencing the

nervoussysteminDM.


21

CAROBFRUIT

Thecarobtree(CeratoniasiliquaL.)belongstotheLeguminosaefamilyandiswidely

cultivatedintheMediterraneanareawhereitisconsideredanimportantcomponent

ofvegetationforeconomicandenvironmentalreasons.Worldproductionisestimated

atabout160,000t/yearproducedfromsome80,000hectareswithveryvariableyields

dependingoncultivar,regionandfarmingpractice.

Thefruit isan indehiscentpod,elongated,compressed,straightorcurved,thickened

at the sutures, 10–30 cm long, 1.5–3.5 cmwideandabout1 cm thickwithbluntor

subacuteapex.Podsarebrownwithawrinkled surfaceandare leatherywhen ripe.

The pulp comprises an outer leathery layer (pericarp) and softer inner region

(mesocarp). Seedsoccur in thepod transversally, separatedbymesocarp.Thecarob

fruit contains two major parts: the pulp (90%) and the seeds (10%). The chemical

compositionofthepulpdependsoncultivar,originandharvestingtime.Carobpulpis

high (48%–56%) in total sugar content (mainly sucrose, glucose and fructose). In

addition, it contains about 18% cellulose and hemicellulose. Ripe carob pods also

containa largeamountofcondensedtannins (16%–20%ofdryweight) .Carobseeds

areusuallyexploitedfortheproductionofcarobbeangum(CBG)orlocustbeangum

(LBG). This gum comes from the endosperm of the seed and chemically is a

galactomannan. It is 2 of 20 added as thickener, stabilizer or flavouring in food. In

additiontothefoodindustry,LBGiswidelyusedforpharmaceuticalpurposesasit is

linkedwiththeinhibitionofgastrointestinaldiseases.Furthermore,LBGisexploitedas

acarrieragentforthecontrolledreleaseofdrugsaloneorincombinationwithother

carrier molecules. Recently, researchers have focused on the valorisation of carob

podssincetheyareanexcellentsourceofbioactivecompoundssuchasdietaryfibre,

polyphenols,andcyclitolsandcontainlowamountsoffat.Inaddition,carobpodscan

beusedasacocoasubstitutesincetheydonotcontaincaffeineandtheobromine.The

whole unprocessed fruit or its by-products, such as the germ, fruit extract, kibbles

without seedsand theseedpeel,havealsobeen investigatedby food technologists.

Different parts of carob fruits have been used as food ingredients in bakery and

confectioneryproducts,aswellasinfermentedandnon-fermentedpastasduetotheir

health-promotingproperties.


22

Carob pods are an ideal substrate for the production of food ingredients exploiting

biotechnology. In particular, they aremainly used to produce citric acid, lactic acid,

mannitol, succinicacidandethanol. In the last twodecades,numerous studieshave

demonstrated interesting findings concerning the bioactivity of carob pulp

constituents.Carobfibreisproducedbywaterextractionofthecarobpulptoremove

themajorityofsolublecarbohydrates;totaldietaryfibrecontentusuallyranged30%–

40%ofcarobpulp(Haber,2002).Thehighproportionofpolyphenolspresentincarob

fibre differentiates it from other dietary fibre sources. In general, carob fibre is

consideredasapredominantlyinsolubleandpracticallynon-fermentabledietaryfibre

(Nasar-Abbas, 2016). On the other hand, the amount of soluble dietary fibre is

significantlylower(max10g100g-1carobfibre)andcontainssimplecarbohydrates

(Goulas,2016).Moreover,theseplantsareaninexhaustibleandinexpensivesourceof

polyphenols. (Imane Lakkab, 2018). The most prevalent categories of phenolic

compoundsincarobfruitarephenolicacids,gallotanninsandflavonoids.Thisgroupof

bioactive compounds havemainly attracted scientific attention due to their links to

healthpromotion.Theconcentrationofpolyphenols incarob fruitsdependsstrongly

ongenetic,environmentalandextractionmethodsandrangesbetween45–5376mg

gallicacidequivalentsper100g.Incarobfruits,phenoliccompoundsarefoundasfree,

asboundorassolubleconjugatedforms;however,themajorityofcarobphenolicsare

covalently bound to the dietary fibers (Dubravka et al., 2014). The polyphenolic

compositionofdifferentpartsofcarobisshowedinFigure8.


23

Figure8:Polyphenoliccompositionofdifferentpartsofcarobfruit(Dubravkaetal.,2014andTheophilouetal.2016).

Healthbenefitsofcarob

Numerousstudieshaverevealedseveralphysiologicalresponsestocarobfruitandits

productsthatmayberelevanttopreventionortreatmentofsomechronicdiseases.

The Carob fruit has several beneficial and health-promoting effects due to its

phytocostituents such as fibre, cyclitols, polyphenols. These groups of bioactive

compoundshavebeen linkedwith thehealth-promotingeffectsofcarob indifferent

therapeutic areas, including anti-cancer, anti-diabetes, anti-diarrheal and anti-

hyperlipidemia. These findings have rendered carob fruit an excellent ingredient for

thedevelopmentoffunctionalfoodandherbalsupplements.Thevalorisationofthese

bioactiveconstituentsismoreattractiveifweconsiderthattheyareusuallydiscarded

as LBG and simple sugars are used by the industry. In addition, experts have

demonstratedthebeneficialeffectsofthecarobfruit(Azadetal.,2015).

Indeed,carobfruitpolyphenolswereshowntohaveantitumor,anti-proliferativeand

proapoptotic activity leading preventive effect, growth decreasing and size reducing


24

action on the cancer, such as osteosarcoma, hepatocellular carcinoma, pancreatic,

colon and breast tumours. Furthermore, by lowering cholesterol and triglycerides

levelscarobfibrehasananti-hyperlipidemiaactionpromotingcardiovascularhealth,

modifyingpostprandiallipemiabyreducingtheextentsoffatdigestionandabsorption

(Macho-Gonzalezetal.,2018).Also,antidiabeticpropertieshavebeendemonstrated.

A recent study showed that CFE considerably reduced postprandial glycemia after a

single administration and enhanced these effects after a week of consumption

(Macho-Gonzalezet al., 2017). Thedecreaseofpost-prandialhyperglycemia through

the inhibition of two key-enzymes coupledwith type 2 diabetesmellitus such as a-

amylaseanda-glucosidaseisanimportanttherapeuticstrategyusedfortheregulation

or the management of diabetes (type 2). Indeed, a-glucosidase, after its catalysis

activity,itreleasesglucoseintothebloodwhichprovokesanincreaseinitslevel.The

reductionof the intestinal carbohydrateabsorptionbya-glucosidase inhibitors limits

the augmentationof blood glucose level. Among these inhibitors,we canquote the

syntheticoralhypoglycemicfactorssuchasacarboseandmiglitol,whichhavesevere

gastrointestinal side effects. Accordingly, the research for natural a-glucosidase

inhibitorswithnosideeffectscouldbean intentionadvanceforthemanagementof

rise in blood glucose rate. Inhibitors of these enzymes delay carbohydrate digestion

andprolongoverallcarbohydratedigestiontime,causingadiminution inthe levelof

glucoseabsorptionandthereforedecreasingthepostprandialplasmaglucoserise. In

thisrespect,theworkofCustódioetal.(2015)reportstheinvitroinhibitoryactivityof

waterdecoctionsofleaves,germflour,pulp,locustbeangumandstembarkofcarob

treeona-amylase,a-glucosidase.Ontheotherhand,alsonewpreventionortherapy

alternativescouldbebasedonstrategiestoreduceortoinhibitnutrientabsorptionin

intestinal section. Hence, the minimization of glucose absorption could be of some

help in controlling hyperglycemia and could represent a novel mechanism for an

antidiabetic agent inpatientswithdiabetes. In this case, numerousmedicinal plants

andtheirextractshavebeenreportedtobeeffectiveinthetreatmentofthisdisease

Theseplantsarerichsourcesofantidiabeticandantioxidantagentssuchasflavonoids,

gallotanninsandotherassociatedpolyphenols.Forthisreason,inaveryrecentstudy,

itwasfoundthattheimmaturecarobbeanprevents intestinalglucoseabsorptionby

the inhibition of electrogenic sodium-dependent glucose transport in mice using a


25

technique of Ussing chamber, who participates in the hypoglycemic effect. More

importantly, immature carob at various doses showed a significant reduction in the

bloodglucoseandbiochemicalprofilesinthediabeticrats(Rtibi,2017).Recently,ithas

beenreportedthattheCarodtreehasmultiplepharmacologicalactivitiesindigestive

tract including antioxidant, antidiarrheal, antibacterial, anti-ulcer and anti-

inflammatory actions (Figure 9). Another study indicates that carob possesses a

laxativeeffectongastrointestinalpropulsion.Rtibietal.,(2017)suggestedthatcarob

tree may be used in preventing free radical-related diseases as a dietary natural

antioxidant supplement and established the beneficial gastrointestinal therapeutic

propertiesof,particularly,theleavesandpodsofCeratoniasiliqua.

Figure9:Pharmacologicaleffectsofcarobtree(CeratoniasiliquaL.)onthegastrointestinaltract.(Rtibietal.,2017).


26

OBJETIVE

We propose to demonstrate the capacity of a diet supplemented with extracts of

carobpulp (Exenterol) (CFE),administeredasapreventiveorantidiabetic treatment,

toalleviatealterationsintheintegrityofthecolonicmucosainanexperimentalanimal

modelofdiabetes

For this, the followingspecificobjectiveshavebeencarriedoutanddescribed in the

introductionsection:

• Toknowwhatdiabetes is:symptoms,ethology, treatment, importanceofthe

disease.

• To study how the colonic epithelial barrier works and is organized and its

interactionwiththemicrobiota.

• Tochecktheeffectofdiabetesonthecolonmucosaandmicrobiota.

• To study the compositionandcharacteristicsofCFEextract, itsbioavailability

anditseffectonthehealth.

TodemonstratethepossibleprotectiveeffectofsupplementedCFEinthedietonthe

integrity of themucosa of altered colon in DMwe proposed to study the following

specificobjectives:

• EffectofDM inductionandconsumptionofCFEon thearchitectureof colon:

weightandlengthofthecolon:distinctionofthedistalandproximalcolon.

• EffectofDMinductionandconsumptionofCFEonthecolonicbarrierintegrity:

morphometric parameters and number of cells of the mucosal layer of the

colon.

• Toassesswhethertheconsumptionofthesupplementinthedietoffibrerich

in CFE reverts or improves the gastrointestinal alterations found in diabetic

animals.


27

MATERIALSANDMETHODS

Carobfruitpulpextract(CFE)Carob fruit pulp extract (CFE) is a natural insoluble dietary fibre obtained from the

carob pulp following the procedure described in PatentWO2004/014150. Once the

seedwasremoves,itwaswashedfor45minwithhotwater(45°C)inconstantstirring,

discarding the finalwashing liquid.Thisprocesswas repeated3 times.Thewetpulp

washomogenizedinwaterina1:2ratio(w/w)andkeptundervigorousstirringfor3

hours at 80 °C. Finish theallotted time, a vacuum filtrationwas carriedout and the

liquidobtainedwascooledthroughadecantingprocessatroomtemperaturefor6h.

Finally,thedecantedsamplewascentrifugedat2500gandthesupernatantremoved.

Resulting residue was dried in a heater at 85 °C for 12 h and 0.2 atmospheres of

pressure.Oncecooled, itwasmilledwithahammermillandsieveduntilobtaininga

productwithsizedlessthan180µm.AccordingtoBastidaetal.(2009)andthePatent

WO2004/014150, the average compositionwas as follows: 4.5−7% proteins, 0.5−1%

fats, 1.5−3.5% sugars, 3−4% ash (0.6−1.1% calcium, 0.02−0.026% sodium,

0.025−0.047%potassium,and0.01−0.016%iron),and74−84%totaldietaryfibre(with

1−3% soluble fibre and 71−81% insoluble fibre). Moreover, the polyphenols are

composed of 34−48% non-extractable condensed tannins and 0.5−1% soluble

extractablepolyphenols.AccordingtoPatentWO2006/000551,themolecularweights

ofCFEtanninsrangedfrom6000to30000Da,thuspermittingestimationofarangeof

thedegreeofpolymerizationof26−133forflavan-3-ol.(Macho-Gonzálezetal.,2018).

DietDietswerepreparedfromapurifieddiet formulation (referenceU8959,version180;

PanlabS.L.).Dietwascomposed(foreachkilogram)bypurifieddietformulation(65%),

lard (10%), cellulose powder (5%) and mixed minced meat lyophilized (25%). The

mixedmincedmeat(50%pork:50%veal)andlardwerepurchasedatalocalstoreand

subsequentlywas lyophilizedandground inachilledmeatcutter (StephanUniversal

Machine UM5; Stephan, Shóne Gmbh and Co.) following a standard procedure to

obtainapowderproduct.Alltheingredientsweremixedandthensubsequentlysieved

for3 timesuntil obtaininga completelyhomogenouspowder. The resultingproduct

wasthedietgiventotheanimals.Somedietscarried1.75%CFEadded,whoseamount


28

wasaddedat theexpenseofcellulose,with theaimofnotmodifying the total fibre

amongthediets,asdetailedintable1.CFEisanatural insolubledietaryfibre,which

was obtained from the carob pulp following the procedure described in

WO2006/000551A1patentandwhosecompositionisdescribedinMacho-Gonzálezet

al.,(2017).

ExperimentaldesignTwenty-four male Wistar rats of two-month-old were obtained from Harlan

Laboratoriesmodels(HarlanS.L.,Barcelona,Spain)andusedinaccordancewithanimal

protocolsapprovedbytheInstitutionalLaboratoryAnimalCareandUseCommitteeat

the Universidad Complutense de Madrid, Spain, and following European Directive

86/609 EEC. The rats were housed in cages and kept in a room at 22±1°C, 60%

humidityandwitha12h light-12hdarkcycle.Animalswerekeptonamaintenance

diet foroneweek.After the acclimatisationperiod, the ratswere fedwith standard

diet (control) or hypercholesterolemic (HC) diet for 8 weeks. Diabetes was induced

from the third week of experiment, by intraperitoneal injection of low-dose

Streptozotocinplusnicotinamide(STZ(65mg/kgb.w.)andNAD(225mg/kgb.w.)and

CFEwas included in thedietofDM-CFE rats.At theendof the study, the ratswere

feed-deprived overnight but allowed free access to water, then were anesthetized

with isoflurane (5%) and euthanized. The colon was removed from the peritoneal

cavityandfrozenat-80°Cuntilprocessing.Thelargeintestinewascollected,weighted

anditslengthwasmeasuredtoobtainthearea.Additionally,segmentsoftheproximal

and distal colon were collected. Some samples were designated to the histological

studyof the intestinalwallbyHematoxilinandeosinstaining (H&E)andthePeriodic

Acid-Schiff(PAS)histochemicaltechniquetoidentifygobletcells.Allexperimentswere

performedincompliancewithDirective86/609/EECof24November1986(amended

by Directive 2003/65/EEC of 22 July 2003) on the protection of scientific research

animals. This study was approved by the Spanish Science and Technology Advisory

Committee (project AGL 2011-29644-C02-02) and by the Ethics Committee of the

UniversidadComplutensedeMadrid(Spain)


29

Figure10:Experimentaldesign

Analyticalmethods

PreparationofparaffinsectionsTissueissectionedanddrop-fixedina10%formalinsolution(2%w/v)foraminimum

48 hours at room temperature. After 48 hours of fixation, tissuesweremoved into

70% ethanol for long term storage. The tissues were washed in distilled water

transferred into the series of alcohol at increasing ratios for dehydration

procedure.Followingdehydration,clearingprocedurewasperformedonthetissuesby

holding inxylenesolution.Thetissuesclearedwithxyleneweretransferred intosoft

paraffin (melting point 46–48°C) and hard paraffin (melting point 56–58°C) baths

respectively.Then,theywereblockedbyembeddingintohardparaffin.Sectionsof7

μmthicknessweretakenfromtheblockedtissuesbyrotarymicrotome(Leitz,Wetzlar,

Germany).


30

Figure11:Preparationofparaffinsections.Fromhttp://www.wesapiens.org/class/4933003/file/7/Histological+techniques%3A

HaematoxylinandEosin(H&E)stainingPrinciplesoftheProcedure:HaematoxylinandEosin(H&E) isoneofthemostwidely

usedhistologicalstainingmethodsofallandisaprimarycontrastmethodinmedical

diagnosis of biopsy specimens. It is used to discriminate between a broad range of

cytoplasmic, nuclear and extracellular matrix features. The H&E staining method

involves application of haematoxylin, a basic dye, to yield a blue-purple contrast on

basophilicstructures.Anacidiceosincounterstainseosinophilicstructuresbrightpink.

ManualProtocolforH&Estain

1. De-paraffinizeslides through threechangesofxylene, incubatingslides10min in

eachchange.

2. Hydrateslidestowaterbydippingthem20-40timesineachofthreechangesof

100%ethanol,twochangesof95%ethanol,oneof70%ethanol.


31

3. Rinsewith2-3changesoftapwater,30-50dipseach.

4. StainwithHematoxylinfor5minutes.

5. Rinse with 3-4 changes of tap water using Hematoxylin rinsing vat, 30-50 dips

each.

6. BlueinAmmoniawaterfor3-4dips.

7. Rinse with 3-4 changes of tap water using Hematoxylin rinsing vat, 30-50 dips

each.

8. Mordantin95%ethanolfor15-20dips.

9. StaininEosinfor10minutes.

10. Dehydratethrough2changesof95%ethanol(15dipseach)followedby3changes

of100%ethanol(30-50dipseach).

11. ClearthroughthreechangesofXylene,30-50dipseach.

12. MountcoverslipwithClear*mount.

PeriodicAcid-Schiffstaining(PAS)Principlesof theProcedure:When treatedwithperiodicacid, glycolsareoxidized to

aldehydes.AfterreactionwithSchiffʹsReagent(amixtureofpararosanilineandsodium

metabisulfite), a pararosaniline adduct is released that stains the glycol-containing

cellularelementspinktored.Cellularelementswhichmaybedemonstratedwiththe

PASprocedureincludeglycogen,fungalwalls,basementmembrane,certainepithelial

sulfomucinsandsialomucins,neutralmucosubstances,colloidmaterialofthethyroid

andtheparsintermediaofthepituitary.

ManualProtocolforPASStain(Figure11)

1.Deparaffinizeandhydratetheslidestowater.

2.Oxidizetheslidesusing250μlofperiodicacidsolutionfor10minutes.

3.Rinseindistilledwater.

4.Apply250μlofSchiffreagentfor15minutes

5.Add250μloflukewarmScottsSolutionfor5minutes.

6.Counterstainusing250μlofMayer'shematoxylinfor1minute.


32

7.Washintapwaterfor5minutes.

8. After staining, dehydrate and clear in xylene and apply permanent mounting

medium.

Figure12:ProtocolPeriodicAcid-Schiffstaining

ImagesanalysisImagesoftheobtainedsectionswerecapturedusingahigh-resolutioncamera(digital

LeicaDFC320camera) coupled toa lightmicroscope (DMLB2Leica).Morphometric

and quantitative analyses were performed with the aid of ImageJ image analysis

softwarefromImagesofthesectionsstainedwithH&EandPASstaining.Cryptswere

selected tomeasure intestinal crypt depth and crypt density.Only crypts that could

clearly be seen to extend from the base to the brush border of the luminal surface

were taken into account. The crypt density represented the number of crypts per

millimetre along the luminal surface. Images of sections obtained using the PAS

histochemical method were used to quantify goblet cells present in 50 images per

animal(40×objective).Resultsareexpressedasthenumberofgobletcellspercrypt.

Statisticalanalysis


33

The valueswereexpressedas themeans± standarderror (S.E.M).OnewayANOVA

followedbyBonferronitestswereusedtoassesstheeffectofCFE.Thedataobtained

fromthesemiquantitativescorewerecomparativelyanalyzedusingtheKruskal-Wallis

test followed by the Bonferroni post hoc test. Results were considered statistically

significant at P<0.05. The calculations were carried out by using the SPSS program

(SPSSInc.,Chicago, IL,USA)andGraphPadPrismsoftware (GraphPadPrism6.01,San

Diego,CA).


34

RESULTS

Effect of ingestion of extracts of carob pulp (Exenterol) (CFE) on the architecture of

colonincontrolanddiabeticrats.

Both full colon weight (20.3%) (P<0.001) (Figure 13A) and colon length (20.9%)

(P<0.0001) (Figure 13B) increased in the DM-CFE group when compared with the

controlandDMgroups.AsshowninFigure13C,themacroscopicaspectofcolonfrom

DM-CFEratsfedweremarkedlylongerthanthosefromcontrolandDMgroups.

Effect of ingestion of extracts of carob pulp (Exenterol) (CFE) on the colonic barrier

integrityinproximalanddistalcolon.

Histological sections of proximal and distal colon from the control group showed to

haveawell-organized liningof crypts,with thepresenceof columnarepithelial cells

(with microvilli) and goblet cells. Furthermore,the colon layer from control rats

revealednoinflammatorychangessuchasulcerations,cryptabscesses,changesinthe

lamina propia, or alterations in the surface epithelium and crypt epithelium (Figure

14A).OnlythedistalDMcolonshowedlossoftheregularcryptstructures,thinningof

themucosa,shortenedcrypts, infiltrationofinflammatoryimmunecells,depletionof

goblet cells, disruption of the brush border (Figures 14A, 15). Morphometric

measurements showed thatDMsignificantlydecreasedall themeasurementsof the

histologicparameterscomparedwiththecontrol rats,suchasdecreases inthecrypt

depth and the number of crypts per millimeter (Figures 14A,B). Thus, the loss was

20.2% (P<0.01) for mucosal thickness, 13.5% (P<0.001) for crypt depth, 21.5%

(P<0.001) for crypt density and a 35% (P<0.0001) for the number of goblet cells.

Following CFE treatment, the rat colonic mucosa and intestinal crypts, and cells

structure,bothenterocytesandgobletcells,graduallyreturnedtonormal,presenting

noinflammatorycellinvasion(Figures14and15).


35

(A)

(B)

(C)

Figure13:Effectofextractsofcarobpulp(Exenterol) (CFE)oncolonicweight (A)and

length (B) in diabetic rats. (C) Photograph taken from experiment showing caecum,

proximal and distal colon in control, DM and DM-CFE rats. Data are mean±SEM

Control DM DM-CFE0.0

0.5

1.0

1.5

2.0

2.5ControlDMDM-CFEa a

b

colo

nic

wei

ght (

g)

Control DM DM-CFE0

5

10

15

20ControlDMDM-CFE

a ab

colo

nic

leng

ht (c

m)


36

(n=8/group). Letters indicate significant different groups,*means significant different

betweenproximalanddistalsections(P<0.05).ANOVAwithBonferroniposthoctest.

(A)

(B)

(C)

Figure14:Anti-diabeticeffectofingestionofextractsofcarobpulp(Exenterol)(CFE)on

depth crypts and crypts density (crypts/mm) in proximal and distal colon. (A)

HistochemicalH&E–stainedcrosssectionofproximalanddistalcolon(x200).(B)Height

Proximal Distal0

10

20

30

40

50Control

DM

DM-CFE

a a

a*

ab*

a*

Num

ber o

f cel

ls/h

emic

ryts

Proximal Distal0

5

10

15

20

25 Control

DM

DM-CFEaa

aa* a*

b

Cry

pts

dens

ity (c

rypt

s/m

m)


37

crypts(numberofcellsperhemicrypts.(C)Cryptdensity(numberofcrypts/mm).Data

are mean±SEM (n=8/group). Letters indicate significant different groups, *means

significantdifferentbetweenproximalanddistalsections.ANOVAwithBonferronipost

hoctest(P<0.05).

(A)

(B)

Figure 15: Effect of extract of carob pulp (Exenterol) on number of goblet cells in

proximalanddistalcolonindiabeticrats,(A)HistochemicalPASstainingimages(x400).

(B)Numberofgobletcellspercrypt.Dataaremean±SEM(n=8/group).Lettersindicate

Proximal Distal0

10

20

30

40

50Control

DM

DM-CFE

aa

a

b*

a*b*

PAS

posi

tive

cells

/cry

pt


38

significantdifferentgroups,*meanssignificantdifferentbetweenproximalanddistal

sections.ANOVAwithBonferroniposthoctest(P<0.05).


39

DISCUSION

Impactofdiabetesinductiononthecolonicmorphometry

Diabetesmellitus(DM)hasdeleteriouseffectsonintestinalhealth.DMcanaffectthe

structure and function of the colon promoting commonly encountered lower

gastrointestinal symptoms such as constipation, diarrhea, abdominal distention,

bloating,andabdominalpain(Rtibietal.,2017).Themorphometricparameterssuch

ascryptdepth,width,cryptdensityandnumberofbothtotalcellsperhemicryptand

gobletcellsarefrequentlyusedasreliableindicatorsformonitoringintestinalmucosal

morphology,which plays critical roles in nutrient absorption. In this study, the total

intestinalwallandthemorphometricparameterinproximalcolonofDManimalsdid

not exhibit any significant alterations compared with nondiabetic animals (P<0.05).

However, the experimental diabetes associated with a high fat diet in this study

resultedinabnormalcoloniccryptmorphologyindistalcoloncomparedtohealthyrat.

DM showed significantly shorter and wider crypts of Lieberkühn as indicates the

measurement of crypt density. As the crypts present basal cells capable of dividing

several times by mitosis in the intestinal mucosa tunic, which carries out the

absorption, thus, its number will determine the epithelium absorption capacity

(NaftalinandPedley,1999).Diabetesmayresultinintestinalabsorptiondecreasedue

totheenterocytenumberandheightdecrease.Whenthegobletcellswerequantified,

we verified that they significantly decreased in distal colon of the DM group. The

decreaseofthecryptdepthandthenumberofgobletcellshasprobablyinterferedin

the normal functioning of the mucosa, its regeneration, and nutrient absorption,

demonstratingthattheinductionofdiabetesreflectsharmfullyforthelargeintestine.

Gulhaneetal. (2016)alsoshowedthatHFD-feeding leadstospontaneousgobletcell

dysfunction,impairedmucosalbarrierfunctionandinflammation,andexacerbatesthe

development of pathology in mice predisposed to ER stress-induced colitis. These

stress-induced changes provide a plausible explanation for the altered microbiota,

leakageofmicrobialproductsandinitiationofmucosalinflammationcharacteristicof

metabolicdisease suchasDM.Also,high fatdiet (HFD) feedinghasbeenassociated

withadversemicrobialmodificationsinthemicrobiotaandimpairedintestinalbarrier

function. In turn, disruption of intestinal barrier function plays a role in the


40

pathogenesisofseveraldiseases includingDM(Moreiraetal.,2012).Theenterocyte

heightreductionwithareducedmucusbarrierfunction,leadingtoenhancedcontact

of colonocytes with microbiota and toxic substances. Thus, microorganism at the

lumenmayeasilyinvadetheorganismthroughthetranscellularpathway.So,weinfer

thatthevolumeofthesecretedbythesecells(mucines)wassmallerfortheDMgroup,

whatmaybeunderstoodasawayofendogenprotein losscontentionaswellasthe

assignmentoflessaminoacidintakeforcells,tissues,organs,andmetabolicpathways

essential for animal survival. The DM-induced intestinal histomorphological changes

such asmucosa damagemay also be related to GMmodification and function. The

bidirectionalinterplaybetweenGMandDM-inducedintestinalchangescontributesto

the pathogenesis ofGI disorders inDM (Zhao et al., 2017). Roopchand et al. (2015)

propose that this altered gut microbiota is, in part, responsible for the altered

intestinalgeneexpression,epithelialintegrity,andinflammatorymarkers,whichthen

leadstodecreasedfatdepositionandglucoseabsorption,alongwithincreasedinsulin

secretion inducing DM. Futhermore, intestinal epithelial barrier dysfunction and

increasedpermeabilityhavebeendescribedinDM.The‘leakyguthypothesis'explains

that the intestinalbarrierdysfunction induces thechronic low-grade inflammation in

varioustargetorgansbyvirtueofmicrobialproducts(Fukui,2016).Thaissetal.(2018)

demostratedthatthehyperglycemiamaypredisposetobarrierdysfunctionleadingto

enhanced enteric infection in the setup of themetabolic syndrome. Streptazotozine

treatment also resulted in dysfunction of intestinal epithelial adherence junctions

under steady-state conditions, coupled with systemic dissemination of microbial

productsandenhancedtrans-epithelialflux.Ontheotherhand,thedifferencesfound

betweentwocolonicsectionsstudiedcouldberelated to theslowercell turnover,a

shorter fermentation time, or higher water-holding and fecal-bulking capacities and

higher GM in the distal colon comparedwith the proximal colon (López-Oliva et al.

2006). Therefore, Naftalin and Pedley, (1999) showed that there is a functional

difference between crypts from caecum and descending distal colon. Only the

descending colon can absorb fluid against a high hydraulic resistance in the lumen,

making thedistal colonmorevulnerable tobeingaltered. This funtionaldifferences

betweentwocolonicsectioncouldexplainthecanexplainthatthedistalcolonismore

vulnerabletoDM-stress.


41

Anti-diabeticeffectsofCFEconsumption

Plantpolyphenolsincludingphenolicacids,flavonoids,stilbenesandlignanshavebeen

proposed as effective supplements for diabetes management and prevention of its

long-termcomplications.Dietarypolyphenol-richproductsmodulatecarbohydrateand

lipid metabolism, attenuate hyperglycemia, dyslipidemia and insulin resistance,

improveadiposetissuemetabolism,andalleviateoxidativestressandstress-sensitive

signalingpathwaysandinflammatoryprocesses(Bahadoranetal.,2016).Thus,dietary

polyphenolshavebeensuggestedtolowertheriskandtreatmentofDMandappearto

reduce the insulin response to a glucose load and have anti-inflammatory effects

(Macho-Gonzalezetal.,2018).Carobfruit (CFE) isanexcellentsourceofsolubleand

insoluble fibres and also contains a panel of polyphenols such as gallic acid and its

derivatives, as well as condensed tannins (Goulas et al., 2016). So far, very limited

studieshavefocusedontheeffectsofCFEonDM.Thepresentstudyshowsthatthe

CFE consumption had an impact on large intestinal morphology in diabetic rats, in

particular inthedistalcolon.CFE intake improvedintestinalarchitecture inthedistal

colonicmucosaofDM-CFEratsincludinganincreaseinmucosalthickness,cryptdepth,

andcryptdensitycomparedwithDMrats.Thus,CFEinthedietinducedlongercrypts

and an increase in total number of crypts per millimeter, indicating that CFE diet

modifiescryptpopulation.Intestinalgrowthandcryptincreasingimplyintheintestinal

epithelial cell increasing and further absorption increase (Chedea et al., 2018). In

addition, the high concentration of polyphenols in CFE (34−48% non-extractable

condensed tanninsand0.5−1%solubleextractablepolyphenols)mayalso contribute

to epithelial hyperplasia, because diets rich in polyphenols from varying sources

and/orwithhightannicacidcontenthavebeenshowntoincreasetherateofmucosal

cell turnover and the excretion of endogenous nitrogen (Bahadoran et al., 2013).

Moreover, ithasalsobeensuggestedthattheprotectiveeffectsofcarobextractson

colon adenoma cells provide potential mechanisms of cellular protection against

factors that contribute to oxidative stress (Klenow et al., 2008).On the other hand,

measurements of intestinal morphology showed that DM induced inflammation

significantlydiminishedinCFE-DMrats.Micronutrientsandpolyphenolsincarobpods

havebeenpreviouslyreportedtoplayanimportantroleinthepreventionofintestinal


42

inflammationduetotheiranti-inflammatoryandantioxidantproperties(Kumazawaet

al.,2002).ThepresentexperimentalsoshowsthatfeedingCFEincreasesthenumber

of goblet cells in colonic distal section compared with the DM group, suggesting a

potential associated increase in mucin secretion and perhaps, therefore, in

strengthening of the gut barrier function. In summary, dietary polyphenolic

compounds of CFE improved glucose homeostasis through the improvement of the

integrity of the colonic intestinal barrier, as well as through the possible prebiotic

effectsinthedigestivetract.

CONCLUSION

The antioxidant and anti-inflammatory properties of polyphenols found in CFE have

the aptitude of preserving the integrity of the colonicmucosal barrier and reducing

colonicdamageinducedbydiabetes.CFEmayserveasignificanttherapeuticeffectin

thetreatmentsofgastrointestinaldisordersDMinduced.

Figure16:CFEpolyphenolsenhancetheintegrityofepithelialbarrierimprovingoverallcolonichealthandpreventdiabetesandmetabolicdisorders.


43

ABREVIATIONS

DB:DiabetesMellitus

CFE:CarobFruitpulpExtract

PAS:PeriodicAcidSchiff

HDL:GoodCholesterol

GDM:GestationalDiabetesMellitus

FPG:FastingPlasmaGlucose

OGTT:OralGlucoseToleranceTest

IGT:ImpairedGlucoseTolerance

IFG:ImpairedFastingGlycaemia

IECs:IntestinalEpithelialCells

SCFAs:Short-ChainFattyAcids


44

ACKNOWLEDGEMENTS

WewouldliketothankallourteachersthathavemadethispossiblesuchasGemaMartínwhohasgivenusthedifferentpatternstowritethisproject.CarlosOrtegahasbeenourtutorandourstrengthforbeingabletodothis.WeareespeciallygratefultoDrMaElviraLópez-Oliva(Fac.FarmaciaUCM)forprovidingthepicturesoftheexperimentmadeandforgivingushertime,patienceandeffortandforbeingalwaysthere.


45

BIBLIOGRAPHY

American Diabetes Association. 2. Classification and Diagnosis of Diabetes: Standards ofMedicalCareinDiabetes-2018.DiabetesCare2018;41:S13.

American Diabetes Association. Standards of medical care in diabetes-2012. Diabetes Care2012;35:S11–S63.

Azab A. Carob (Ceratonia siliqua): health, medicine and chemistry. Eur Chem Bull. 2017;10:456–69.BelaiA,LincolnJ,MilnerP,BurnstockG:Differentialeffectofstreptozotocin-induceddiabetesontheinnervationoftheileumanddistalcolon.Gastroenterology1991;100:1024-1032.Bastida,S.;Sánchez-Muniz,FJ.;Olivero,R.;Pérez-Olleros,L.;Ruiz-Roso,B.;Jiménez-Colmenero,F.Antioxidantactivityofcarobfruitextracts incookedporkmeatsystemsduringchilledandfrozenstorage.FoodChem.2009;116,748−54.Bahadoranetal.JournalofDiabetes&MetabolicDisorders2013,12:43Chedea, VS., Palade, LM., Marin, DE., Pelmus, RS., Habeanu, M., Rotar, MC., Taranu, I.IntestinalAbsorptionandAntioxidantActivityofGrapePomacePolyphenols.Nutrients,2018.10,588.Cheng LK,O'GradyG,Du P, Egbuji JU,Windsor JA, PullanAJ. Gastrointestinal system.WileyInterdiscip.Rev.Syst.Biol.Med.2010;2:65–79.

Custodio,L.;Patarra,J.;Albericio,F.;Neng,NR.;Nogueira,J.M.;Romano,A.Invitroantioxidantand inhibitory activity of water decoctions of carob tree (Ceratonia siliqua L.) oncholinesterases,amylaseandglucosidase.Nat.Prod.Res.2015;29,2155–2159.

Desai,MS.et al.Adietary fiber-deprivedgutmicrobiotadegrades the colonicmucusbarrierandenhancespathogensusceptibility.Cell2016;167,1339–1353.e21.

DiagnosisandClassificationofDiabetesMellitus.DiabetesCare2014;37(Supplement_1),S81–S90.

digital.csic.es/bitstream/10261/5220/1/TESIS%20Susana%20Delgado.pdf

Fukui H. Increased Intestinal Permeability and Decreased Barrier Function: Does It ReallyInfluencetheRiskofInflammation?InflammIntestDis2016;1:135-145.

Gulhane, et al. High Fat Diets Induce Colonic Epithelial Cell Stress and Inflammation that isreversedbyIL-22.ScientificReports2016;6,Articlenumber:28990.

HowellJCandWellsJM.Generatingintestinaltissuefromstemcells:potentialforresearchandtherapyRegenMed.2011;6:743–755.


46

http://www.diabetesatlas.org/resources/2015-atlas.html

http://www.gutmicrobiotaforhealth.com/es/inicio/microbiota-intestinal/

http://www.innerbody.com/anatomy/digestive/large-intestin

https://courses.lumenlearning.com/boundless-ap/chapter/the-large-intestine/

https://mejorconsalud.com/fisiologia-del-intestino-grueso/

https://www.investigacionyciencia.es/blogs/medicina-y-biologia/43/posts/da-mundial-del-microbioma-16476?utm_source=Twitter&utm_medium=Social&utm_campaign=tw+web

https://www.ncbi.nlm.nih.gov/pubmed/1996606

https://www.news-medical.net/health/What-Does-the-Large-Intestine-Do-(Spanish).aspx

InternationalDiabetesFederation.IDFDiabetesAtlas.6thed.Brussels:IDF;2013.

InternationalExpertCommittee.InternationalExpertCommitteereportontheroleoftheA1Cassayinthediagnosisofdiabetes.DiabetesCare2009;32:1327.

JašarevićE,MorrisonKE,BaleTL.Sexdifferencesinthegutmicrobiome–brainaxisacrossthelifespan.Phil.Trans.R.Soc.B2016;371,20150122.

Jašarević,E,A.B.Rodgers,T.L.BaleAnovelroleformaternalstressandmicrobialtransmissioninearlylifeprogrammingandneurodevelopmentNeurobiol.Stress,2015;1:81-88.

Keshavarzian, A S.J. Green, P.A. Engen, R.M. Voigt, A. Naqib, C.B. Forsyth, E. Mutlu, K.M.Shannon.Colonicbacterial composition inParkinson’sdiseaseMov.Disord., 2015;30:1351-1360.KlenowS,JahnsF,Pool-ZobelBL,GleiM.DoesanExtractofCarob(CeratoniasiliquaL.)HaveChemopreventivePotentialRelatedToOxidativeStressandDrugMetabolisminHumanColonCells?JournalofAgriculturalandFoodChemistry.2009;57:2999-3004.KumazawaS,TaniguchiM,SuzukiY,ShimuraM,KwonMS,NakayamaT.Antioxidantactivityofpolyphenolsincarobpods.JAgricFoodChem.2002;50:373-377.López-Oliva,ME,AgisA,Garcia-Palencia P,Goñi I,Muñoz-Martínez E. Inductionof epithelialhypoplasia in ratcecalanddistalcolonicmucosabygrapeantioxidantdietary fiberNutritionResearch2006;26:651–658.Macho-Gonzalez, A.; Garcimartín, A.; Lopez-Oliva, M. E.; Bertocco, G.; Naes, F.; Bastida, S.;Sanchez-Muniz, F. J.; Benedí, J. Fiber purified extracts of carob fruit decrease carbohydrateabsorption.FoodFunct.2017;8,2258−65.


47

Macho-González, A. A. Garcimartín, F. Naes, M. E. López-Oliva, A. Amores-Arrojo, M. J.González-Muñoz,S.Bastida,J.Benedí,F.J.Sánchez-Muniz.EffectsofFiberPurifiedExtractofCarobFruitonFatDigestionandPostprandial Lipemia inHealthyRats JournalofAgriculturalandFoodChemistry2018-06-21,DOI:10.1021/acs.jafc.8b01476.Maynard CL, Elson CO, Hatton RD, Weaver CT. Reciprocal interactions of the intestinalmicrobiotaandimmunesystem.Nature2012;489(7415):231–241.Mehta,S.S.Ahlawat,D.P.Sharma,R.S.DaburNoveltrendsindevelopmentofdietaryfiberrichmeat products-a critical review. Journal of Food Science and Technology 2013;10.1007/s13197-013-1010-2

Moreira,A.P.;Texeira,T.F.;Ferreira,A.B.;PeluzioMdo,C.;AlfenasRde,C.Influenceofahigh-fat diet on gutmicrobiota, intestinal permeability andmetabolic endotoxaemia. Br. J. Nutr.2012,108,801–809.

Mukherjee PK, Sendid B, Hoarau G, et al. Mycobiota in gastrointestinal diseases. Nat RevGastroenterolHepatol.2015;12:77–87.

MussoG,GambinoR,CassaderM.Interactionsbetweengutmicrobiotaandhostmetabolismpredisposingtoobesityanddiabetes.AnnuRevMed.2011;62:361–380.

Naftalin RJ and Pedley KR. Regional crypt function in rat large intestine in relation to fluidabsorptionandgrowthofthepericryptalsheath,JournalofPhysiology1999;51:.211—227.

OlokobaAB,ObateruOA,Olokoba LB. Type 2 diabetesmellitus: a reviewof current trends.OmanMedJ.2012;27:269–273.

PattersonE,RyanPM,CryanJF,DinanTG,RossRP,FitzgeraldGF,StantonC.Gutmicrobiota,obesityanddiabetes.PostgradMedJ.2016;92:286–300.

Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immunehomeostasis.NatRevImmunol.2014;14:141-53.doi:10.1038/nri3608.

Piper,M.S.&Saad,R.J.DiabetesMellitusandtheColon.CurrTreatOptionsGastro2017;15:460.

Qin J, Li Y, Cai Z. et al. A metagenome-wide association study of gut microbiota in type 2diabetes.Nature.2012;490(7418):55–60.

RtibiK,SelmiS,GramiD,AmriM,EtoB.Chemicalconstituentsandpharmacologicalactionsofcarob pods and leaves (Ceratonia siliqua L.) on the gastrointestinal tract: A review. BiomedPharmacother.2017;93:522–28.

SudesnaC,KamleshKhunti,MelanieJDavies:“Type2diabetes”.Lancet2017;389:2239–51

Thaissetal.,Hyperglycemiadrivesintestinalbarrierdysfunctionandriskforentericinfection.Science2018;.10.1126/science.aar3318.


48

TranTT,NaigamwallaD,OprescuAI.etal.Hyperinsulinemia,butnototherfactorsassociatedwith insulin resistance, acutely enhances colorectal epithelial proliferation in vivo.Endocrinology.2006;147:1830–1837.

Turner,JR. Intestinalmucosalbarrierfunctioninhealthanddisease.NatRevImmunol.2009;9:799–809.

WildS,RoglicG,GreenA.etal.Globalprevalenceofdiabetes:estimatefortheyear2000andprojectionsfor2030.DiabetesCare.2004;127:1047–1053.

WorldHealthOrganization.GlobalReportonDiabetes.2016,88

Wu Y, Ding Y, Tanaka Y, ZhangW. Risk factors contributing to type 2 diabetes and recentadvancesinthetreatmentandprevention.IntJMedSci.2014;11:1185-200.

Zatalia SR, Sanusi H. The role of antioxidants in the pathophysiology, complications, andmanagementofdiabetesmellitus.ActaMedIndones.2013;45:141–147.

Zeng,H.,Lazarova,D.L.,Bordonaro,M.,Mechanismslinkingdietaryfibre,gutmicrobiotaandcoloncancerprevention.WorldJ.Gastrointest.Oncol.2014;6,41–51.

ZhaoJ,ChenP,GregersenH.Stress-strainanalysisof jejunalcontractility inresponsetoflowand ramp distension in type 2 diabetic GK rats: effect of carbachol stimulation. J Biomech.2013;46:2469–2476.

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