Research ArticleMultiple Mechanisms of Flaxseed Effectiveness in InflammatoryBowel Disease

Amber Hanif Palla 12 Anwar-ul-Hassan Gilani 23 Samra Bashir 4

and Najeeb Ur Rehman5

1Faculty of Pharmacy Department of Basic Medical Sciences Barrett Hodgson University Karachi Pakistan2Department of Biological and Biomedical Sciences e Aga Khan University Medical College Karachi Pakistan3e University of Haripur Haripur Khyber Pakhtunkhwa Pakistan4Department of Pharmacy Capital University of Science and Technology Islamabad Pakistan5Department of Pharmacology College of Pharmacy Prince Sattam Bin Abdulaziz University Al-Kharj 11942 Saudi Arabia

Correspondence should be addressed to Amber Hanif Palla amberpallabhuedupk

Received 1 April 2020 Accepted 17 June 2020 Published 13 July 2020

Academic Editor Francesca Mancianti

Copyright copy 2020 Amber Hanif Palla et al (is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Ethnopharmacological Relevance Natural products like Flaxseed (Linum usitatissimum) have traditionally been used ininflammatory bowel disease (IBD) It is known to contain multiple constituents which may account for its effectiveness asIBD is a multifaceted disease Aim of the Study In the current study we aimed to assess pharmacological basis for themedicinal use of Flaxseed in IBD Materials and Methods Aqueous-methanolic crude extracts of Flaxseed (FsCr) andFlaxseed oil were tested against 6 acetic acid- (AA-) induced colitis in BALBc mice Microscopic damage parameters of thehematoxylin and eosin-stained and periodic acid-Schiff-alcian blue-stained sections of the colon were scored to be assessedPossible antispasmodic mechanism was studied on isolated rabbit jejunum while antibacterial activity was assessed in vitrofor microbes implicated in IBD Results In AA-induced colitis Flaxseed oil was found to be more effective in reducingmortality and colonic ulcers than FsCr at 500mgkg dose FsCr was more efficacious in increasing mucin content ascompared to oil exhibiting slightly greater anti-inflammatory effect (50 vs 35) and reducing depth of lesion (55 vs4231 respectively) Antispasmodic activity of FsCr (003 and 01mgml) was mediated by phosphodiesterase inhibitors(PDEI possibly PDE-4 subtype) with a resultant increase in cAMP levels Flaxseed oil PDEI activity was mild (1 and 3mgml) FsCr (01 and 03 mgml) was potent in exhibiting anticholinergic activity similar to dicyclomine whereas Flaxseed oilshowed anticholinergic effect at 1 and 3mgml Flaxseed oil (9 and 14 microgml) was bactericidal against enteropathogenic Ecoli(EPEC) enterotoxigenic Ecoli (ETEC) and enteroaggregative Ecoli (EAEC) whereas FsCr exhibited bactericidal effectagainst EPEC at 100 microgml Conclusions Results of this study taken together with previous studies suggest that Flaxseedpossesses anti-inflammatory antibacterial and antispasmodic action through multiple pathways and thus offers promisingpotential to be developed for IBD

1 Introduction

Inflammatory bowel disease (IBD) is a heterogeneous dis-ease in which multiple triggers act simultaneously (emajor maneuvering targets in IBD are immune dysregula-tion barrier and microbial defects along with abdominalspasms and colic as accompanying symptoms [1] Despiteadvancement in IBD therapeutics rate of failure to attain the

remission remains high possibly due to multifactorial eti-ology of the disease

Flaxseed (scientific name Linum usitatissimum) hastraditionally been used for IBD [2 3] In our earlier studieswe provided some evidence for its traditional use by showingthat the aqueous-methanolic crude extract of Flaxseed(FsCr) ameliorated the severity in mice model of colitis byreducing mediators of inflammation (myeloperoxidase and

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2020 Article ID 7974835 16 pageshttpsdoiorg10115520207974835

cytokines) and inducers of oxidative stress [4] (e extractthat we studied comprised predominantly of polar com-ponents whereas Flaxseed as a whole is a combination ofboth the polar and nonpolar constituents [5] (erefore togain an insight into the holistic potential of Flaxseed in IBDone of the aims of the current study was to assess the effect ofFlaxseed oil (nonpolar constituents) for its effectivenessagainst IBDmodel of mice and subsequently compare theseparameters with FsCr

Knowing that antispasmodics have an important role inIBD therapeutics as an adjunctive therapy [1] we also aimedto explore the precise mode of antispasmodic mechanisms ofFlaxseed (both the extract and oil) using isolated gut tissuesfrom experimental animals Earlier we have reported thatboth FsCr and Flaxseed oil exhibited antidiarrheal activitywith possible involvement of Ca+2 channel inhibition andnonspecific K+ channel activation respectively [6 7]However the possibility of other potential mechanisms wasthere as the inhibitory effect in isolated jejunal tissues againstcarbachol- (CCh 1 μM) induced contractions was notstudied which can help in identifying the presence of an-ticholinergic andor phosphodiesterase inhibitory mecha-nisms [8 9] both of which have potential for IBDtherapeutics [10 11]

(e third aim of our study was to assess the antibacterialactivity of Flaxseed against the bacteria implicated in IBD asmicrobes have an important role in IBD pathogenesis [12]Amongst them enteropathogenic E coli (EPEC) enter-otoxigenic Ecoli (ETEC) and enteroaggregative Ecoli(EAEC) are implicated in IBD as they share virulenceproperties with adherent invasive Ecoli (AIEC) [13] a strainisolated from biopsies of ileal Crohnrsquos disease (CD) patientsFlaxseed has not been studied for its activity against thesepathogens Hence in the current study efficacy and potencyof FsCr and Flaxseed oil against EPEC ETEC and EAECwere tested in vitro

To sum up our objective in the current study is to assessthe holistic potential of Flaxseed by examining and com-paring both the extract and the oil for their protective effectagainst acetic acid (AA)-induced colitis in mice antispas-modic mechanisms and antibacterial activity thusencompassing the different triggers of IBD

2 Materials and Methods

21 Standard Drugs Acetic acid acetylcholine chloridecarbamoyl choline chloride DPX periodic acid predniso-lone Schiff reagent and verapamil hydrochloride werepurchased from Sigma Chemicals Co St Louis MO USANutrient agar and Luria-Bertani broth powders used forantibacterial assays were also purchased from SigmaChemicals Chemicals used for making physiological saltsolutions including potassium chloride calcium chlorideglucose magnesium chloride magnesium sulfate potassiumchloride sodium bicarbonate sodium dihydrogen phos-phate EGTA and sodium chloride were obtained fromMerck Darmstadt Germany DMSO and Tween-80 used forsolubilization were purchased from Merck DarmstadtGermany and Scharlau Chemicals Barcelona Spain

respectively All chemicals used were of the available ana-lytical grade Cenogenics single strip kit was used for theoccult blood test cAMP enzyme immunoassay kit formultiple species was purchased from Arbor Assays AnnArbor MI USA (e reference drugs gentamycin waspurchased from Sigma Chemicals Co St Louis MO USANutrient agar and Luria-Bertani broth powders used forantimicrobial assays were purchased from Sigma Chemicalsand were of the available analytical grade

22 Preparation of Plant Material (Flaxseed Extract and Oil)Linum usitatissimum L (Flaxseed) was bought from anauthentic herb supplier in the local market of KarachiPakistan (e plant name and physical characteristicsmatched the description when checked with httpwwwtheplantlistorg It appeared as deep brown flat and oval seedwith a pointed tip and measured about 4mm A sample ofFlaxseed was deposited in the herbarium of the Departmentof Biological and Biomedical Sciences of the Aga KhanUniversity with a voucher number LU-SE-0812-106 (eseeds were made free of dirt and other adulterants and weregrounded finely into powder by an electrical chopper

(e aqueous-methanolic crude extract of Flaxseed(FsCr) was prepared from approximately 1 kg groundFlaxseed by the process of cold maceration using aqueousmethanol (30 70 vv) as a solvent for extraction [7] For oilextraction previously defined methods were used [14]Briefly approximately 1 kg of ground Flaxseed was soaked inpetroleum ether and kept on a water bath at 45degC for 7 dayswith occasional shaking Petroleum ether was evaporatedusing Rota vapor and the oil was filtered to clarity Re-spective yields of the FsCr and the oil were 8 and 27 wwwith reference to dried seeds Both the extract and the oilwere plated on nutrient agar and incubated at 37degC for24 hrs (ere was no evident growth on the plate

23 Animals (e study protocol (005-Ani-BBS-13) wasapproved by ECACU (Ethics Committee for Animal Careand Use) of the Aga Khan University which is in accordancewith the international guidelines for laboratory animal useand care [15] Rabbits (1ndash15 kg) of either sex and of the localbreed were used for the ex vivo study whereas SpragueDawley rats (150ndash200 g) were used for the in vivo studyFemale BALBc mice 6ndash8 weeks old (22ndash30 g) were used forthe colitis study and kept in the animal house of the AgaKhan University (Karachi) in rectangular cages(47times 34times18 cm3) with sawdust (changed every 48 hrs) Allanimals were given a standard diet and maintained understandard conditions (temperature 22ndash25degC 70ndash75 hu-midity and 12 hr light and dark cycle) Animals also had freeaccess to water and food ad libitum throughout the studyexcept in case of induction of colitis where animalsrsquo foodwas withdrawn before 18 hrs and rabbits were kept onfasting for 24 hrs before sacrificing them for ex vivo study

Mice used for colitis study were sacrificed by cervicaldislocation 24 hrs after induction of colitis whereas rabbitsstarved for 24 hrs were anaesthetized with thiopental sodium(200mgkg ip) and sacrificed by cervical dislocation

2 Evidence-Based Complementary and Alternative Medicine

Bacterial strains used in this study included enteropatho-genic E coli (234869 EPEC) enterotoxigenic E coli(H10407 ETEC) and enteroaggregative E coli (042EAEC) grown aerobically at 37degC in Luria-Bertani broth(LB) for 16 hrs

24Working Stocks For the in vivo and ex vivo experimentsthe extract and the oil were solubilized in 10 DMSO-5Tween-80 and subsequent dilutions were made in distilledwater on the day of the experiment

For determining the effect against enteric pathogens theFsCr stock was prepared as 1000mgml in 50 methanoland then centrifuged at 14000 g for 60min(e supernatantwas microfiltered and subsequently a working stock wasprepared into 1mgml From this working stock FsCr 10and 20 μl were picked for the antimicrobial assay (assayvolume 200 μl) to get the total concentration to be 50 μgmland 100 μgml respectively

For positive control groups (100 kill) 10 μl of microbeswas suspended with 100 μgml of vancomycin (for Gram-negative) whereas for negative control 10 μl of inoculumwas suspended with the corresponding volume of the vehiclesolvent (MeOH) 125methanol corresponding to the finalconcentration of MeOH in FsCr working solutions wasused as vehicle control At this concentration there was noeffect on bacteria kill

Flaxseed Oil was already in the liquid form and could beeasily drawn in minutest quantities hence no dilution wasrequired 1ml (1000 μl) of oil corresponding to 1mg(1000 μg) was centrifuged at 14000 g for 60min and sub-sequently microfiltered From this 18 μl and 28 μl werepicked for assay (assay volume 200 μl) that correspondedto 9 μgml and 14 μgml respectively (18 μg in 200 μl for1000 μl or 1ml 18200lowast1000 9 μgml)

25 Phytochemical Analysis (e aqueous-methanolic crude(30 70) extract of Flaxseed (FsCr) was qualitatively ana-lyzed according to standard methods for the presence orabsence of alkaloids anthraquinones coumarins flavo-noids saponins sterols tannins and terpenes [16]

26 Experimental Protocol (e study design was similar toour previously reported study [4] with a sham control groupdiseased control (untreated) positive control (predniso-lone) and treatment groups (ere were 12 animals in eachgroup (ree treatment groups were administered 150300 and 500mgkg FsCr and another three groups weregiven similar doses of Flaxseed oil intraperitoneally (ip) for5 days prior to the induction of colitis On the 6th day allgroups except the sham control group were induced withcolitis under anaesthesia All the groups continued to receivethe respective treatments till the last day when mice were tobe sacrificed

Acetic acid- (AA-) induced colitis model by intrarectal(IR) administration was adopted to decipher the effect ofFsCr and oil as reported previously [4 17] Briefly BALBcmice fasted for 18 hrs with access to water ad libitum were

anaesthetized and administered 01ml of 6 IR acetic acid(vv in 09 saline) through feeding needle held verticallyfor 45 sec and then flushed with 01ml of saline Mice weresacrificed 24 hrs after the induction of colitis

27 Evaluation of AA-Induced Colitis

271 Mortality Rate and DAI (e numbers of mice dyingin the respective groups were compared to the total animalspresent in the same group before start of the experiment andmortality rate was calculated as follows

mortality Rate mice died in a group

total number of mice in that groupx 100

(1)

Disease activity index (DAI) was assessed after 24 hrswhich was the average of loss in body weight effect on stoolconsistency and type of rectal bleed [18] (e semiquanti-tative scoring details are elaborated in Table 1

272 Histological Assessment After assessing DAI micewere sacrificed by cervical dislocation and the colon tissues5 cm proximal to anus were excised and stored in neutralbuffered formalin for 24 hrs Histological processing of colontissues was done to assess colonic damage using hematoxylinand eosin (H and E) staining and mucus content wasassessed by periodic acid-Schiff-alcian blue (PAS-AB)staining For quantifying themicroscopic damage of H and Eand PAS-AB stained sections of the colon were scored in asingle blinded fashion by a histopathologist using Neurathet al [19] and Vilaseca score [20] whereas goblet cells wereassessed on the basis of level of the mucin staining accordingto Dorofeyev et al [21] the details of which are presented inSupplementary Table 1

28AntispasmodicMechanisms FsCr and Flaxseed oil weretested for their antispasmodic mechanisms using isolatedrabbit jejunum preparations in a tissue bath assembly set-upfor their inhibitory effect against CCh- (1 μM) inducedcontractions and for their previously reported effect onspontaneous and high K+- [7] and low K+-induced con-tractions [6]

281 PDE Inhibitory Activity To assess PDE inhibitoryeffect of test materials isoprenaline-induced inhibitoryCRCs were constructed against CCh- (1 microM) induced sus-tained contractions in isolated rabbit jejunum in the absenceand presence of the FsCr and Flaxseed oil as describedpreviously [22] Since the PDEI activity of FsCr was strongeras compared to oil FsCr was further studied for its in-hibitory effect against the subtype of PDE enzyme along withcAMP levels in FsCr tested tissues For this purpose arelationship between inhibitory effects of different PDEenzyme inhibitors was examined in the presence and ab-sence of FsCr [23 24] Briefly isolated rabbit jejunal tissueswere contracted by CCh (1 microM) Once these contractionswere sustained concentration-dependent inhibitory

Evidence-Based Complementary and Alternative Medicine 3

responses were constructed by cumulative dosing of PDEenzyme subtypes 3 4 and 5 inhibitors cilostazol rolipramand zaprinast respectively [25] (is was followed byrestabilization of the jejunal tissues and then the same typesof concentration-dependent inhibitory responses of each ofthe PDE subtypes 3 4 and 5 inhibitors were constructed inthe presence of 003mgml of FsCr or one of the other typesof PDE inhibitors cilostazol (100 μM) rolipram (3 μM) andzaprinast (100 μM) (administered before inducing CCh-induced contractions)(e results were interpreted based onthe potentiation of PDE inhibitorrsquos concentration-depen-dent inhibitory responses with or without pretreatment ofFsCr

To confirm the PDE inhibitory effect of FsCr cAMPcontent of the jejunum was measured by enzyme immu-noassay [26] using cAMP enzyme immunoassay kit formultiple species (Arbor Assays DetectX Direct cAMP En-zyme Immunoassay kit Ann Arbor MI USA) For thispurpose CCh-induced contraction of the jejunum wasinhibited with FsCr in respective different doses (en thejejunal tissues were snap-frozen in liquid nitrogen subse-quently homogenized with 1ml of sample diluent for every100mg of tissue on ice kept like this for 10min and thencentrifuged at ge6000 g at 4degC for 15min (e supernatantwas collected and either analyzed immediately or storedfrozen at minus80degC Similar steps were repeated for tissuestreated with different doses of papaverine which served as apositive control and tissues without administration of anydrug and only contracted with CCh which served as anegative control (e cAMP content was expressed as pi-comole per ml

282 Anticholinergic Activity In order to confirm theinvolvement of anticholinergic mechanisms first controlconcentration-response curves (CRCs) of Ach wereconstructed in guinea pig ileum up to the maximal effectand then these CRCs were reconstructed in the presenceof an increasing concentration of the plant material(FsCr and oil) (e rightward parallel shift in the CRCsof Ach indicated the competitive antagonism [27] whilenonparallel displacement with suppression of the max-imal response confirmed the nonspecific antagonism[28]

29 Antibacterial Assay EPEC ETEC and EAEC wereassessed using the quantitative antibacterial assays aspreviously reported [29] At first optical density (OD) ofbacteria was adjusted through Luria Broth (LB) to 022corresponding to approximately 106 colony forming units(cfu) followed by making a serial dilution of bacteria at

10minus1ndash10minus6 and plating them this became the inoculumcontrol Simultaneously in other sets of tubes 10 microl ofrespective bacterial inoculum was suspended with dif-ferent doses of Flaxseed oil (9 μgml and 14 μgml) andFsCr (50 and 100 microgml) the total assay volume was200 microl and LB was used as an assay buffer For negativecontrol 10 microl of original inoculum (OI) was suspendedwith corresponding volumes of LB instead of Flaxseed oilFsCr Additional negative control of the correspondingconcentration of methanol (MeOH) was used for FsCr asit was solubilized in MeOH For positive control groups(100 kill) 10 microl of inoculum was suspended with 100 microgml gentamycin (e assay tubes were then incubated inCO2 incubator for 2 hrs on shaking After incubationmicrobial cultures were 10-fold serially diluted in LB(10minus1ndash10minus6) plated on nutrient agar plates and incubatedfurther at 37degC overnight and microbial cfu wereenumerated

(e effect of test material was compared to the originallyincubated number of bacteria in terms of reduction in cfuin respective treatment groups (e effect was considered asbactericidal if the concentration of bacteria in the treatedgroups was less than OI(e percent bactericidal effects weredetermined as follows

100 minuscf u in extractoriginal inoculum

1113888 1113889lowast1001113890 1113891 (2)

(e conclusions were drawn based on colony count asfollows

(1) If the microbial cfu was less than the cfu in OI itwas considered as a bactericidal effect

(2) If the microbial cfu was similar to OI or greaterthan OI it was inferred that test material has noeffect

210 Statistical Analysis (e data expressed are mean-s plusmn standard error of the mean (SEM n number of ex-periments) One-way ANOVA was applied to differentiatethe results in cyclic AMP (cAMP) assay A probability ofplt 005 was considered significant (e concentration-response curves (CRCs) were analyzed by nonlinear re-gression using the GraphPad program (GraphPad SanDiego CA USA) (e antibacterial analysis was deter-mined by unpaired t-test (two-tailed) or one-way ANOVAfollowed by Tukeyrsquos posttest All other graphing calcula-tions and statistical analyses were also carried out usingGraphPad software (GraphPad San Diego CaliforniaUSA)

Table 1 Disease activity index (DAI) scoring criteria

Parameter Scoring criteriaWeight loss 1ndash5 6ndash10 11ndash20gt 21 0 1 2 3Stool Normal soft but still formed very soft diarrhoea 0 1 2 3Bleedoccult Normal hemoccult positive blood traces in stool visible gross bleeding = 0 1 2 3DAI (Weight loss + stool consistency + rectal bleeding)3

4 Evidence-Based Complementary and Alternative Medicine

3 Results

31 Phytochemistry Preliminary phytochemical analysis ofthe aqueous-methanolic crude extract of Flaxseed (FsCr)revealed the presence of tannins flavonoids triterpenesalkaloids and coumarins whereas saponins were weak andanthraquinones were not detected

32 Evaluation of the Effect on the Ulcerative Colitis Model(e effects of Flaxseed oil and FsCr at the doses of 150 300and 500mgkg were tested against AA-induced colitis

321 Mortality Rate and DAI (emortality rate of 6 AA-induced colitis mice (untreated) was 58 whereas pre-treatment with Flaxseed oil and extract reduced the mor-tality rate significantly (plt 001 vs plt 0001 at 300 and500mgkg doses respectively) (e optimal effect of FsCrwas at 300mgkg dose whereas Flaxseed oilrsquos optimal effectwas evident at 500mgkg dose (e effect of oil at 500mgkgdose was superior to that of the extract (833plusmn 5 vs33plusmn 6)

Overall both the extract and oil improved the DAIsignificantly as compared to diseased control FsCr im-provement in DAI started at 150mgkg whereas Flaxseed oilimproved DAI at 300mgkg dose Both Flaxseed oil andextract were effective at 300mgkg doses with FsCr beingmore effective at this dose whereas Flaxseed oil exhibitedmaximum improvement in DAI at 500mgkg dose (Table 2)

322 Microscopic Damage Both the extract and oil wereequally efficacious in improving the microscopic damageparameters when compared to diseased control Howeverthe type of protective effect was different for both FsCr andFlaxseed oil

Both Flaxseed oil and FsCr were equally efficacious inreducing neutrophil infiltration (Neurath score) except thatFsCr was more potent than Flaxseed oil (e colonicdamage scores showed significant dose-dependent reductionby FsCr with maximum reduction by 5518 at 300mgkgdose and the oil also showed similar efficacy (5589 re-duction in Neurath score) but at 500mgkg dose When themicroscopic damage was quantified using Vilaseca score[20] which is the sum of the size of ulceration inflam-mation and depth of lesion Flaxseed oil was more effica-cious in reducing ulcers (max effect 775 at 500mgkgdose) as compared to FsCr (max effect 56 at 300mgkgdose) FsCr was slightly more efficacious than Flaxseed oil inreducing the inflammation (50 vs 3546 respectively) anddepth of lesion (55 vs 4231 respectively)

(e effects of Flaxseed extract and oil were compared fortheir effectiveness in increasing the mucin score as com-pared to diseased control FsCr was both potent and effi-cacious (92 mucin increase) in promoting mucinproduction as compared to oil (maximum mucin produc-tion 4228) (e percentage difference is elaborated inTable 3 whereas the scores are illustrated in SupplementaryTable 2

33AntispasmodicMechanisms Both FsCr and Flaxseed oilwere studied for their effect against CCh- high K+- and lowK+-induced contractions in a spontaneously contractingrabbit jejunum to assess and confirm their possible anti-spasmodic mechanisms

When compared for inhibitory effect against differentspasmogens FsCr showed selectivity for CCh-inducedcontractions (Figure 1(a)) exhibiting inhibitory effect atlower doses similar to that of papaverine (Figures 1(c) and1(d)) whereas verapamil showed higher potency againsthigh K+ when compared with carbachol (Figure 1(d))Similarly Flaxseed oil exhibited inhibitory effect againstCCh- (1 μM) induced contractions at a higher concentrationof 5mgml (Figure 1(b)) hence FsCr was more potentagainst CCh- (1 μM) induced contractions (plt 001) ascompared to Flaxseed oil In contrast Flaxseed oil was foundselective at the activation of K+ channels which was absentin FsCr at the tested doses whereas its effect against high K+

was negligible as previously reported [6] (Figure 1(b))Any material that will inhibit CCh-induced contraction

is either PDEI anticholinergic or both [26] Hence FsCrwas further explored for its anticholinergic and PDEImechanisms

331 PDEI Activity of FsCr and Flaxseed Oil To test for thepresence or absence of PDEI-like effect inhibitory CRCs ofisoprenaline were constructed against CCh-induced sus-tained contractions (Figures 2(a)ndash2(d)) in the presence andabsence of FsCr (Figure 2(a)) and Flaxseed oil (Figure 2(b))in rabbit jejunal tissues Pretreatment of the jejunal tissueswith increasing concentrations of FsCr (001 and 003mgml) and Flaxseed oil (1mgml and 3mgml) shifted theisoprenaline-induced inhibitory CRCs against CCh (1 μM)to the left indicating PDEI similar to papaverine at 1 and3 μM (Figure 2(c)) whereas there was no shift in the case ofverapamil (003 and 01 μM) (Figure 2(d)) Due to the strongPDEI effect of FsCr it was further explored for in-depthmechanisms for its subtype and the associated increase incAMP levels

Since PDE inhibitory effect was the predominantmechanism for the antispasmodic activity of FsCr it wasfurther studied for the possible subtype of PDE involvedPDEI are expected to inhibit CCh-induced sustained con-tractions Hence at first the effect of different PDE in-hibitorsrsquo subtypes against CCh-induced contraction withand without pretreatment with FsCr was recorded(Figures 3(a)ndash3(c))

Cilostazol a selective inhibitor of PDE-3 showed aconcentration-dependent inhibitory response against CCh-induced contraction as evident in Figures 3(a) Pretreatmentwith FsCr potentiated the inhibitory effect of cilostazol(plt 0001) as evident by a leftward shift in the curve alongwith a significant reduction in EC50 values (additive effect)(Table 4) A similar pattern was evident when effect ofcilostazol was measured with preincubation of rolipram(PDE-4 subtype inhibitor) and zaprinast (PDE-5 subtypeinhibitor) indicating that FsCr may have inhibited a PDEsubtype other than cilostazol A similar leftward shift was

Evidence-Based Complementary and Alternative Medicine 5

evident when inhibitory CRC was constructed againstzaprinast in presence of FsCr and other standard PDEinhibitors

Rolipram a selective inhibitor of PDE-4 inhibited thecontraction induced by CCh in a concentration-dependentmanner Interestingly the inhibitory response of rolipramwas not potentiated by pretreatment with FsCr whereas itwas significantly potentiated by pretreatment with cilostazol(plt 0001) and also zaprinast (plt 0001) as evident inFigures 3(b) Hence it is concluded that FsCr might beinhibiting PDE-4 subtype similar to rolipram a standardPDE-4 inhibitor

Since cAMP levels are increased when PDE enzyme isinhibited in the tissues [30] we also estimated the cAMPlevels in the control CCh-treated tissues and compared

them with FsCr- and papaverine-pretreated jejunaltissues Untreated tissues showed 1689 plusmn 142 pmol ofcAMPml of tissue homogenate Compared to this FsCrafter treatment with 1 3 and 5mgml of FsCr againstCCh-induced contraction showed cAMP levels up to2911 plusmn 162 (plt 005) 6599 plusmn 1 (plt 0001) and7873 plusmn 117 (plt 0001) pmolml respectively as evidentin Figure 4(a) (e papaverine control showed similarpattern of the rise in the cAMP levels with control CCh-treated tissues showing cAMP levels of2101 plusmn 175 pmolml whereas treatment with 1 μM and3 μM of papaverine in CCh-contracted tissues increasedthe cAMP levels to 587 plusmn 42 pmolml (plt 005) and9189 plusmn 34 (plt 0001) pmolml respectively as evidentin Figure 4(b)

Table 3 Comparison of histopathological scoring for Flaxseed oil and extract

Oil reduction from control Extract reduction from control Oil vs extract

Vilaseca score

UlcerationUntreated150mgkg 313 24 lowastlowastlowast

300mgkg 25 56lowast lowastlowastlowast

500mgkg 775lowastlowast 3314 lowastlowastlowast

Inflammation150mgkg 227 85 lowastlowastlowast

300mgkg 1364 50lowastlowastlowast lowastlowastlowast

500mgkg 3546lowastlowast 30 NSDepth of lesion1

150mgkg 769 2307lowast300mgkg 2308lowastlowast 55lowastlowastlowast lowastlowastlowast

500mgkg 4231lowastlowastlowast 3847lowastlowastlowast NS

Neurath score150mgkg 882 3304 lowastlowast

300mgkg 2647lowastlowast 5518lowast lowastlowastlowast

500mgkg 5589lowastlowastlowast 5115lowastlowast NS

Mucin score ()150mgkg 285 25 lowastlowastlowast

300mgkg 3571lowast 75lowastlowastlowast lowastlowastlowast

500mgkg 4228lowastlowast 9166lowastlowastlowast lowastlowastlowast

Neurath score [19] score 0 no leukocyte infiltration score 1 low level of leukocyte infiltration score 2moderate level of leukocyte infiltration score3moderate level of leukocyte infiltration high vascular density and thickening of colon wall score 4 transmural leukocyte infiltration loss of goblet cellshigh vascular density and thickening of the colon wall Vilaseca Score [20] total score 9 ulceration 1 [no ulcer epithelialization small ulcerslt 3mm largeulcersge 3mm score 0 1 2 respectively] inflammation 3 [none mild moderate severe 0 1 2 3] depth of lesion 3 [none mucosa muscularis propriaserosa 0 1 2 3] mucin score [21] score 4 [lt1 of stained cells 1ndash30 of stained cells (low level of staining) 30ndash80 of stained cells (medium level ofstaining) up to 80 of stained cells score 0 1 2 3] One-way ANOVA followed by Tukeyrsquos posttest was used for analysis Values expressed are meansplusmn SEM(n 6) lowastplt 005 lowastlowast plt 001 lowastlowastlowast plt 0001 vs AA Diseased control group Two-way ANOVA followed by Bonferronirsquos posttest was used for comparingthe effect of corresponding doses of Flaxseed oil and extract All the drugs were administered ip Colitis was induced by IR administration of 01ml 6 aceticacid during anaesthesia Tissues were embedded in formalin for another 24 hrs then processed and stained with H and E and PAS-AB staining ipintraperitoneally IR intrarectal H and E hematoxylin and eosin PAS-AB periodic acid-Schiff-Alcian blue

Table 2 Comparison of mortality rate and DAI of acetic acid-induced colitis in mice

Mortality rate () DAI ()Oil Extract Oil Extract

Untreated 5833plusmn 423 50plusmn 501 217plusmn 006 211plusmn 003150mgkg 55plusmn 12 3333plusmn 5lowast 21plusmn 008 117plusmn 001lowast300mgkg 3333plusmn 6lowastlowast 25plusmn 8lowastlowast 180plusmn 002lowastlowast 071plusmn 021lowastlowast500mgkg 833plusmn 5lowastlowastlowast 33plusmn 6lowastlowastlowast 093plusmn 001lowastlowastlowast 097plusmn 023lowastlowast

Values given are meansplusmn SEM of 3 experiments (n 12) One-way ANOVAwas used for statistical analysis followed by Tukeyrsquos posttest lowastplt 005 lowastlowastplt 001lowastlowastlowastplt 0001 compared with the untreated group () all the drugs were administered intraperitoneally for 7 days Colitis was induced on the 6th day withintrarectal administration of 01ml 6 acetic acid Mice were assessed 24 hrs after induction of colitis DAI [18] (weight loss + stool consistency + rectalbleeding)3 mortality rate (mice died in a grouptotal number of mice in that group)times 100 DAI disease activity index

6 Evidence-Based Complementary and Alternative Medicine

332 Anticholinergic Activity of FsCr and Flaxseed OilBesides PDEI CCh-induced contraction is also inhibited byanticholinergics therefore Flaxseed extract and oil werefurther tested in rabbit jejunal tissues to see if anticholinergicactivity exists Pretreatment of FsCr (01 and 03mgml)shifted Ach-induced bolus CRCs to the right in parallelmanner without suppression of the maximal response in aconcentration-dependent manner as shown in Figure 5(a)With further increase in the dose of extract at 1mgml therightward shift in Ach CRCs was nonparallel with sup-pression in maximal response Dicyclomine which is knownto exhibit a cholinergic antagonist effect at a lower dose and

CCB at a higher dose was used as a standard and showed asimilar rightward parallel shift of Ach CRCs at 003 μM(plt 005) and a rightward nonparallel shift due to sup-pression of the maximal response at 01 μM as shown inFigure 5(c)

Flaxseed oil shifted Ach-induced bolus CRCs to the rightin parallel manner without suppression of the maximalresponse at higher doses of 1 3 and 5mgml in a con-centration-dependent manner as shown in Figure 5(b)similar to atropine as shown in Figure 5(d) (is furtherindicates that FsCr was more potent anticholinergic thanFlaxseed oil

Flaxseed extract (mgml)

K+ (80mM)Spontaneous

CCh (1μM)K+ (25mM)

o

f con

trol

100

75

50

25

0

003 01 03 1 3 5

(a)

CCh (1μM)K+ (25mM)K+ (80mM)

Spontaneous

Flaxseed oil (mgml)

o

f con

trol

150

125

100

75

50

25

0

003 01 03 1 3 5001

(b)

Papaverine (μM)

K+ (80mM)Spontaneous

CCh (1μM)K+ (25mM)

o

f con

trol

001 03 3 30

100

75

50

25

0

(c)

K+ (80mM)Spontaneous

Verapamil (μM)

CCh (1μM)K+ (25mM)

o

f con

trol

100

75

50

25

003 01 03 1001

(d)

Figure 1 Inhibitory effect of aqueous-methanolic crude extract of Flaxseed (FsCr) (a) Flaxseed oil (b) papaverine (c) and verapamil (d)against CCh- high K+- (80mM) and low K+- (25mM) induced contractions in isolated rabbit jejunal preparations

Evidence-Based Complementary and Alternative Medicine 7

34 Antibacterial Activity Both Flaxseed extract (FsCr)and oil were tested in vitro for their effect against EPECETEC and EAEC Flaxseed oil was more potent and ef-ficacious against all the tested bacteria as compared toFsCr (e bactericidal effect of Flaxseed oil increaseddose-dependently at 9 and 14 μgml (Figures 6(a)ndash6(c))whereas the effect of FsCr was only evident against EPECat 100 μgml and no effect was observed against ETEC andEAEC (Figures 6(d)ndash6(f )) (e colony counts (cfu) areelaborated in Supplementary Table 3 Hence the minimaleffective dose for antibacterial activity against thesebacteria is almost 100 μgml for FsCr and 9 μgml forFlaxseed oil

Since FsCr did not exhibit bactericidal activity againstETEC and EAEC a possibility of bacteriostatic mechanismwas tested by increasing the incubation time from 2 to 16 hrs(to provide time for bacteria to grow) Doses of 5 and125mgml of FsCr were used for assay time of 16 hrswhereas doses of 005 and 01mg (50 and 100 microgml) of FsCrwere used when assay time was 2 hrs (e analysis is shownin Supplementary Figures 2ndash4 (A-B)

4 Discussion

Inflammatory bowel disease (IBD) is a manifestation ofmultiple dysregulations acting simultaneously therefore it

Isoprenaline alone

+FsCr (003mgml)+FsCr (001mgml)

Isoprenaline (μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1 3

(a)

Isoprenaline alone+Flaxseed oil (1mgml)+Flaxseed oil (3mgml)

Isoprenaline (microg)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0003 03 1 3 5

(b)

Isoprenaline (μM)

Isoprenaline alone+Papaverine (1μM)+Papaverine (3μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(c)

Isoprenaline (μM)

Isoprenaline alone

+Verapamil (01μM)+Verapamil (003μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(d)

Figure 2 Concentration-response curves (CRCs) of isoprenaline

8 Evidence-Based Complementary and Alternative Medicine

Cilostazol control+FsCr (003mgml) +Zaprinast (100μM)

+Rolipram (3μM)

Cilostazol (μM)

CCh-

indu

ced

cont

ract

ion

()

0003 03 30 3000

100

75

50

25

0

(a)

Zaprinast control+FsCr (003mgml)

+Rolipram (3μM)+Cilostazol (100μM)

Zaprinast (μM)00003 03 30 1000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(b)

Rolipram control+FsCr (003mgml)

+Cilostazol (100μM)+Zaprinast (100μM)

Rolipram (μM)0003 03 30 3000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(c)

Figure 3 ((a)ndash(c)) Effect of FsCr on the relaxant responses of cilostazol (a) zaprinast (b) and rolipram (c) in rabbit jejunum precontractedwith CCh (1 μM)

Table 4 EC50 values of different PDE-subtype inhibitors with and without FsCr pretreatmentPDE inhibitorCilostazol (microM) Control +FsCr +Rolipram +ZaprinastEC50 1563 091lowastlowastlowast 015lowastlowastlowast 283lowastlowastlowastCI 7632 to 3199 035 to 235 006 to 039 148 to 541Rolipram (microM) Control +FsCr +Cilostazol (microM) +ZaprinastEC50 1241 1034 2421lowastlowastlowast 161lowastlowastlowastCI 642 to 2398 549 to 1946 121 to 483 096 to 270Zaprinast (microM) Control +FsCr +Rolipram +CilostazolEC50 1463 359lowastlowastlowast 1928lowastlowastlowast 038lowastlowastCI 7445 to 2874 1381 to 937 1191 to 3121 015 to 096EC50 effective concentration to produce 50 response CI confidence interval FsCr aqueous-methanolic crude extract of Flaxseed (70 30) All con-centrations are in microM lowastlowastlowastplt 0001 lowastlowastplt 001 one-way ANOVA was done followed by Tukeyrsquos posttest to compare the significance of meansplusmn SEM ofcontrols with other groups All treatment groups are compared with Control

Evidence-Based Complementary and Alternative Medicine 9

0

25

50

75

100

FsCr (mgml)

CCh (1μM)

+1mgml

+3mgml

+5mgml

cAM

P (p

mol

ml)

Sham

lowast

lowastlowastlowast

(a)

0

50

100

150

200

Papaverine

CCh (1μM)

+100μM

+300μM

+1000μMcA

MP

(pm

olm

l)

Sham

lowastlowastlowast

lowast

(b)

Figure 4 Effect of FsCr (a) and papaverine (b) on the cyclic nucleotide content of rabbit jejunum

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

FsCr (01mgmL)Control FsCr (03mgmL)

FsCr (3mgmL)

(a)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash45 ndash35 ndash25ndash85Log [ACh] M

Fs oil (1mgmL)Control Fs oil (3mgmL)

Fs oil (5mgmL)

(b)

Figure 5 Continued

10 Evidence-Based Complementary and Alternative Medicine

needs to be catered from different ends for an effectivedisease control Multitargeted approach has gained a lot ofinterest in IBD therapeutics [1] therefore we explored thevarious pharmacological targets of Flaxseed in order torationalize its medicinal use in IBD

Our preliminary studies have shown that Flaxseed ex-tract (FsCr) was effective against 6 AA-induced colitis inmice due to its antioxidant and anti-inflammatory activities[4] However this does not represent the potential of wholeFlaxseed as the extract so prepared mainly constitutespolar compounds of Flaxseed [5] Hence we studied dif-ferent mechanisms of both Flaxseed extract (known to

contain polar constituents) and oil (known to containnonpolar constituents) and then compared their effects onvarious parameters implicated in IBD

(e first step was to identify the effectiveness of Flaxseedoil against IBD and compare it with Flaxseed extract (FsCr)It was evident with parameters such as mortality rate diseaseactivity index (DAI) and microscopic scoring that Flaxseedoil was as effective in ameliorating the severity of IBD inmice model as FsCr however there were some differencesOne of the differences was that FsCr was more potent thanFlaxseed oil in producing these effects Flaxseed oil was moreefficacious in reducing ulcer size as compared to FsCr

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

Dicyclomine (003μM)Dicyclomine (01μM)

Control

(c)

Atropine (0003μM)Control Atropine (001μM)

Atropine (003μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

(d)

ControlVerapamil (003μM)Verapamil (01μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55ndash85Log [ACh] M

(e)

Figure 5 Concentration-response curves of Ach in the absence and presence of the increasing concentrations of (a) FsCr FS oil (b)dicyclomine (c) atropine (d) and verapamil (e) in isolated rabbit jejunum preparations

Evidence-Based Complementary and Alternative Medicine 11

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Flaxseed oil

Num

ber o

f EPE

C cf

u

Original inoculumUntreated (EPEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(a)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Original inoculumUntreated EPEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

lowast

(b)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Flaxseed oil

Original inoculumUntreated (ETEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(c)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f ETE

C cf

u

Original inoculumUntreated ETEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(d)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Flaxseed oil

Original inoculumUntreated (EAEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(e)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Original inoculumUntreated EAEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(f )

Figure 6 Bar charts show colony counts of sim106 EPEC (a d) ETEC (b e) and EAEC (c f ) incubated with and without Flaxseed oil (andashc)and Flaxseed extract (FsCr) (dndashf)

12 Evidence-Based Complementary and Alternative Medicine

reduction in neutrophil and lymphocytic infiltration wasslightly better with FsCr than Flaxseed oil FsCr was alsomore potent and efficacious in promoting mucin productionthan Flaxseed oil Despite these advancements the mortalityrate of Flaxseed oil was less as compared to FsCr Differencesin inflammatory and mucosal protective parameters in theextract and oil indicate that both the polar and nonpolarfractions of Flaxseed have the potential to mediate mucosalprotective effect in the IBDmodel of mice andmay provide asynergistic effect when given as a whole seed(is seems trueas Flaxseed and its oil showed clinical improvement in ul-cerative colitis patients in one of the latest reported studies[31] On the contrary our current findings are in contrast toan earlier report where 10 Flaxseed diet aggravated theseverity of dextran sodium sulfate- (DSS-) induced colitismodel of mice [32] One of the possibilities for this could bethat Flaxseed meal has a laxative effect [33] which is knownto aggravate colitis Nevertheless improvement in clinicalstudies in UC patients shows that this may not be the case orthis aspect may vary depending on the severity of the diseaseIn the clinical study patients who were not on corticoste-roids andor immune modulators were opted for whichmeans that the recruited UC patients have limited diseaseseverity More animal and clinical studies are needed toclarify this aspect

(e other part of our study aimed to identify themechanisms of Flaxseed in terms of its antispasmodic effectInterestingly not only did we find the antispasmodicmechanisms but we also got some lead for its anti-in-flammatory effect We found that both FsCr and Flaxseedoil caused complete inhibition of CCh-induced contractioncompared with other spasmogens which is indicative ofPDE inhibitory activity andor anticholinergic activity[26 34] Upon further experimentation it was evident thatFsCr not only exhibited a stronger PDEI but also increasedthe tissue cAMP levels Hence we further aimed to identifywhich subtype of PDEI receptor was inhibited It was foundthat FsCr mediated the similar inhibitory pathway for PDEenzyme as rolipram a standard PDE4 inhibitor (is wasidentified because FsCr and rolipram given simultaneouslycould not produce synergistic inhibitory effect against CCh-induced contraction whereas other PDEI when coad-ministered with FsCr potentiated the effect indicating thatperhaps rolipram and FsCr worked through the samesubtype of PDE enzyme (is interpretation is furthersupported by previous studies where the effect of one of thesubtypes of PDE inhibitors has been potentiated againstCCh-induced contraction when pretreated with the othersubtypes of PDE inhibitor [23 24 30] (is finding has avery important implication because blocking PDE-4 enzymehas been proposed as one of the novel approaches to targetIBD PDE-4 enzymes are present on inflammatory cells andtheir inhibition prevents mucosal damage and subsequentinflammatory cell infiltration by increasing cAMP [35] Ourfindings are also relatable because subsequently we were ableto show the increased level of cAMP in the FsCr-treatedtissues which is the end result of PDE-4 subtype inhibitionInterestingly in the HPLC fingerprints of FsCr (unpub-lished) one of the peaks matched quercetin a flavonoid that

has lately been identified as a potential PDE-4 inhibitor [36]and has been shown to ameliorate the severity of acetic acidcolitis in rats [37]

FsCr was the most potent against CCh-induced con-traction resembling papaverine It was however different inthat it showed some selectivity against high K+ likedicyclomine whereas papaverine showed similar inhibi-tion against both spontaneous and high K+-induced con-tractions (is raised the possibility of an additionalanticholinergic along with Ca+2 antagonist-like activitysimilar to dicyclomine component at a higher dose In-terestingly when tested for its anticholinergic activityFsCr at 03 and 1mgml concentrations caused therightward shift of the CRCs of Ach without suppression ofthe maximum response a classic pattern of a competitiveantagonist [27] such as dicyclomine Expectedly at thehigher concentration of 3mgml FsCr further shifted theCRCs of Ach to the right but suppressed the maximalresponse possibly due to the Ca+ channel antagonist-likemechanism which has also been reported earlier [7]Hence FsCr has been proved to exhibit multiple anti-spasmodic mechanisms ranging from PDEI-like effect at alower concentration followed by anticholinergic at a rel-atively higher concentration and Ca+ antagonist-like effectat a further higher concentration Contrary to the extractFlaxseed oil mediated antispasmodic effect predominatelythrough K+ channel opener (KCO) activity [6] Both PDEIand anticholinergic activity were evident in oil at a rela-tively higher concentration whereas CCB activity couldnot be observed However the possibility of some addi-tional mechanisms cannot be ruled out

With current findings Flaxseed as a whole mediatesantispasmodic action through multiple mechanisms in-cluding PDEI (subtype PDE-4) anticholinergic KCO andCa+2 antagonist-like activities All of these mechanisms areknown to have a protective role in IBD [10 11 13] and thusprovide an additional pharmacological basis for the effec-tiveness of Flaxseedrsquos protective effect in IBD

Besides therapeutic use of antispasmodics in IBD an-tibacterials also are used as an adjunct because reducing theaggressive microbial load has shown improvement in clinicaloutcomes in patients with IBD [38 39] (erefore in thecurrent study we targeted a few of the E coli strains im-plicated in IBD Flaxseed oil showed a bactericidal activityagainst all the three strains tested but FsCr was bactericidalagainst EPEC (is difference in efficacy and potency in-dicates that Flaxseed oil may have a different antibacterialmechanism or additional chemical constituent(s) with an-tibacterial activity

(e bactericidal effect of both FsCr and Flaxseed oilagainst EPEC is a very important finding because EPECrsquosinvasive level is more or less similar to AIEC strain (LF82)[13] Moreover EPEC has also been shown to sustainiodoacetamide-induced UC-like colitis in rats [40] HenceFlaxseed may reduce the invasive ability of Ecoli in IBDpatients and possibly its consequent manifestation thatincludes upregulation of NF-kβ [41] modulation of IL-1βIL-6 TNF-α COX-2 and apoptosis hence reducing in-flammatory cascades (is effect coupled with the earlier

Evidence-Based Complementary and Alternative Medicine 13

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

responses were constructed by cumulative dosing of PDEenzyme subtypes 3 4 and 5 inhibitors cilostazol rolipramand zaprinast respectively [25] (is was followed byrestabilization of the jejunal tissues and then the same typesof concentration-dependent inhibitory responses of each ofthe PDE subtypes 3 4 and 5 inhibitors were constructed inthe presence of 003mgml of FsCr or one of the other typesof PDE inhibitors cilostazol (100 μM) rolipram (3 μM) andzaprinast (100 μM) (administered before inducing CCh-induced contractions)(e results were interpreted based onthe potentiation of PDE inhibitorrsquos concentration-depen-dent inhibitory responses with or without pretreatment ofFsCr

To confirm the PDE inhibitory effect of FsCr cAMPcontent of the jejunum was measured by enzyme immu-noassay [26] using cAMP enzyme immunoassay kit formultiple species (Arbor Assays DetectX Direct cAMP En-zyme Immunoassay kit Ann Arbor MI USA) For thispurpose CCh-induced contraction of the jejunum wasinhibited with FsCr in respective different doses (en thejejunal tissues were snap-frozen in liquid nitrogen subse-quently homogenized with 1ml of sample diluent for every100mg of tissue on ice kept like this for 10min and thencentrifuged at ge6000 g at 4degC for 15min (e supernatantwas collected and either analyzed immediately or storedfrozen at minus80degC Similar steps were repeated for tissuestreated with different doses of papaverine which served as apositive control and tissues without administration of anydrug and only contracted with CCh which served as anegative control (e cAMP content was expressed as pi-comole per ml

282 Anticholinergic Activity In order to confirm theinvolvement of anticholinergic mechanisms first controlconcentration-response curves (CRCs) of Ach wereconstructed in guinea pig ileum up to the maximal effectand then these CRCs were reconstructed in the presenceof an increasing concentration of the plant material(FsCr and oil) (e rightward parallel shift in the CRCsof Ach indicated the competitive antagonism [27] whilenonparallel displacement with suppression of the max-imal response confirmed the nonspecific antagonism[28]

29 Antibacterial Assay EPEC ETEC and EAEC wereassessed using the quantitative antibacterial assays aspreviously reported [29] At first optical density (OD) ofbacteria was adjusted through Luria Broth (LB) to 022corresponding to approximately 106 colony forming units(cfu) followed by making a serial dilution of bacteria at

10minus1ndash10minus6 and plating them this became the inoculumcontrol Simultaneously in other sets of tubes 10 microl ofrespective bacterial inoculum was suspended with dif-ferent doses of Flaxseed oil (9 μgml and 14 μgml) andFsCr (50 and 100 microgml) the total assay volume was200 microl and LB was used as an assay buffer For negativecontrol 10 microl of original inoculum (OI) was suspendedwith corresponding volumes of LB instead of Flaxseed oilFsCr Additional negative control of the correspondingconcentration of methanol (MeOH) was used for FsCr asit was solubilized in MeOH For positive control groups(100 kill) 10 microl of inoculum was suspended with 100 microgml gentamycin (e assay tubes were then incubated inCO2 incubator for 2 hrs on shaking After incubationmicrobial cultures were 10-fold serially diluted in LB(10minus1ndash10minus6) plated on nutrient agar plates and incubatedfurther at 37degC overnight and microbial cfu wereenumerated

(e effect of test material was compared to the originallyincubated number of bacteria in terms of reduction in cfuin respective treatment groups (e effect was considered asbactericidal if the concentration of bacteria in the treatedgroups was less than OI(e percent bactericidal effects weredetermined as follows

100 minuscf u in extractoriginal inoculum

1113888 1113889lowast1001113890 1113891 (2)

(e conclusions were drawn based on colony count asfollows

(1) If the microbial cfu was less than the cfu in OI itwas considered as a bactericidal effect

(2) If the microbial cfu was similar to OI or greaterthan OI it was inferred that test material has noeffect

210 Statistical Analysis (e data expressed are mean-s plusmn standard error of the mean (SEM n number of ex-periments) One-way ANOVA was applied to differentiatethe results in cyclic AMP (cAMP) assay A probability ofplt 005 was considered significant (e concentration-response curves (CRCs) were analyzed by nonlinear re-gression using the GraphPad program (GraphPad SanDiego CA USA) (e antibacterial analysis was deter-mined by unpaired t-test (two-tailed) or one-way ANOVAfollowed by Tukeyrsquos posttest All other graphing calcula-tions and statistical analyses were also carried out usingGraphPad software (GraphPad San Diego CaliforniaUSA)

Table 1 Disease activity index (DAI) scoring criteria

Parameter Scoring criteriaWeight loss 1ndash5 6ndash10 11ndash20gt 21 0 1 2 3Stool Normal soft but still formed very soft diarrhoea 0 1 2 3Bleedoccult Normal hemoccult positive blood traces in stool visible gross bleeding = 0 1 2 3DAI (Weight loss + stool consistency + rectal bleeding)3

4 Evidence-Based Complementary and Alternative Medicine

3 Results

31 Phytochemistry Preliminary phytochemical analysis ofthe aqueous-methanolic crude extract of Flaxseed (FsCr)revealed the presence of tannins flavonoids triterpenesalkaloids and coumarins whereas saponins were weak andanthraquinones were not detected

32 Evaluation of the Effect on the Ulcerative Colitis Model(e effects of Flaxseed oil and FsCr at the doses of 150 300and 500mgkg were tested against AA-induced colitis

321 Mortality Rate and DAI (emortality rate of 6 AA-induced colitis mice (untreated) was 58 whereas pre-treatment with Flaxseed oil and extract reduced the mor-tality rate significantly (plt 001 vs plt 0001 at 300 and500mgkg doses respectively) (e optimal effect of FsCrwas at 300mgkg dose whereas Flaxseed oilrsquos optimal effectwas evident at 500mgkg dose (e effect of oil at 500mgkgdose was superior to that of the extract (833plusmn 5 vs33plusmn 6)

Overall both the extract and oil improved the DAIsignificantly as compared to diseased control FsCr im-provement in DAI started at 150mgkg whereas Flaxseed oilimproved DAI at 300mgkg dose Both Flaxseed oil andextract were effective at 300mgkg doses with FsCr beingmore effective at this dose whereas Flaxseed oil exhibitedmaximum improvement in DAI at 500mgkg dose (Table 2)

322 Microscopic Damage Both the extract and oil wereequally efficacious in improving the microscopic damageparameters when compared to diseased control Howeverthe type of protective effect was different for both FsCr andFlaxseed oil

Both Flaxseed oil and FsCr were equally efficacious inreducing neutrophil infiltration (Neurath score) except thatFsCr was more potent than Flaxseed oil (e colonicdamage scores showed significant dose-dependent reductionby FsCr with maximum reduction by 5518 at 300mgkgdose and the oil also showed similar efficacy (5589 re-duction in Neurath score) but at 500mgkg dose When themicroscopic damage was quantified using Vilaseca score[20] which is the sum of the size of ulceration inflam-mation and depth of lesion Flaxseed oil was more effica-cious in reducing ulcers (max effect 775 at 500mgkgdose) as compared to FsCr (max effect 56 at 300mgkgdose) FsCr was slightly more efficacious than Flaxseed oil inreducing the inflammation (50 vs 3546 respectively) anddepth of lesion (55 vs 4231 respectively)

(e effects of Flaxseed extract and oil were compared fortheir effectiveness in increasing the mucin score as com-pared to diseased control FsCr was both potent and effi-cacious (92 mucin increase) in promoting mucinproduction as compared to oil (maximum mucin produc-tion 4228) (e percentage difference is elaborated inTable 3 whereas the scores are illustrated in SupplementaryTable 2

33AntispasmodicMechanisms Both FsCr and Flaxseed oilwere studied for their effect against CCh- high K+- and lowK+-induced contractions in a spontaneously contractingrabbit jejunum to assess and confirm their possible anti-spasmodic mechanisms

When compared for inhibitory effect against differentspasmogens FsCr showed selectivity for CCh-inducedcontractions (Figure 1(a)) exhibiting inhibitory effect atlower doses similar to that of papaverine (Figures 1(c) and1(d)) whereas verapamil showed higher potency againsthigh K+ when compared with carbachol (Figure 1(d))Similarly Flaxseed oil exhibited inhibitory effect againstCCh- (1 μM) induced contractions at a higher concentrationof 5mgml (Figure 1(b)) hence FsCr was more potentagainst CCh- (1 μM) induced contractions (plt 001) ascompared to Flaxseed oil In contrast Flaxseed oil was foundselective at the activation of K+ channels which was absentin FsCr at the tested doses whereas its effect against high K+

was negligible as previously reported [6] (Figure 1(b))Any material that will inhibit CCh-induced contraction

is either PDEI anticholinergic or both [26] Hence FsCrwas further explored for its anticholinergic and PDEImechanisms

331 PDEI Activity of FsCr and Flaxseed Oil To test for thepresence or absence of PDEI-like effect inhibitory CRCs ofisoprenaline were constructed against CCh-induced sus-tained contractions (Figures 2(a)ndash2(d)) in the presence andabsence of FsCr (Figure 2(a)) and Flaxseed oil (Figure 2(b))in rabbit jejunal tissues Pretreatment of the jejunal tissueswith increasing concentrations of FsCr (001 and 003mgml) and Flaxseed oil (1mgml and 3mgml) shifted theisoprenaline-induced inhibitory CRCs against CCh (1 μM)to the left indicating PDEI similar to papaverine at 1 and3 μM (Figure 2(c)) whereas there was no shift in the case ofverapamil (003 and 01 μM) (Figure 2(d)) Due to the strongPDEI effect of FsCr it was further explored for in-depthmechanisms for its subtype and the associated increase incAMP levels

Since PDE inhibitory effect was the predominantmechanism for the antispasmodic activity of FsCr it wasfurther studied for the possible subtype of PDE involvedPDEI are expected to inhibit CCh-induced sustained con-tractions Hence at first the effect of different PDE in-hibitorsrsquo subtypes against CCh-induced contraction withand without pretreatment with FsCr was recorded(Figures 3(a)ndash3(c))

Cilostazol a selective inhibitor of PDE-3 showed aconcentration-dependent inhibitory response against CCh-induced contraction as evident in Figures 3(a) Pretreatmentwith FsCr potentiated the inhibitory effect of cilostazol(plt 0001) as evident by a leftward shift in the curve alongwith a significant reduction in EC50 values (additive effect)(Table 4) A similar pattern was evident when effect ofcilostazol was measured with preincubation of rolipram(PDE-4 subtype inhibitor) and zaprinast (PDE-5 subtypeinhibitor) indicating that FsCr may have inhibited a PDEsubtype other than cilostazol A similar leftward shift was

Evidence-Based Complementary and Alternative Medicine 5

evident when inhibitory CRC was constructed againstzaprinast in presence of FsCr and other standard PDEinhibitors

Rolipram a selective inhibitor of PDE-4 inhibited thecontraction induced by CCh in a concentration-dependentmanner Interestingly the inhibitory response of rolipramwas not potentiated by pretreatment with FsCr whereas itwas significantly potentiated by pretreatment with cilostazol(plt 0001) and also zaprinast (plt 0001) as evident inFigures 3(b) Hence it is concluded that FsCr might beinhibiting PDE-4 subtype similar to rolipram a standardPDE-4 inhibitor

Since cAMP levels are increased when PDE enzyme isinhibited in the tissues [30] we also estimated the cAMPlevels in the control CCh-treated tissues and compared

them with FsCr- and papaverine-pretreated jejunaltissues Untreated tissues showed 1689 plusmn 142 pmol ofcAMPml of tissue homogenate Compared to this FsCrafter treatment with 1 3 and 5mgml of FsCr againstCCh-induced contraction showed cAMP levels up to2911 plusmn 162 (plt 005) 6599 plusmn 1 (plt 0001) and7873 plusmn 117 (plt 0001) pmolml respectively as evidentin Figure 4(a) (e papaverine control showed similarpattern of the rise in the cAMP levels with control CCh-treated tissues showing cAMP levels of2101 plusmn 175 pmolml whereas treatment with 1 μM and3 μM of papaverine in CCh-contracted tissues increasedthe cAMP levels to 587 plusmn 42 pmolml (plt 005) and9189 plusmn 34 (plt 0001) pmolml respectively as evidentin Figure 4(b)

Table 3 Comparison of histopathological scoring for Flaxseed oil and extract

Oil reduction from control Extract reduction from control Oil vs extract

Vilaseca score

UlcerationUntreated150mgkg 313 24 lowastlowastlowast

300mgkg 25 56lowast lowastlowastlowast

500mgkg 775lowastlowast 3314 lowastlowastlowast

Inflammation150mgkg 227 85 lowastlowastlowast

300mgkg 1364 50lowastlowastlowast lowastlowastlowast

500mgkg 3546lowastlowast 30 NSDepth of lesion1

150mgkg 769 2307lowast300mgkg 2308lowastlowast 55lowastlowastlowast lowastlowastlowast

500mgkg 4231lowastlowastlowast 3847lowastlowastlowast NS

Neurath score150mgkg 882 3304 lowastlowast

300mgkg 2647lowastlowast 5518lowast lowastlowastlowast

500mgkg 5589lowastlowastlowast 5115lowastlowast NS

Mucin score ()150mgkg 285 25 lowastlowastlowast

300mgkg 3571lowast 75lowastlowastlowast lowastlowastlowast

500mgkg 4228lowastlowast 9166lowastlowastlowast lowastlowastlowast

Neurath score [19] score 0 no leukocyte infiltration score 1 low level of leukocyte infiltration score 2moderate level of leukocyte infiltration score3moderate level of leukocyte infiltration high vascular density and thickening of colon wall score 4 transmural leukocyte infiltration loss of goblet cellshigh vascular density and thickening of the colon wall Vilaseca Score [20] total score 9 ulceration 1 [no ulcer epithelialization small ulcerslt 3mm largeulcersge 3mm score 0 1 2 respectively] inflammation 3 [none mild moderate severe 0 1 2 3] depth of lesion 3 [none mucosa muscularis propriaserosa 0 1 2 3] mucin score [21] score 4 [lt1 of stained cells 1ndash30 of stained cells (low level of staining) 30ndash80 of stained cells (medium level ofstaining) up to 80 of stained cells score 0 1 2 3] One-way ANOVA followed by Tukeyrsquos posttest was used for analysis Values expressed are meansplusmn SEM(n 6) lowastplt 005 lowastlowast plt 001 lowastlowastlowast plt 0001 vs AA Diseased control group Two-way ANOVA followed by Bonferronirsquos posttest was used for comparingthe effect of corresponding doses of Flaxseed oil and extract All the drugs were administered ip Colitis was induced by IR administration of 01ml 6 aceticacid during anaesthesia Tissues were embedded in formalin for another 24 hrs then processed and stained with H and E and PAS-AB staining ipintraperitoneally IR intrarectal H and E hematoxylin and eosin PAS-AB periodic acid-Schiff-Alcian blue

Table 2 Comparison of mortality rate and DAI of acetic acid-induced colitis in mice

Mortality rate () DAI ()Oil Extract Oil Extract

Untreated 5833plusmn 423 50plusmn 501 217plusmn 006 211plusmn 003150mgkg 55plusmn 12 3333plusmn 5lowast 21plusmn 008 117plusmn 001lowast300mgkg 3333plusmn 6lowastlowast 25plusmn 8lowastlowast 180plusmn 002lowastlowast 071plusmn 021lowastlowast500mgkg 833plusmn 5lowastlowastlowast 33plusmn 6lowastlowastlowast 093plusmn 001lowastlowastlowast 097plusmn 023lowastlowast

Values given are meansplusmn SEM of 3 experiments (n 12) One-way ANOVAwas used for statistical analysis followed by Tukeyrsquos posttest lowastplt 005 lowastlowastplt 001lowastlowastlowastplt 0001 compared with the untreated group () all the drugs were administered intraperitoneally for 7 days Colitis was induced on the 6th day withintrarectal administration of 01ml 6 acetic acid Mice were assessed 24 hrs after induction of colitis DAI [18] (weight loss + stool consistency + rectalbleeding)3 mortality rate (mice died in a grouptotal number of mice in that group)times 100 DAI disease activity index

6 Evidence-Based Complementary and Alternative Medicine

332 Anticholinergic Activity of FsCr and Flaxseed OilBesides PDEI CCh-induced contraction is also inhibited byanticholinergics therefore Flaxseed extract and oil werefurther tested in rabbit jejunal tissues to see if anticholinergicactivity exists Pretreatment of FsCr (01 and 03mgml)shifted Ach-induced bolus CRCs to the right in parallelmanner without suppression of the maximal response in aconcentration-dependent manner as shown in Figure 5(a)With further increase in the dose of extract at 1mgml therightward shift in Ach CRCs was nonparallel with sup-pression in maximal response Dicyclomine which is knownto exhibit a cholinergic antagonist effect at a lower dose and

CCB at a higher dose was used as a standard and showed asimilar rightward parallel shift of Ach CRCs at 003 μM(plt 005) and a rightward nonparallel shift due to sup-pression of the maximal response at 01 μM as shown inFigure 5(c)

Flaxseed oil shifted Ach-induced bolus CRCs to the rightin parallel manner without suppression of the maximalresponse at higher doses of 1 3 and 5mgml in a con-centration-dependent manner as shown in Figure 5(b)similar to atropine as shown in Figure 5(d) (is furtherindicates that FsCr was more potent anticholinergic thanFlaxseed oil

Flaxseed extract (mgml)

K+ (80mM)Spontaneous

CCh (1μM)K+ (25mM)

o

f con

trol

100

75

50

25

0

003 01 03 1 3 5

(a)

CCh (1μM)K+ (25mM)K+ (80mM)

Spontaneous

Flaxseed oil (mgml)

o

f con

trol

150

125

100

75

50

25

0

003 01 03 1 3 5001

(b)

Papaverine (μM)

K+ (80mM)Spontaneous

CCh (1μM)K+ (25mM)

o

f con

trol

001 03 3 30

100

75

50

25

0

(c)

K+ (80mM)Spontaneous

Verapamil (μM)

CCh (1μM)K+ (25mM)

o

f con

trol

100

75

50

25

003 01 03 1001

(d)

Figure 1 Inhibitory effect of aqueous-methanolic crude extract of Flaxseed (FsCr) (a) Flaxseed oil (b) papaverine (c) and verapamil (d)against CCh- high K+- (80mM) and low K+- (25mM) induced contractions in isolated rabbit jejunal preparations

Evidence-Based Complementary and Alternative Medicine 7

34 Antibacterial Activity Both Flaxseed extract (FsCr)and oil were tested in vitro for their effect against EPECETEC and EAEC Flaxseed oil was more potent and ef-ficacious against all the tested bacteria as compared toFsCr (e bactericidal effect of Flaxseed oil increaseddose-dependently at 9 and 14 μgml (Figures 6(a)ndash6(c))whereas the effect of FsCr was only evident against EPECat 100 μgml and no effect was observed against ETEC andEAEC (Figures 6(d)ndash6(f )) (e colony counts (cfu) areelaborated in Supplementary Table 3 Hence the minimaleffective dose for antibacterial activity against thesebacteria is almost 100 μgml for FsCr and 9 μgml forFlaxseed oil

Since FsCr did not exhibit bactericidal activity againstETEC and EAEC a possibility of bacteriostatic mechanismwas tested by increasing the incubation time from 2 to 16 hrs(to provide time for bacteria to grow) Doses of 5 and125mgml of FsCr were used for assay time of 16 hrswhereas doses of 005 and 01mg (50 and 100 microgml) of FsCrwere used when assay time was 2 hrs (e analysis is shownin Supplementary Figures 2ndash4 (A-B)

4 Discussion

Inflammatory bowel disease (IBD) is a manifestation ofmultiple dysregulations acting simultaneously therefore it

Isoprenaline alone

+FsCr (003mgml)+FsCr (001mgml)

Isoprenaline (μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1 3

(a)

Isoprenaline alone+Flaxseed oil (1mgml)+Flaxseed oil (3mgml)

Isoprenaline (microg)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0003 03 1 3 5

(b)

Isoprenaline (μM)

Isoprenaline alone+Papaverine (1μM)+Papaverine (3μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(c)

Isoprenaline (μM)

Isoprenaline alone

+Verapamil (01μM)+Verapamil (003μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(d)

Figure 2 Concentration-response curves (CRCs) of isoprenaline

8 Evidence-Based Complementary and Alternative Medicine

Cilostazol control+FsCr (003mgml) +Zaprinast (100μM)

+Rolipram (3μM)

Cilostazol (μM)

CCh-

indu

ced

cont

ract

ion

()

0003 03 30 3000

100

75

50

25

0

(a)

Zaprinast control+FsCr (003mgml)

+Rolipram (3μM)+Cilostazol (100μM)

Zaprinast (μM)00003 03 30 1000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(b)

Rolipram control+FsCr (003mgml)

+Cilostazol (100μM)+Zaprinast (100μM)

Rolipram (μM)0003 03 30 3000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(c)

Figure 3 ((a)ndash(c)) Effect of FsCr on the relaxant responses of cilostazol (a) zaprinast (b) and rolipram (c) in rabbit jejunum precontractedwith CCh (1 μM)

Table 4 EC50 values of different PDE-subtype inhibitors with and without FsCr pretreatmentPDE inhibitorCilostazol (microM) Control +FsCr +Rolipram +ZaprinastEC50 1563 091lowastlowastlowast 015lowastlowastlowast 283lowastlowastlowastCI 7632 to 3199 035 to 235 006 to 039 148 to 541Rolipram (microM) Control +FsCr +Cilostazol (microM) +ZaprinastEC50 1241 1034 2421lowastlowastlowast 161lowastlowastlowastCI 642 to 2398 549 to 1946 121 to 483 096 to 270Zaprinast (microM) Control +FsCr +Rolipram +CilostazolEC50 1463 359lowastlowastlowast 1928lowastlowastlowast 038lowastlowastCI 7445 to 2874 1381 to 937 1191 to 3121 015 to 096EC50 effective concentration to produce 50 response CI confidence interval FsCr aqueous-methanolic crude extract of Flaxseed (70 30) All con-centrations are in microM lowastlowastlowastplt 0001 lowastlowastplt 001 one-way ANOVA was done followed by Tukeyrsquos posttest to compare the significance of meansplusmn SEM ofcontrols with other groups All treatment groups are compared with Control

Evidence-Based Complementary and Alternative Medicine 9

0

25

50

75

100

FsCr (mgml)

CCh (1μM)

+1mgml

+3mgml

+5mgml

cAM

P (p

mol

ml)

Sham

lowast

lowastlowastlowast

(a)

0

50

100

150

200

Papaverine

CCh (1μM)

+100μM

+300μM

+1000μMcA

MP

(pm

olm

l)

Sham

lowastlowastlowast

lowast

(b)

Figure 4 Effect of FsCr (a) and papaverine (b) on the cyclic nucleotide content of rabbit jejunum

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

FsCr (01mgmL)Control FsCr (03mgmL)

FsCr (3mgmL)

(a)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash45 ndash35 ndash25ndash85Log [ACh] M

Fs oil (1mgmL)Control Fs oil (3mgmL)

Fs oil (5mgmL)

(b)

Figure 5 Continued

10 Evidence-Based Complementary and Alternative Medicine

needs to be catered from different ends for an effectivedisease control Multitargeted approach has gained a lot ofinterest in IBD therapeutics [1] therefore we explored thevarious pharmacological targets of Flaxseed in order torationalize its medicinal use in IBD

Our preliminary studies have shown that Flaxseed ex-tract (FsCr) was effective against 6 AA-induced colitis inmice due to its antioxidant and anti-inflammatory activities[4] However this does not represent the potential of wholeFlaxseed as the extract so prepared mainly constitutespolar compounds of Flaxseed [5] Hence we studied dif-ferent mechanisms of both Flaxseed extract (known to

contain polar constituents) and oil (known to containnonpolar constituents) and then compared their effects onvarious parameters implicated in IBD

(e first step was to identify the effectiveness of Flaxseedoil against IBD and compare it with Flaxseed extract (FsCr)It was evident with parameters such as mortality rate diseaseactivity index (DAI) and microscopic scoring that Flaxseedoil was as effective in ameliorating the severity of IBD inmice model as FsCr however there were some differencesOne of the differences was that FsCr was more potent thanFlaxseed oil in producing these effects Flaxseed oil was moreefficacious in reducing ulcer size as compared to FsCr

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

Dicyclomine (003μM)Dicyclomine (01μM)

Control

(c)

Atropine (0003μM)Control Atropine (001μM)

Atropine (003μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

(d)

ControlVerapamil (003μM)Verapamil (01μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55ndash85Log [ACh] M

(e)

Figure 5 Concentration-response curves of Ach in the absence and presence of the increasing concentrations of (a) FsCr FS oil (b)dicyclomine (c) atropine (d) and verapamil (e) in isolated rabbit jejunum preparations

Evidence-Based Complementary and Alternative Medicine 11

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Flaxseed oil

Num

ber o

f EPE

C cf

u

Original inoculumUntreated (EPEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(a)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Original inoculumUntreated EPEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

lowast

(b)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Flaxseed oil

Original inoculumUntreated (ETEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(c)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f ETE

C cf

u

Original inoculumUntreated ETEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(d)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Flaxseed oil

Original inoculumUntreated (EAEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(e)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Original inoculumUntreated EAEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(f )

Figure 6 Bar charts show colony counts of sim106 EPEC (a d) ETEC (b e) and EAEC (c f ) incubated with and without Flaxseed oil (andashc)and Flaxseed extract (FsCr) (dndashf)

12 Evidence-Based Complementary and Alternative Medicine

reduction in neutrophil and lymphocytic infiltration wasslightly better with FsCr than Flaxseed oil FsCr was alsomore potent and efficacious in promoting mucin productionthan Flaxseed oil Despite these advancements the mortalityrate of Flaxseed oil was less as compared to FsCr Differencesin inflammatory and mucosal protective parameters in theextract and oil indicate that both the polar and nonpolarfractions of Flaxseed have the potential to mediate mucosalprotective effect in the IBDmodel of mice andmay provide asynergistic effect when given as a whole seed(is seems trueas Flaxseed and its oil showed clinical improvement in ul-cerative colitis patients in one of the latest reported studies[31] On the contrary our current findings are in contrast toan earlier report where 10 Flaxseed diet aggravated theseverity of dextran sodium sulfate- (DSS-) induced colitismodel of mice [32] One of the possibilities for this could bethat Flaxseed meal has a laxative effect [33] which is knownto aggravate colitis Nevertheless improvement in clinicalstudies in UC patients shows that this may not be the case orthis aspect may vary depending on the severity of the diseaseIn the clinical study patients who were not on corticoste-roids andor immune modulators were opted for whichmeans that the recruited UC patients have limited diseaseseverity More animal and clinical studies are needed toclarify this aspect

(e other part of our study aimed to identify themechanisms of Flaxseed in terms of its antispasmodic effectInterestingly not only did we find the antispasmodicmechanisms but we also got some lead for its anti-in-flammatory effect We found that both FsCr and Flaxseedoil caused complete inhibition of CCh-induced contractioncompared with other spasmogens which is indicative ofPDE inhibitory activity andor anticholinergic activity[26 34] Upon further experimentation it was evident thatFsCr not only exhibited a stronger PDEI but also increasedthe tissue cAMP levels Hence we further aimed to identifywhich subtype of PDEI receptor was inhibited It was foundthat FsCr mediated the similar inhibitory pathway for PDEenzyme as rolipram a standard PDE4 inhibitor (is wasidentified because FsCr and rolipram given simultaneouslycould not produce synergistic inhibitory effect against CCh-induced contraction whereas other PDEI when coad-ministered with FsCr potentiated the effect indicating thatperhaps rolipram and FsCr worked through the samesubtype of PDE enzyme (is interpretation is furthersupported by previous studies where the effect of one of thesubtypes of PDE inhibitors has been potentiated againstCCh-induced contraction when pretreated with the othersubtypes of PDE inhibitor [23 24 30] (is finding has avery important implication because blocking PDE-4 enzymehas been proposed as one of the novel approaches to targetIBD PDE-4 enzymes are present on inflammatory cells andtheir inhibition prevents mucosal damage and subsequentinflammatory cell infiltration by increasing cAMP [35] Ourfindings are also relatable because subsequently we were ableto show the increased level of cAMP in the FsCr-treatedtissues which is the end result of PDE-4 subtype inhibitionInterestingly in the HPLC fingerprints of FsCr (unpub-lished) one of the peaks matched quercetin a flavonoid that

has lately been identified as a potential PDE-4 inhibitor [36]and has been shown to ameliorate the severity of acetic acidcolitis in rats [37]

FsCr was the most potent against CCh-induced con-traction resembling papaverine It was however different inthat it showed some selectivity against high K+ likedicyclomine whereas papaverine showed similar inhibi-tion against both spontaneous and high K+-induced con-tractions (is raised the possibility of an additionalanticholinergic along with Ca+2 antagonist-like activitysimilar to dicyclomine component at a higher dose In-terestingly when tested for its anticholinergic activityFsCr at 03 and 1mgml concentrations caused therightward shift of the CRCs of Ach without suppression ofthe maximum response a classic pattern of a competitiveantagonist [27] such as dicyclomine Expectedly at thehigher concentration of 3mgml FsCr further shifted theCRCs of Ach to the right but suppressed the maximalresponse possibly due to the Ca+ channel antagonist-likemechanism which has also been reported earlier [7]Hence FsCr has been proved to exhibit multiple anti-spasmodic mechanisms ranging from PDEI-like effect at alower concentration followed by anticholinergic at a rel-atively higher concentration and Ca+ antagonist-like effectat a further higher concentration Contrary to the extractFlaxseed oil mediated antispasmodic effect predominatelythrough K+ channel opener (KCO) activity [6] Both PDEIand anticholinergic activity were evident in oil at a rela-tively higher concentration whereas CCB activity couldnot be observed However the possibility of some addi-tional mechanisms cannot be ruled out

With current findings Flaxseed as a whole mediatesantispasmodic action through multiple mechanisms in-cluding PDEI (subtype PDE-4) anticholinergic KCO andCa+2 antagonist-like activities All of these mechanisms areknown to have a protective role in IBD [10 11 13] and thusprovide an additional pharmacological basis for the effec-tiveness of Flaxseedrsquos protective effect in IBD

Besides therapeutic use of antispasmodics in IBD an-tibacterials also are used as an adjunct because reducing theaggressive microbial load has shown improvement in clinicaloutcomes in patients with IBD [38 39] (erefore in thecurrent study we targeted a few of the E coli strains im-plicated in IBD Flaxseed oil showed a bactericidal activityagainst all the three strains tested but FsCr was bactericidalagainst EPEC (is difference in efficacy and potency in-dicates that Flaxseed oil may have a different antibacterialmechanism or additional chemical constituent(s) with an-tibacterial activity

(e bactericidal effect of both FsCr and Flaxseed oilagainst EPEC is a very important finding because EPECrsquosinvasive level is more or less similar to AIEC strain (LF82)[13] Moreover EPEC has also been shown to sustainiodoacetamide-induced UC-like colitis in rats [40] HenceFlaxseed may reduce the invasive ability of Ecoli in IBDpatients and possibly its consequent manifestation thatincludes upregulation of NF-kβ [41] modulation of IL-1βIL-6 TNF-α COX-2 and apoptosis hence reducing in-flammatory cascades (is effect coupled with the earlier

Evidence-Based Complementary and Alternative Medicine 13

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

3 Results

31 Phytochemistry Preliminary phytochemical analysis ofthe aqueous-methanolic crude extract of Flaxseed (FsCr)revealed the presence of tannins flavonoids triterpenesalkaloids and coumarins whereas saponins were weak andanthraquinones were not detected

32 Evaluation of the Effect on the Ulcerative Colitis Model(e effects of Flaxseed oil and FsCr at the doses of 150 300and 500mgkg were tested against AA-induced colitis

321 Mortality Rate and DAI (emortality rate of 6 AA-induced colitis mice (untreated) was 58 whereas pre-treatment with Flaxseed oil and extract reduced the mor-tality rate significantly (plt 001 vs plt 0001 at 300 and500mgkg doses respectively) (e optimal effect of FsCrwas at 300mgkg dose whereas Flaxseed oilrsquos optimal effectwas evident at 500mgkg dose (e effect of oil at 500mgkgdose was superior to that of the extract (833plusmn 5 vs33plusmn 6)

Overall both the extract and oil improved the DAIsignificantly as compared to diseased control FsCr im-provement in DAI started at 150mgkg whereas Flaxseed oilimproved DAI at 300mgkg dose Both Flaxseed oil andextract were effective at 300mgkg doses with FsCr beingmore effective at this dose whereas Flaxseed oil exhibitedmaximum improvement in DAI at 500mgkg dose (Table 2)

322 Microscopic Damage Both the extract and oil wereequally efficacious in improving the microscopic damageparameters when compared to diseased control Howeverthe type of protective effect was different for both FsCr andFlaxseed oil

Both Flaxseed oil and FsCr were equally efficacious inreducing neutrophil infiltration (Neurath score) except thatFsCr was more potent than Flaxseed oil (e colonicdamage scores showed significant dose-dependent reductionby FsCr with maximum reduction by 5518 at 300mgkgdose and the oil also showed similar efficacy (5589 re-duction in Neurath score) but at 500mgkg dose When themicroscopic damage was quantified using Vilaseca score[20] which is the sum of the size of ulceration inflam-mation and depth of lesion Flaxseed oil was more effica-cious in reducing ulcers (max effect 775 at 500mgkgdose) as compared to FsCr (max effect 56 at 300mgkgdose) FsCr was slightly more efficacious than Flaxseed oil inreducing the inflammation (50 vs 3546 respectively) anddepth of lesion (55 vs 4231 respectively)

(e effects of Flaxseed extract and oil were compared fortheir effectiveness in increasing the mucin score as com-pared to diseased control FsCr was both potent and effi-cacious (92 mucin increase) in promoting mucinproduction as compared to oil (maximum mucin produc-tion 4228) (e percentage difference is elaborated inTable 3 whereas the scores are illustrated in SupplementaryTable 2

33AntispasmodicMechanisms Both FsCr and Flaxseed oilwere studied for their effect against CCh- high K+- and lowK+-induced contractions in a spontaneously contractingrabbit jejunum to assess and confirm their possible anti-spasmodic mechanisms

When compared for inhibitory effect against differentspasmogens FsCr showed selectivity for CCh-inducedcontractions (Figure 1(a)) exhibiting inhibitory effect atlower doses similar to that of papaverine (Figures 1(c) and1(d)) whereas verapamil showed higher potency againsthigh K+ when compared with carbachol (Figure 1(d))Similarly Flaxseed oil exhibited inhibitory effect againstCCh- (1 μM) induced contractions at a higher concentrationof 5mgml (Figure 1(b)) hence FsCr was more potentagainst CCh- (1 μM) induced contractions (plt 001) ascompared to Flaxseed oil In contrast Flaxseed oil was foundselective at the activation of K+ channels which was absentin FsCr at the tested doses whereas its effect against high K+

was negligible as previously reported [6] (Figure 1(b))Any material that will inhibit CCh-induced contraction

is either PDEI anticholinergic or both [26] Hence FsCrwas further explored for its anticholinergic and PDEImechanisms

331 PDEI Activity of FsCr and Flaxseed Oil To test for thepresence or absence of PDEI-like effect inhibitory CRCs ofisoprenaline were constructed against CCh-induced sus-tained contractions (Figures 2(a)ndash2(d)) in the presence andabsence of FsCr (Figure 2(a)) and Flaxseed oil (Figure 2(b))in rabbit jejunal tissues Pretreatment of the jejunal tissueswith increasing concentrations of FsCr (001 and 003mgml) and Flaxseed oil (1mgml and 3mgml) shifted theisoprenaline-induced inhibitory CRCs against CCh (1 μM)to the left indicating PDEI similar to papaverine at 1 and3 μM (Figure 2(c)) whereas there was no shift in the case ofverapamil (003 and 01 μM) (Figure 2(d)) Due to the strongPDEI effect of FsCr it was further explored for in-depthmechanisms for its subtype and the associated increase incAMP levels

Since PDE inhibitory effect was the predominantmechanism for the antispasmodic activity of FsCr it wasfurther studied for the possible subtype of PDE involvedPDEI are expected to inhibit CCh-induced sustained con-tractions Hence at first the effect of different PDE in-hibitorsrsquo subtypes against CCh-induced contraction withand without pretreatment with FsCr was recorded(Figures 3(a)ndash3(c))

Cilostazol a selective inhibitor of PDE-3 showed aconcentration-dependent inhibitory response against CCh-induced contraction as evident in Figures 3(a) Pretreatmentwith FsCr potentiated the inhibitory effect of cilostazol(plt 0001) as evident by a leftward shift in the curve alongwith a significant reduction in EC50 values (additive effect)(Table 4) A similar pattern was evident when effect ofcilostazol was measured with preincubation of rolipram(PDE-4 subtype inhibitor) and zaprinast (PDE-5 subtypeinhibitor) indicating that FsCr may have inhibited a PDEsubtype other than cilostazol A similar leftward shift was

Evidence-Based Complementary and Alternative Medicine 5

evident when inhibitory CRC was constructed againstzaprinast in presence of FsCr and other standard PDEinhibitors

Rolipram a selective inhibitor of PDE-4 inhibited thecontraction induced by CCh in a concentration-dependentmanner Interestingly the inhibitory response of rolipramwas not potentiated by pretreatment with FsCr whereas itwas significantly potentiated by pretreatment with cilostazol(plt 0001) and also zaprinast (plt 0001) as evident inFigures 3(b) Hence it is concluded that FsCr might beinhibiting PDE-4 subtype similar to rolipram a standardPDE-4 inhibitor

Since cAMP levels are increased when PDE enzyme isinhibited in the tissues [30] we also estimated the cAMPlevels in the control CCh-treated tissues and compared

them with FsCr- and papaverine-pretreated jejunaltissues Untreated tissues showed 1689 plusmn 142 pmol ofcAMPml of tissue homogenate Compared to this FsCrafter treatment with 1 3 and 5mgml of FsCr againstCCh-induced contraction showed cAMP levels up to2911 plusmn 162 (plt 005) 6599 plusmn 1 (plt 0001) and7873 plusmn 117 (plt 0001) pmolml respectively as evidentin Figure 4(a) (e papaverine control showed similarpattern of the rise in the cAMP levels with control CCh-treated tissues showing cAMP levels of2101 plusmn 175 pmolml whereas treatment with 1 μM and3 μM of papaverine in CCh-contracted tissues increasedthe cAMP levels to 587 plusmn 42 pmolml (plt 005) and9189 plusmn 34 (plt 0001) pmolml respectively as evidentin Figure 4(b)

Table 3 Comparison of histopathological scoring for Flaxseed oil and extract

Oil reduction from control Extract reduction from control Oil vs extract

Vilaseca score

UlcerationUntreated150mgkg 313 24 lowastlowastlowast

300mgkg 25 56lowast lowastlowastlowast

500mgkg 775lowastlowast 3314 lowastlowastlowast

Inflammation150mgkg 227 85 lowastlowastlowast

300mgkg 1364 50lowastlowastlowast lowastlowastlowast

500mgkg 3546lowastlowast 30 NSDepth of lesion1

150mgkg 769 2307lowast300mgkg 2308lowastlowast 55lowastlowastlowast lowastlowastlowast

500mgkg 4231lowastlowastlowast 3847lowastlowastlowast NS

Neurath score150mgkg 882 3304 lowastlowast

300mgkg 2647lowastlowast 5518lowast lowastlowastlowast

500mgkg 5589lowastlowastlowast 5115lowastlowast NS

Mucin score ()150mgkg 285 25 lowastlowastlowast

300mgkg 3571lowast 75lowastlowastlowast lowastlowastlowast

500mgkg 4228lowastlowast 9166lowastlowastlowast lowastlowastlowast

Neurath score [19] score 0 no leukocyte infiltration score 1 low level of leukocyte infiltration score 2moderate level of leukocyte infiltration score3moderate level of leukocyte infiltration high vascular density and thickening of colon wall score 4 transmural leukocyte infiltration loss of goblet cellshigh vascular density and thickening of the colon wall Vilaseca Score [20] total score 9 ulceration 1 [no ulcer epithelialization small ulcerslt 3mm largeulcersge 3mm score 0 1 2 respectively] inflammation 3 [none mild moderate severe 0 1 2 3] depth of lesion 3 [none mucosa muscularis propriaserosa 0 1 2 3] mucin score [21] score 4 [lt1 of stained cells 1ndash30 of stained cells (low level of staining) 30ndash80 of stained cells (medium level ofstaining) up to 80 of stained cells score 0 1 2 3] One-way ANOVA followed by Tukeyrsquos posttest was used for analysis Values expressed are meansplusmn SEM(n 6) lowastplt 005 lowastlowast plt 001 lowastlowastlowast plt 0001 vs AA Diseased control group Two-way ANOVA followed by Bonferronirsquos posttest was used for comparingthe effect of corresponding doses of Flaxseed oil and extract All the drugs were administered ip Colitis was induced by IR administration of 01ml 6 aceticacid during anaesthesia Tissues were embedded in formalin for another 24 hrs then processed and stained with H and E and PAS-AB staining ipintraperitoneally IR intrarectal H and E hematoxylin and eosin PAS-AB periodic acid-Schiff-Alcian blue

Table 2 Comparison of mortality rate and DAI of acetic acid-induced colitis in mice

Mortality rate () DAI ()Oil Extract Oil Extract

Untreated 5833plusmn 423 50plusmn 501 217plusmn 006 211plusmn 003150mgkg 55plusmn 12 3333plusmn 5lowast 21plusmn 008 117plusmn 001lowast300mgkg 3333plusmn 6lowastlowast 25plusmn 8lowastlowast 180plusmn 002lowastlowast 071plusmn 021lowastlowast500mgkg 833plusmn 5lowastlowastlowast 33plusmn 6lowastlowastlowast 093plusmn 001lowastlowastlowast 097plusmn 023lowastlowast

Values given are meansplusmn SEM of 3 experiments (n 12) One-way ANOVAwas used for statistical analysis followed by Tukeyrsquos posttest lowastplt 005 lowastlowastplt 001lowastlowastlowastplt 0001 compared with the untreated group () all the drugs were administered intraperitoneally for 7 days Colitis was induced on the 6th day withintrarectal administration of 01ml 6 acetic acid Mice were assessed 24 hrs after induction of colitis DAI [18] (weight loss + stool consistency + rectalbleeding)3 mortality rate (mice died in a grouptotal number of mice in that group)times 100 DAI disease activity index

6 Evidence-Based Complementary and Alternative Medicine

332 Anticholinergic Activity of FsCr and Flaxseed OilBesides PDEI CCh-induced contraction is also inhibited byanticholinergics therefore Flaxseed extract and oil werefurther tested in rabbit jejunal tissues to see if anticholinergicactivity exists Pretreatment of FsCr (01 and 03mgml)shifted Ach-induced bolus CRCs to the right in parallelmanner without suppression of the maximal response in aconcentration-dependent manner as shown in Figure 5(a)With further increase in the dose of extract at 1mgml therightward shift in Ach CRCs was nonparallel with sup-pression in maximal response Dicyclomine which is knownto exhibit a cholinergic antagonist effect at a lower dose and

CCB at a higher dose was used as a standard and showed asimilar rightward parallel shift of Ach CRCs at 003 μM(plt 005) and a rightward nonparallel shift due to sup-pression of the maximal response at 01 μM as shown inFigure 5(c)

Flaxseed oil shifted Ach-induced bolus CRCs to the rightin parallel manner without suppression of the maximalresponse at higher doses of 1 3 and 5mgml in a con-centration-dependent manner as shown in Figure 5(b)similar to atropine as shown in Figure 5(d) (is furtherindicates that FsCr was more potent anticholinergic thanFlaxseed oil

Flaxseed extract (mgml)

K+ (80mM)Spontaneous

CCh (1μM)K+ (25mM)

o

f con

trol

100

75

50

25

0

003 01 03 1 3 5

(a)

CCh (1μM)K+ (25mM)K+ (80mM)

Spontaneous

Flaxseed oil (mgml)

o

f con

trol

150

125

100

75

50

25

0

003 01 03 1 3 5001

(b)

Papaverine (μM)

K+ (80mM)Spontaneous

CCh (1μM)K+ (25mM)

o

f con

trol

001 03 3 30

100

75

50

25

0

(c)

K+ (80mM)Spontaneous

Verapamil (μM)

CCh (1μM)K+ (25mM)

o

f con

trol

100

75

50

25

003 01 03 1001

(d)

Figure 1 Inhibitory effect of aqueous-methanolic crude extract of Flaxseed (FsCr) (a) Flaxseed oil (b) papaverine (c) and verapamil (d)against CCh- high K+- (80mM) and low K+- (25mM) induced contractions in isolated rabbit jejunal preparations

Evidence-Based Complementary and Alternative Medicine 7

34 Antibacterial Activity Both Flaxseed extract (FsCr)and oil were tested in vitro for their effect against EPECETEC and EAEC Flaxseed oil was more potent and ef-ficacious against all the tested bacteria as compared toFsCr (e bactericidal effect of Flaxseed oil increaseddose-dependently at 9 and 14 μgml (Figures 6(a)ndash6(c))whereas the effect of FsCr was only evident against EPECat 100 μgml and no effect was observed against ETEC andEAEC (Figures 6(d)ndash6(f )) (e colony counts (cfu) areelaborated in Supplementary Table 3 Hence the minimaleffective dose for antibacterial activity against thesebacteria is almost 100 μgml for FsCr and 9 μgml forFlaxseed oil

Since FsCr did not exhibit bactericidal activity againstETEC and EAEC a possibility of bacteriostatic mechanismwas tested by increasing the incubation time from 2 to 16 hrs(to provide time for bacteria to grow) Doses of 5 and125mgml of FsCr were used for assay time of 16 hrswhereas doses of 005 and 01mg (50 and 100 microgml) of FsCrwere used when assay time was 2 hrs (e analysis is shownin Supplementary Figures 2ndash4 (A-B)

4 Discussion

Inflammatory bowel disease (IBD) is a manifestation ofmultiple dysregulations acting simultaneously therefore it

Isoprenaline alone

+FsCr (003mgml)+FsCr (001mgml)

Isoprenaline (μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1 3

(a)

Isoprenaline alone+Flaxseed oil (1mgml)+Flaxseed oil (3mgml)

Isoprenaline (microg)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0003 03 1 3 5

(b)

Isoprenaline (μM)

Isoprenaline alone+Papaverine (1μM)+Papaverine (3μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(c)

Isoprenaline (μM)

Isoprenaline alone

+Verapamil (01μM)+Verapamil (003μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(d)

Figure 2 Concentration-response curves (CRCs) of isoprenaline

8 Evidence-Based Complementary and Alternative Medicine

Cilostazol control+FsCr (003mgml) +Zaprinast (100μM)

+Rolipram (3μM)

Cilostazol (μM)

CCh-

indu

ced

cont

ract

ion

()

0003 03 30 3000

100

75

50

25

0

(a)

Zaprinast control+FsCr (003mgml)

+Rolipram (3μM)+Cilostazol (100μM)

Zaprinast (μM)00003 03 30 1000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(b)

Rolipram control+FsCr (003mgml)

+Cilostazol (100μM)+Zaprinast (100μM)

Rolipram (μM)0003 03 30 3000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(c)

Figure 3 ((a)ndash(c)) Effect of FsCr on the relaxant responses of cilostazol (a) zaprinast (b) and rolipram (c) in rabbit jejunum precontractedwith CCh (1 μM)

Table 4 EC50 values of different PDE-subtype inhibitors with and without FsCr pretreatmentPDE inhibitorCilostazol (microM) Control +FsCr +Rolipram +ZaprinastEC50 1563 091lowastlowastlowast 015lowastlowastlowast 283lowastlowastlowastCI 7632 to 3199 035 to 235 006 to 039 148 to 541Rolipram (microM) Control +FsCr +Cilostazol (microM) +ZaprinastEC50 1241 1034 2421lowastlowastlowast 161lowastlowastlowastCI 642 to 2398 549 to 1946 121 to 483 096 to 270Zaprinast (microM) Control +FsCr +Rolipram +CilostazolEC50 1463 359lowastlowastlowast 1928lowastlowastlowast 038lowastlowastCI 7445 to 2874 1381 to 937 1191 to 3121 015 to 096EC50 effective concentration to produce 50 response CI confidence interval FsCr aqueous-methanolic crude extract of Flaxseed (70 30) All con-centrations are in microM lowastlowastlowastplt 0001 lowastlowastplt 001 one-way ANOVA was done followed by Tukeyrsquos posttest to compare the significance of meansplusmn SEM ofcontrols with other groups All treatment groups are compared with Control

Evidence-Based Complementary and Alternative Medicine 9

0

25

50

75

100

FsCr (mgml)

CCh (1μM)

+1mgml

+3mgml

+5mgml

cAM

P (p

mol

ml)

Sham

lowast

lowastlowastlowast

(a)

0

50

100

150

200

Papaverine

CCh (1μM)

+100μM

+300μM

+1000μMcA

MP

(pm

olm

l)

Sham

lowastlowastlowast

lowast

(b)

Figure 4 Effect of FsCr (a) and papaverine (b) on the cyclic nucleotide content of rabbit jejunum

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

FsCr (01mgmL)Control FsCr (03mgmL)

FsCr (3mgmL)

(a)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash45 ndash35 ndash25ndash85Log [ACh] M

Fs oil (1mgmL)Control Fs oil (3mgmL)

Fs oil (5mgmL)

(b)

Figure 5 Continued

10 Evidence-Based Complementary and Alternative Medicine

needs to be catered from different ends for an effectivedisease control Multitargeted approach has gained a lot ofinterest in IBD therapeutics [1] therefore we explored thevarious pharmacological targets of Flaxseed in order torationalize its medicinal use in IBD

Our preliminary studies have shown that Flaxseed ex-tract (FsCr) was effective against 6 AA-induced colitis inmice due to its antioxidant and anti-inflammatory activities[4] However this does not represent the potential of wholeFlaxseed as the extract so prepared mainly constitutespolar compounds of Flaxseed [5] Hence we studied dif-ferent mechanisms of both Flaxseed extract (known to

contain polar constituents) and oil (known to containnonpolar constituents) and then compared their effects onvarious parameters implicated in IBD

(e first step was to identify the effectiveness of Flaxseedoil against IBD and compare it with Flaxseed extract (FsCr)It was evident with parameters such as mortality rate diseaseactivity index (DAI) and microscopic scoring that Flaxseedoil was as effective in ameliorating the severity of IBD inmice model as FsCr however there were some differencesOne of the differences was that FsCr was more potent thanFlaxseed oil in producing these effects Flaxseed oil was moreefficacious in reducing ulcer size as compared to FsCr

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

Dicyclomine (003μM)Dicyclomine (01μM)

Control

(c)

Atropine (0003μM)Control Atropine (001μM)

Atropine (003μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

(d)

ControlVerapamil (003μM)Verapamil (01μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55ndash85Log [ACh] M

(e)

Figure 5 Concentration-response curves of Ach in the absence and presence of the increasing concentrations of (a) FsCr FS oil (b)dicyclomine (c) atropine (d) and verapamil (e) in isolated rabbit jejunum preparations

Evidence-Based Complementary and Alternative Medicine 11

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Flaxseed oil

Num

ber o

f EPE

C cf

u

Original inoculumUntreated (EPEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(a)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Original inoculumUntreated EPEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

lowast

(b)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Flaxseed oil

Original inoculumUntreated (ETEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(c)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f ETE

C cf

u

Original inoculumUntreated ETEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(d)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Flaxseed oil

Original inoculumUntreated (EAEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(e)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Original inoculumUntreated EAEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(f )

Figure 6 Bar charts show colony counts of sim106 EPEC (a d) ETEC (b e) and EAEC (c f ) incubated with and without Flaxseed oil (andashc)and Flaxseed extract (FsCr) (dndashf)

12 Evidence-Based Complementary and Alternative Medicine

reduction in neutrophil and lymphocytic infiltration wasslightly better with FsCr than Flaxseed oil FsCr was alsomore potent and efficacious in promoting mucin productionthan Flaxseed oil Despite these advancements the mortalityrate of Flaxseed oil was less as compared to FsCr Differencesin inflammatory and mucosal protective parameters in theextract and oil indicate that both the polar and nonpolarfractions of Flaxseed have the potential to mediate mucosalprotective effect in the IBDmodel of mice andmay provide asynergistic effect when given as a whole seed(is seems trueas Flaxseed and its oil showed clinical improvement in ul-cerative colitis patients in one of the latest reported studies[31] On the contrary our current findings are in contrast toan earlier report where 10 Flaxseed diet aggravated theseverity of dextran sodium sulfate- (DSS-) induced colitismodel of mice [32] One of the possibilities for this could bethat Flaxseed meal has a laxative effect [33] which is knownto aggravate colitis Nevertheless improvement in clinicalstudies in UC patients shows that this may not be the case orthis aspect may vary depending on the severity of the diseaseIn the clinical study patients who were not on corticoste-roids andor immune modulators were opted for whichmeans that the recruited UC patients have limited diseaseseverity More animal and clinical studies are needed toclarify this aspect

(e other part of our study aimed to identify themechanisms of Flaxseed in terms of its antispasmodic effectInterestingly not only did we find the antispasmodicmechanisms but we also got some lead for its anti-in-flammatory effect We found that both FsCr and Flaxseedoil caused complete inhibition of CCh-induced contractioncompared with other spasmogens which is indicative ofPDE inhibitory activity andor anticholinergic activity[26 34] Upon further experimentation it was evident thatFsCr not only exhibited a stronger PDEI but also increasedthe tissue cAMP levels Hence we further aimed to identifywhich subtype of PDEI receptor was inhibited It was foundthat FsCr mediated the similar inhibitory pathway for PDEenzyme as rolipram a standard PDE4 inhibitor (is wasidentified because FsCr and rolipram given simultaneouslycould not produce synergistic inhibitory effect against CCh-induced contraction whereas other PDEI when coad-ministered with FsCr potentiated the effect indicating thatperhaps rolipram and FsCr worked through the samesubtype of PDE enzyme (is interpretation is furthersupported by previous studies where the effect of one of thesubtypes of PDE inhibitors has been potentiated againstCCh-induced contraction when pretreated with the othersubtypes of PDE inhibitor [23 24 30] (is finding has avery important implication because blocking PDE-4 enzymehas been proposed as one of the novel approaches to targetIBD PDE-4 enzymes are present on inflammatory cells andtheir inhibition prevents mucosal damage and subsequentinflammatory cell infiltration by increasing cAMP [35] Ourfindings are also relatable because subsequently we were ableto show the increased level of cAMP in the FsCr-treatedtissues which is the end result of PDE-4 subtype inhibitionInterestingly in the HPLC fingerprints of FsCr (unpub-lished) one of the peaks matched quercetin a flavonoid that

has lately been identified as a potential PDE-4 inhibitor [36]and has been shown to ameliorate the severity of acetic acidcolitis in rats [37]

FsCr was the most potent against CCh-induced con-traction resembling papaverine It was however different inthat it showed some selectivity against high K+ likedicyclomine whereas papaverine showed similar inhibi-tion against both spontaneous and high K+-induced con-tractions (is raised the possibility of an additionalanticholinergic along with Ca+2 antagonist-like activitysimilar to dicyclomine component at a higher dose In-terestingly when tested for its anticholinergic activityFsCr at 03 and 1mgml concentrations caused therightward shift of the CRCs of Ach without suppression ofthe maximum response a classic pattern of a competitiveantagonist [27] such as dicyclomine Expectedly at thehigher concentration of 3mgml FsCr further shifted theCRCs of Ach to the right but suppressed the maximalresponse possibly due to the Ca+ channel antagonist-likemechanism which has also been reported earlier [7]Hence FsCr has been proved to exhibit multiple anti-spasmodic mechanisms ranging from PDEI-like effect at alower concentration followed by anticholinergic at a rel-atively higher concentration and Ca+ antagonist-like effectat a further higher concentration Contrary to the extractFlaxseed oil mediated antispasmodic effect predominatelythrough K+ channel opener (KCO) activity [6] Both PDEIand anticholinergic activity were evident in oil at a rela-tively higher concentration whereas CCB activity couldnot be observed However the possibility of some addi-tional mechanisms cannot be ruled out

With current findings Flaxseed as a whole mediatesantispasmodic action through multiple mechanisms in-cluding PDEI (subtype PDE-4) anticholinergic KCO andCa+2 antagonist-like activities All of these mechanisms areknown to have a protective role in IBD [10 11 13] and thusprovide an additional pharmacological basis for the effec-tiveness of Flaxseedrsquos protective effect in IBD

Besides therapeutic use of antispasmodics in IBD an-tibacterials also are used as an adjunct because reducing theaggressive microbial load has shown improvement in clinicaloutcomes in patients with IBD [38 39] (erefore in thecurrent study we targeted a few of the E coli strains im-plicated in IBD Flaxseed oil showed a bactericidal activityagainst all the three strains tested but FsCr was bactericidalagainst EPEC (is difference in efficacy and potency in-dicates that Flaxseed oil may have a different antibacterialmechanism or additional chemical constituent(s) with an-tibacterial activity

(e bactericidal effect of both FsCr and Flaxseed oilagainst EPEC is a very important finding because EPECrsquosinvasive level is more or less similar to AIEC strain (LF82)[13] Moreover EPEC has also been shown to sustainiodoacetamide-induced UC-like colitis in rats [40] HenceFlaxseed may reduce the invasive ability of Ecoli in IBDpatients and possibly its consequent manifestation thatincludes upregulation of NF-kβ [41] modulation of IL-1βIL-6 TNF-α COX-2 and apoptosis hence reducing in-flammatory cascades (is effect coupled with the earlier

Evidence-Based Complementary and Alternative Medicine 13

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

evident when inhibitory CRC was constructed againstzaprinast in presence of FsCr and other standard PDEinhibitors

Rolipram a selective inhibitor of PDE-4 inhibited thecontraction induced by CCh in a concentration-dependentmanner Interestingly the inhibitory response of rolipramwas not potentiated by pretreatment with FsCr whereas itwas significantly potentiated by pretreatment with cilostazol(plt 0001) and also zaprinast (plt 0001) as evident inFigures 3(b) Hence it is concluded that FsCr might beinhibiting PDE-4 subtype similar to rolipram a standardPDE-4 inhibitor

Since cAMP levels are increased when PDE enzyme isinhibited in the tissues [30] we also estimated the cAMPlevels in the control CCh-treated tissues and compared

them with FsCr- and papaverine-pretreated jejunaltissues Untreated tissues showed 1689 plusmn 142 pmol ofcAMPml of tissue homogenate Compared to this FsCrafter treatment with 1 3 and 5mgml of FsCr againstCCh-induced contraction showed cAMP levels up to2911 plusmn 162 (plt 005) 6599 plusmn 1 (plt 0001) and7873 plusmn 117 (plt 0001) pmolml respectively as evidentin Figure 4(a) (e papaverine control showed similarpattern of the rise in the cAMP levels with control CCh-treated tissues showing cAMP levels of2101 plusmn 175 pmolml whereas treatment with 1 μM and3 μM of papaverine in CCh-contracted tissues increasedthe cAMP levels to 587 plusmn 42 pmolml (plt 005) and9189 plusmn 34 (plt 0001) pmolml respectively as evidentin Figure 4(b)

Table 3 Comparison of histopathological scoring for Flaxseed oil and extract

Oil reduction from control Extract reduction from control Oil vs extract

Vilaseca score

UlcerationUntreated150mgkg 313 24 lowastlowastlowast

300mgkg 25 56lowast lowastlowastlowast

500mgkg 775lowastlowast 3314 lowastlowastlowast

Inflammation150mgkg 227 85 lowastlowastlowast

300mgkg 1364 50lowastlowastlowast lowastlowastlowast

500mgkg 3546lowastlowast 30 NSDepth of lesion1

150mgkg 769 2307lowast300mgkg 2308lowastlowast 55lowastlowastlowast lowastlowastlowast

500mgkg 4231lowastlowastlowast 3847lowastlowastlowast NS

Neurath score150mgkg 882 3304 lowastlowast

300mgkg 2647lowastlowast 5518lowast lowastlowastlowast

500mgkg 5589lowastlowastlowast 5115lowastlowast NS

Mucin score ()150mgkg 285 25 lowastlowastlowast

300mgkg 3571lowast 75lowastlowastlowast lowastlowastlowast

500mgkg 4228lowastlowast 9166lowastlowastlowast lowastlowastlowast

Neurath score [19] score 0 no leukocyte infiltration score 1 low level of leukocyte infiltration score 2moderate level of leukocyte infiltration score3moderate level of leukocyte infiltration high vascular density and thickening of colon wall score 4 transmural leukocyte infiltration loss of goblet cellshigh vascular density and thickening of the colon wall Vilaseca Score [20] total score 9 ulceration 1 [no ulcer epithelialization small ulcerslt 3mm largeulcersge 3mm score 0 1 2 respectively] inflammation 3 [none mild moderate severe 0 1 2 3] depth of lesion 3 [none mucosa muscularis propriaserosa 0 1 2 3] mucin score [21] score 4 [lt1 of stained cells 1ndash30 of stained cells (low level of staining) 30ndash80 of stained cells (medium level ofstaining) up to 80 of stained cells score 0 1 2 3] One-way ANOVA followed by Tukeyrsquos posttest was used for analysis Values expressed are meansplusmn SEM(n 6) lowastplt 005 lowastlowast plt 001 lowastlowastlowast plt 0001 vs AA Diseased control group Two-way ANOVA followed by Bonferronirsquos posttest was used for comparingthe effect of corresponding doses of Flaxseed oil and extract All the drugs were administered ip Colitis was induced by IR administration of 01ml 6 aceticacid during anaesthesia Tissues were embedded in formalin for another 24 hrs then processed and stained with H and E and PAS-AB staining ipintraperitoneally IR intrarectal H and E hematoxylin and eosin PAS-AB periodic acid-Schiff-Alcian blue

Table 2 Comparison of mortality rate and DAI of acetic acid-induced colitis in mice

Mortality rate () DAI ()Oil Extract Oil Extract

Untreated 5833plusmn 423 50plusmn 501 217plusmn 006 211plusmn 003150mgkg 55plusmn 12 3333plusmn 5lowast 21plusmn 008 117plusmn 001lowast300mgkg 3333plusmn 6lowastlowast 25plusmn 8lowastlowast 180plusmn 002lowastlowast 071plusmn 021lowastlowast500mgkg 833plusmn 5lowastlowastlowast 33plusmn 6lowastlowastlowast 093plusmn 001lowastlowastlowast 097plusmn 023lowastlowast

Values given are meansplusmn SEM of 3 experiments (n 12) One-way ANOVAwas used for statistical analysis followed by Tukeyrsquos posttest lowastplt 005 lowastlowastplt 001lowastlowastlowastplt 0001 compared with the untreated group () all the drugs were administered intraperitoneally for 7 days Colitis was induced on the 6th day withintrarectal administration of 01ml 6 acetic acid Mice were assessed 24 hrs after induction of colitis DAI [18] (weight loss + stool consistency + rectalbleeding)3 mortality rate (mice died in a grouptotal number of mice in that group)times 100 DAI disease activity index

6 Evidence-Based Complementary and Alternative Medicine

332 Anticholinergic Activity of FsCr and Flaxseed OilBesides PDEI CCh-induced contraction is also inhibited byanticholinergics therefore Flaxseed extract and oil werefurther tested in rabbit jejunal tissues to see if anticholinergicactivity exists Pretreatment of FsCr (01 and 03mgml)shifted Ach-induced bolus CRCs to the right in parallelmanner without suppression of the maximal response in aconcentration-dependent manner as shown in Figure 5(a)With further increase in the dose of extract at 1mgml therightward shift in Ach CRCs was nonparallel with sup-pression in maximal response Dicyclomine which is knownto exhibit a cholinergic antagonist effect at a lower dose and

CCB at a higher dose was used as a standard and showed asimilar rightward parallel shift of Ach CRCs at 003 μM(plt 005) and a rightward nonparallel shift due to sup-pression of the maximal response at 01 μM as shown inFigure 5(c)

Flaxseed oil shifted Ach-induced bolus CRCs to the rightin parallel manner without suppression of the maximalresponse at higher doses of 1 3 and 5mgml in a con-centration-dependent manner as shown in Figure 5(b)similar to atropine as shown in Figure 5(d) (is furtherindicates that FsCr was more potent anticholinergic thanFlaxseed oil

Flaxseed extract (mgml)

K+ (80mM)Spontaneous

CCh (1μM)K+ (25mM)

o

f con

trol

100

75

50

25

0

003 01 03 1 3 5

(a)

CCh (1μM)K+ (25mM)K+ (80mM)

Spontaneous

Flaxseed oil (mgml)

o

f con

trol

150

125

100

75

50

25

0

003 01 03 1 3 5001

(b)

Papaverine (μM)

K+ (80mM)Spontaneous

CCh (1μM)K+ (25mM)

o

f con

trol

001 03 3 30

100

75

50

25

0

(c)

K+ (80mM)Spontaneous

Verapamil (μM)

CCh (1μM)K+ (25mM)

o

f con

trol

100

75

50

25

003 01 03 1001

(d)

Figure 1 Inhibitory effect of aqueous-methanolic crude extract of Flaxseed (FsCr) (a) Flaxseed oil (b) papaverine (c) and verapamil (d)against CCh- high K+- (80mM) and low K+- (25mM) induced contractions in isolated rabbit jejunal preparations

Evidence-Based Complementary and Alternative Medicine 7

34 Antibacterial Activity Both Flaxseed extract (FsCr)and oil were tested in vitro for their effect against EPECETEC and EAEC Flaxseed oil was more potent and ef-ficacious against all the tested bacteria as compared toFsCr (e bactericidal effect of Flaxseed oil increaseddose-dependently at 9 and 14 μgml (Figures 6(a)ndash6(c))whereas the effect of FsCr was only evident against EPECat 100 μgml and no effect was observed against ETEC andEAEC (Figures 6(d)ndash6(f )) (e colony counts (cfu) areelaborated in Supplementary Table 3 Hence the minimaleffective dose for antibacterial activity against thesebacteria is almost 100 μgml for FsCr and 9 μgml forFlaxseed oil

Since FsCr did not exhibit bactericidal activity againstETEC and EAEC a possibility of bacteriostatic mechanismwas tested by increasing the incubation time from 2 to 16 hrs(to provide time for bacteria to grow) Doses of 5 and125mgml of FsCr were used for assay time of 16 hrswhereas doses of 005 and 01mg (50 and 100 microgml) of FsCrwere used when assay time was 2 hrs (e analysis is shownin Supplementary Figures 2ndash4 (A-B)

4 Discussion

Inflammatory bowel disease (IBD) is a manifestation ofmultiple dysregulations acting simultaneously therefore it

Isoprenaline alone

+FsCr (003mgml)+FsCr (001mgml)

Isoprenaline (μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1 3

(a)

Isoprenaline alone+Flaxseed oil (1mgml)+Flaxseed oil (3mgml)

Isoprenaline (microg)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0003 03 1 3 5

(b)

Isoprenaline (μM)

Isoprenaline alone+Papaverine (1μM)+Papaverine (3μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(c)

Isoprenaline (μM)

Isoprenaline alone

+Verapamil (01μM)+Verapamil (003μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(d)

Figure 2 Concentration-response curves (CRCs) of isoprenaline

8 Evidence-Based Complementary and Alternative Medicine

Cilostazol control+FsCr (003mgml) +Zaprinast (100μM)

+Rolipram (3μM)

Cilostazol (μM)

CCh-

indu

ced

cont

ract

ion

()

0003 03 30 3000

100

75

50

25

0

(a)

Zaprinast control+FsCr (003mgml)

+Rolipram (3μM)+Cilostazol (100μM)

Zaprinast (μM)00003 03 30 1000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(b)

Rolipram control+FsCr (003mgml)

+Cilostazol (100μM)+Zaprinast (100μM)

Rolipram (μM)0003 03 30 3000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(c)

Figure 3 ((a)ndash(c)) Effect of FsCr on the relaxant responses of cilostazol (a) zaprinast (b) and rolipram (c) in rabbit jejunum precontractedwith CCh (1 μM)

Table 4 EC50 values of different PDE-subtype inhibitors with and without FsCr pretreatmentPDE inhibitorCilostazol (microM) Control +FsCr +Rolipram +ZaprinastEC50 1563 091lowastlowastlowast 015lowastlowastlowast 283lowastlowastlowastCI 7632 to 3199 035 to 235 006 to 039 148 to 541Rolipram (microM) Control +FsCr +Cilostazol (microM) +ZaprinastEC50 1241 1034 2421lowastlowastlowast 161lowastlowastlowastCI 642 to 2398 549 to 1946 121 to 483 096 to 270Zaprinast (microM) Control +FsCr +Rolipram +CilostazolEC50 1463 359lowastlowastlowast 1928lowastlowastlowast 038lowastlowastCI 7445 to 2874 1381 to 937 1191 to 3121 015 to 096EC50 effective concentration to produce 50 response CI confidence interval FsCr aqueous-methanolic crude extract of Flaxseed (70 30) All con-centrations are in microM lowastlowastlowastplt 0001 lowastlowastplt 001 one-way ANOVA was done followed by Tukeyrsquos posttest to compare the significance of meansplusmn SEM ofcontrols with other groups All treatment groups are compared with Control

Evidence-Based Complementary and Alternative Medicine 9

0

25

50

75

100

FsCr (mgml)

CCh (1μM)

+1mgml

+3mgml

+5mgml

cAM

P (p

mol

ml)

Sham

lowast

lowastlowastlowast

(a)

0

50

100

150

200

Papaverine

CCh (1μM)

+100μM

+300μM

+1000μMcA

MP

(pm

olm

l)

Sham

lowastlowastlowast

lowast

(b)

Figure 4 Effect of FsCr (a) and papaverine (b) on the cyclic nucleotide content of rabbit jejunum

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

FsCr (01mgmL)Control FsCr (03mgmL)

FsCr (3mgmL)

(a)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash45 ndash35 ndash25ndash85Log [ACh] M

Fs oil (1mgmL)Control Fs oil (3mgmL)

Fs oil (5mgmL)

(b)

Figure 5 Continued

10 Evidence-Based Complementary and Alternative Medicine

needs to be catered from different ends for an effectivedisease control Multitargeted approach has gained a lot ofinterest in IBD therapeutics [1] therefore we explored thevarious pharmacological targets of Flaxseed in order torationalize its medicinal use in IBD

Our preliminary studies have shown that Flaxseed ex-tract (FsCr) was effective against 6 AA-induced colitis inmice due to its antioxidant and anti-inflammatory activities[4] However this does not represent the potential of wholeFlaxseed as the extract so prepared mainly constitutespolar compounds of Flaxseed [5] Hence we studied dif-ferent mechanisms of both Flaxseed extract (known to

contain polar constituents) and oil (known to containnonpolar constituents) and then compared their effects onvarious parameters implicated in IBD

(e first step was to identify the effectiveness of Flaxseedoil against IBD and compare it with Flaxseed extract (FsCr)It was evident with parameters such as mortality rate diseaseactivity index (DAI) and microscopic scoring that Flaxseedoil was as effective in ameliorating the severity of IBD inmice model as FsCr however there were some differencesOne of the differences was that FsCr was more potent thanFlaxseed oil in producing these effects Flaxseed oil was moreefficacious in reducing ulcer size as compared to FsCr

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

Dicyclomine (003μM)Dicyclomine (01μM)

Control

(c)

Atropine (0003μM)Control Atropine (001μM)

Atropine (003μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

(d)

ControlVerapamil (003μM)Verapamil (01μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55ndash85Log [ACh] M

(e)

Figure 5 Concentration-response curves of Ach in the absence and presence of the increasing concentrations of (a) FsCr FS oil (b)dicyclomine (c) atropine (d) and verapamil (e) in isolated rabbit jejunum preparations

Evidence-Based Complementary and Alternative Medicine 11

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Flaxseed oil

Num

ber o

f EPE

C cf

u

Original inoculumUntreated (EPEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(a)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Original inoculumUntreated EPEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

lowast

(b)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Flaxseed oil

Original inoculumUntreated (ETEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(c)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f ETE

C cf

u

Original inoculumUntreated ETEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(d)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Flaxseed oil

Original inoculumUntreated (EAEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(e)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Original inoculumUntreated EAEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(f )

Figure 6 Bar charts show colony counts of sim106 EPEC (a d) ETEC (b e) and EAEC (c f ) incubated with and without Flaxseed oil (andashc)and Flaxseed extract (FsCr) (dndashf)

12 Evidence-Based Complementary and Alternative Medicine

reduction in neutrophil and lymphocytic infiltration wasslightly better with FsCr than Flaxseed oil FsCr was alsomore potent and efficacious in promoting mucin productionthan Flaxseed oil Despite these advancements the mortalityrate of Flaxseed oil was less as compared to FsCr Differencesin inflammatory and mucosal protective parameters in theextract and oil indicate that both the polar and nonpolarfractions of Flaxseed have the potential to mediate mucosalprotective effect in the IBDmodel of mice andmay provide asynergistic effect when given as a whole seed(is seems trueas Flaxseed and its oil showed clinical improvement in ul-cerative colitis patients in one of the latest reported studies[31] On the contrary our current findings are in contrast toan earlier report where 10 Flaxseed diet aggravated theseverity of dextran sodium sulfate- (DSS-) induced colitismodel of mice [32] One of the possibilities for this could bethat Flaxseed meal has a laxative effect [33] which is knownto aggravate colitis Nevertheless improvement in clinicalstudies in UC patients shows that this may not be the case orthis aspect may vary depending on the severity of the diseaseIn the clinical study patients who were not on corticoste-roids andor immune modulators were opted for whichmeans that the recruited UC patients have limited diseaseseverity More animal and clinical studies are needed toclarify this aspect

(e other part of our study aimed to identify themechanisms of Flaxseed in terms of its antispasmodic effectInterestingly not only did we find the antispasmodicmechanisms but we also got some lead for its anti-in-flammatory effect We found that both FsCr and Flaxseedoil caused complete inhibition of CCh-induced contractioncompared with other spasmogens which is indicative ofPDE inhibitory activity andor anticholinergic activity[26 34] Upon further experimentation it was evident thatFsCr not only exhibited a stronger PDEI but also increasedthe tissue cAMP levels Hence we further aimed to identifywhich subtype of PDEI receptor was inhibited It was foundthat FsCr mediated the similar inhibitory pathway for PDEenzyme as rolipram a standard PDE4 inhibitor (is wasidentified because FsCr and rolipram given simultaneouslycould not produce synergistic inhibitory effect against CCh-induced contraction whereas other PDEI when coad-ministered with FsCr potentiated the effect indicating thatperhaps rolipram and FsCr worked through the samesubtype of PDE enzyme (is interpretation is furthersupported by previous studies where the effect of one of thesubtypes of PDE inhibitors has been potentiated againstCCh-induced contraction when pretreated with the othersubtypes of PDE inhibitor [23 24 30] (is finding has avery important implication because blocking PDE-4 enzymehas been proposed as one of the novel approaches to targetIBD PDE-4 enzymes are present on inflammatory cells andtheir inhibition prevents mucosal damage and subsequentinflammatory cell infiltration by increasing cAMP [35] Ourfindings are also relatable because subsequently we were ableto show the increased level of cAMP in the FsCr-treatedtissues which is the end result of PDE-4 subtype inhibitionInterestingly in the HPLC fingerprints of FsCr (unpub-lished) one of the peaks matched quercetin a flavonoid that

has lately been identified as a potential PDE-4 inhibitor [36]and has been shown to ameliorate the severity of acetic acidcolitis in rats [37]

FsCr was the most potent against CCh-induced con-traction resembling papaverine It was however different inthat it showed some selectivity against high K+ likedicyclomine whereas papaverine showed similar inhibi-tion against both spontaneous and high K+-induced con-tractions (is raised the possibility of an additionalanticholinergic along with Ca+2 antagonist-like activitysimilar to dicyclomine component at a higher dose In-terestingly when tested for its anticholinergic activityFsCr at 03 and 1mgml concentrations caused therightward shift of the CRCs of Ach without suppression ofthe maximum response a classic pattern of a competitiveantagonist [27] such as dicyclomine Expectedly at thehigher concentration of 3mgml FsCr further shifted theCRCs of Ach to the right but suppressed the maximalresponse possibly due to the Ca+ channel antagonist-likemechanism which has also been reported earlier [7]Hence FsCr has been proved to exhibit multiple anti-spasmodic mechanisms ranging from PDEI-like effect at alower concentration followed by anticholinergic at a rel-atively higher concentration and Ca+ antagonist-like effectat a further higher concentration Contrary to the extractFlaxseed oil mediated antispasmodic effect predominatelythrough K+ channel opener (KCO) activity [6] Both PDEIand anticholinergic activity were evident in oil at a rela-tively higher concentration whereas CCB activity couldnot be observed However the possibility of some addi-tional mechanisms cannot be ruled out

With current findings Flaxseed as a whole mediatesantispasmodic action through multiple mechanisms in-cluding PDEI (subtype PDE-4) anticholinergic KCO andCa+2 antagonist-like activities All of these mechanisms areknown to have a protective role in IBD [10 11 13] and thusprovide an additional pharmacological basis for the effec-tiveness of Flaxseedrsquos protective effect in IBD

Besides therapeutic use of antispasmodics in IBD an-tibacterials also are used as an adjunct because reducing theaggressive microbial load has shown improvement in clinicaloutcomes in patients with IBD [38 39] (erefore in thecurrent study we targeted a few of the E coli strains im-plicated in IBD Flaxseed oil showed a bactericidal activityagainst all the three strains tested but FsCr was bactericidalagainst EPEC (is difference in efficacy and potency in-dicates that Flaxseed oil may have a different antibacterialmechanism or additional chemical constituent(s) with an-tibacterial activity

(e bactericidal effect of both FsCr and Flaxseed oilagainst EPEC is a very important finding because EPECrsquosinvasive level is more or less similar to AIEC strain (LF82)[13] Moreover EPEC has also been shown to sustainiodoacetamide-induced UC-like colitis in rats [40] HenceFlaxseed may reduce the invasive ability of Ecoli in IBDpatients and possibly its consequent manifestation thatincludes upregulation of NF-kβ [41] modulation of IL-1βIL-6 TNF-α COX-2 and apoptosis hence reducing in-flammatory cascades (is effect coupled with the earlier

Evidence-Based Complementary and Alternative Medicine 13

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

332 Anticholinergic Activity of FsCr and Flaxseed OilBesides PDEI CCh-induced contraction is also inhibited byanticholinergics therefore Flaxseed extract and oil werefurther tested in rabbit jejunal tissues to see if anticholinergicactivity exists Pretreatment of FsCr (01 and 03mgml)shifted Ach-induced bolus CRCs to the right in parallelmanner without suppression of the maximal response in aconcentration-dependent manner as shown in Figure 5(a)With further increase in the dose of extract at 1mgml therightward shift in Ach CRCs was nonparallel with sup-pression in maximal response Dicyclomine which is knownto exhibit a cholinergic antagonist effect at a lower dose and

CCB at a higher dose was used as a standard and showed asimilar rightward parallel shift of Ach CRCs at 003 μM(plt 005) and a rightward nonparallel shift due to sup-pression of the maximal response at 01 μM as shown inFigure 5(c)

Flaxseed oil shifted Ach-induced bolus CRCs to the rightin parallel manner without suppression of the maximalresponse at higher doses of 1 3 and 5mgml in a con-centration-dependent manner as shown in Figure 5(b)similar to atropine as shown in Figure 5(d) (is furtherindicates that FsCr was more potent anticholinergic thanFlaxseed oil

Flaxseed extract (mgml)

K+ (80mM)Spontaneous

CCh (1μM)K+ (25mM)

o

f con

trol

100

75

50

25

0

003 01 03 1 3 5

(a)

CCh (1μM)K+ (25mM)K+ (80mM)

Spontaneous

Flaxseed oil (mgml)

o

f con

trol

150

125

100

75

50

25

0

003 01 03 1 3 5001

(b)

Papaverine (μM)

K+ (80mM)Spontaneous

CCh (1μM)K+ (25mM)

o

f con

trol

001 03 3 30

100

75

50

25

0

(c)

K+ (80mM)Spontaneous

Verapamil (μM)

CCh (1μM)K+ (25mM)

o

f con

trol

100

75

50

25

003 01 03 1001

(d)

Figure 1 Inhibitory effect of aqueous-methanolic crude extract of Flaxseed (FsCr) (a) Flaxseed oil (b) papaverine (c) and verapamil (d)against CCh- high K+- (80mM) and low K+- (25mM) induced contractions in isolated rabbit jejunal preparations

Evidence-Based Complementary and Alternative Medicine 7

34 Antibacterial Activity Both Flaxseed extract (FsCr)and oil were tested in vitro for their effect against EPECETEC and EAEC Flaxseed oil was more potent and ef-ficacious against all the tested bacteria as compared toFsCr (e bactericidal effect of Flaxseed oil increaseddose-dependently at 9 and 14 μgml (Figures 6(a)ndash6(c))whereas the effect of FsCr was only evident against EPECat 100 μgml and no effect was observed against ETEC andEAEC (Figures 6(d)ndash6(f )) (e colony counts (cfu) areelaborated in Supplementary Table 3 Hence the minimaleffective dose for antibacterial activity against thesebacteria is almost 100 μgml for FsCr and 9 μgml forFlaxseed oil

Since FsCr did not exhibit bactericidal activity againstETEC and EAEC a possibility of bacteriostatic mechanismwas tested by increasing the incubation time from 2 to 16 hrs(to provide time for bacteria to grow) Doses of 5 and125mgml of FsCr were used for assay time of 16 hrswhereas doses of 005 and 01mg (50 and 100 microgml) of FsCrwere used when assay time was 2 hrs (e analysis is shownin Supplementary Figures 2ndash4 (A-B)

4 Discussion

Inflammatory bowel disease (IBD) is a manifestation ofmultiple dysregulations acting simultaneously therefore it

Isoprenaline alone

+FsCr (003mgml)+FsCr (001mgml)

Isoprenaline (μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1 3

(a)

Isoprenaline alone+Flaxseed oil (1mgml)+Flaxseed oil (3mgml)

Isoprenaline (microg)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0003 03 1 3 5

(b)

Isoprenaline (μM)

Isoprenaline alone+Papaverine (1μM)+Papaverine (3μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(c)

Isoprenaline (μM)

Isoprenaline alone

+Verapamil (01μM)+Verapamil (003μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(d)

Figure 2 Concentration-response curves (CRCs) of isoprenaline

8 Evidence-Based Complementary and Alternative Medicine

Cilostazol control+FsCr (003mgml) +Zaprinast (100μM)

+Rolipram (3μM)

Cilostazol (μM)

CCh-

indu

ced

cont

ract

ion

()

0003 03 30 3000

100

75

50

25

0

(a)

Zaprinast control+FsCr (003mgml)

+Rolipram (3μM)+Cilostazol (100μM)

Zaprinast (μM)00003 03 30 1000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(b)

Rolipram control+FsCr (003mgml)

+Cilostazol (100μM)+Zaprinast (100μM)

Rolipram (μM)0003 03 30 3000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(c)

Figure 3 ((a)ndash(c)) Effect of FsCr on the relaxant responses of cilostazol (a) zaprinast (b) and rolipram (c) in rabbit jejunum precontractedwith CCh (1 μM)

Table 4 EC50 values of different PDE-subtype inhibitors with and without FsCr pretreatmentPDE inhibitorCilostazol (microM) Control +FsCr +Rolipram +ZaprinastEC50 1563 091lowastlowastlowast 015lowastlowastlowast 283lowastlowastlowastCI 7632 to 3199 035 to 235 006 to 039 148 to 541Rolipram (microM) Control +FsCr +Cilostazol (microM) +ZaprinastEC50 1241 1034 2421lowastlowastlowast 161lowastlowastlowastCI 642 to 2398 549 to 1946 121 to 483 096 to 270Zaprinast (microM) Control +FsCr +Rolipram +CilostazolEC50 1463 359lowastlowastlowast 1928lowastlowastlowast 038lowastlowastCI 7445 to 2874 1381 to 937 1191 to 3121 015 to 096EC50 effective concentration to produce 50 response CI confidence interval FsCr aqueous-methanolic crude extract of Flaxseed (70 30) All con-centrations are in microM lowastlowastlowastplt 0001 lowastlowastplt 001 one-way ANOVA was done followed by Tukeyrsquos posttest to compare the significance of meansplusmn SEM ofcontrols with other groups All treatment groups are compared with Control

Evidence-Based Complementary and Alternative Medicine 9

0

25

50

75

100

FsCr (mgml)

CCh (1μM)

+1mgml

+3mgml

+5mgml

cAM

P (p

mol

ml)

Sham

lowast

lowastlowastlowast

(a)

0

50

100

150

200

Papaverine

CCh (1μM)

+100μM

+300μM

+1000μMcA

MP

(pm

olm

l)

Sham

lowastlowastlowast

lowast

(b)

Figure 4 Effect of FsCr (a) and papaverine (b) on the cyclic nucleotide content of rabbit jejunum

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

FsCr (01mgmL)Control FsCr (03mgmL)

FsCr (3mgmL)

(a)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash45 ndash35 ndash25ndash85Log [ACh] M

Fs oil (1mgmL)Control Fs oil (3mgmL)

Fs oil (5mgmL)

(b)

Figure 5 Continued

10 Evidence-Based Complementary and Alternative Medicine

needs to be catered from different ends for an effectivedisease control Multitargeted approach has gained a lot ofinterest in IBD therapeutics [1] therefore we explored thevarious pharmacological targets of Flaxseed in order torationalize its medicinal use in IBD

Our preliminary studies have shown that Flaxseed ex-tract (FsCr) was effective against 6 AA-induced colitis inmice due to its antioxidant and anti-inflammatory activities[4] However this does not represent the potential of wholeFlaxseed as the extract so prepared mainly constitutespolar compounds of Flaxseed [5] Hence we studied dif-ferent mechanisms of both Flaxseed extract (known to

contain polar constituents) and oil (known to containnonpolar constituents) and then compared their effects onvarious parameters implicated in IBD

(e first step was to identify the effectiveness of Flaxseedoil against IBD and compare it with Flaxseed extract (FsCr)It was evident with parameters such as mortality rate diseaseactivity index (DAI) and microscopic scoring that Flaxseedoil was as effective in ameliorating the severity of IBD inmice model as FsCr however there were some differencesOne of the differences was that FsCr was more potent thanFlaxseed oil in producing these effects Flaxseed oil was moreefficacious in reducing ulcer size as compared to FsCr

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

Dicyclomine (003μM)Dicyclomine (01μM)

Control

(c)

Atropine (0003μM)Control Atropine (001μM)

Atropine (003μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

(d)

ControlVerapamil (003μM)Verapamil (01μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55ndash85Log [ACh] M

(e)

Figure 5 Concentration-response curves of Ach in the absence and presence of the increasing concentrations of (a) FsCr FS oil (b)dicyclomine (c) atropine (d) and verapamil (e) in isolated rabbit jejunum preparations

Evidence-Based Complementary and Alternative Medicine 11

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Flaxseed oil

Num

ber o

f EPE

C cf

u

Original inoculumUntreated (EPEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(a)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Original inoculumUntreated EPEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

lowast

(b)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Flaxseed oil

Original inoculumUntreated (ETEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(c)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f ETE

C cf

u

Original inoculumUntreated ETEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(d)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Flaxseed oil

Original inoculumUntreated (EAEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(e)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Original inoculumUntreated EAEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(f )

Figure 6 Bar charts show colony counts of sim106 EPEC (a d) ETEC (b e) and EAEC (c f ) incubated with and without Flaxseed oil (andashc)and Flaxseed extract (FsCr) (dndashf)

12 Evidence-Based Complementary and Alternative Medicine

reduction in neutrophil and lymphocytic infiltration wasslightly better with FsCr than Flaxseed oil FsCr was alsomore potent and efficacious in promoting mucin productionthan Flaxseed oil Despite these advancements the mortalityrate of Flaxseed oil was less as compared to FsCr Differencesin inflammatory and mucosal protective parameters in theextract and oil indicate that both the polar and nonpolarfractions of Flaxseed have the potential to mediate mucosalprotective effect in the IBDmodel of mice andmay provide asynergistic effect when given as a whole seed(is seems trueas Flaxseed and its oil showed clinical improvement in ul-cerative colitis patients in one of the latest reported studies[31] On the contrary our current findings are in contrast toan earlier report where 10 Flaxseed diet aggravated theseverity of dextran sodium sulfate- (DSS-) induced colitismodel of mice [32] One of the possibilities for this could bethat Flaxseed meal has a laxative effect [33] which is knownto aggravate colitis Nevertheless improvement in clinicalstudies in UC patients shows that this may not be the case orthis aspect may vary depending on the severity of the diseaseIn the clinical study patients who were not on corticoste-roids andor immune modulators were opted for whichmeans that the recruited UC patients have limited diseaseseverity More animal and clinical studies are needed toclarify this aspect

(e other part of our study aimed to identify themechanisms of Flaxseed in terms of its antispasmodic effectInterestingly not only did we find the antispasmodicmechanisms but we also got some lead for its anti-in-flammatory effect We found that both FsCr and Flaxseedoil caused complete inhibition of CCh-induced contractioncompared with other spasmogens which is indicative ofPDE inhibitory activity andor anticholinergic activity[26 34] Upon further experimentation it was evident thatFsCr not only exhibited a stronger PDEI but also increasedthe tissue cAMP levels Hence we further aimed to identifywhich subtype of PDEI receptor was inhibited It was foundthat FsCr mediated the similar inhibitory pathway for PDEenzyme as rolipram a standard PDE4 inhibitor (is wasidentified because FsCr and rolipram given simultaneouslycould not produce synergistic inhibitory effect against CCh-induced contraction whereas other PDEI when coad-ministered with FsCr potentiated the effect indicating thatperhaps rolipram and FsCr worked through the samesubtype of PDE enzyme (is interpretation is furthersupported by previous studies where the effect of one of thesubtypes of PDE inhibitors has been potentiated againstCCh-induced contraction when pretreated with the othersubtypes of PDE inhibitor [23 24 30] (is finding has avery important implication because blocking PDE-4 enzymehas been proposed as one of the novel approaches to targetIBD PDE-4 enzymes are present on inflammatory cells andtheir inhibition prevents mucosal damage and subsequentinflammatory cell infiltration by increasing cAMP [35] Ourfindings are also relatable because subsequently we were ableto show the increased level of cAMP in the FsCr-treatedtissues which is the end result of PDE-4 subtype inhibitionInterestingly in the HPLC fingerprints of FsCr (unpub-lished) one of the peaks matched quercetin a flavonoid that

has lately been identified as a potential PDE-4 inhibitor [36]and has been shown to ameliorate the severity of acetic acidcolitis in rats [37]

FsCr was the most potent against CCh-induced con-traction resembling papaverine It was however different inthat it showed some selectivity against high K+ likedicyclomine whereas papaverine showed similar inhibi-tion against both spontaneous and high K+-induced con-tractions (is raised the possibility of an additionalanticholinergic along with Ca+2 antagonist-like activitysimilar to dicyclomine component at a higher dose In-terestingly when tested for its anticholinergic activityFsCr at 03 and 1mgml concentrations caused therightward shift of the CRCs of Ach without suppression ofthe maximum response a classic pattern of a competitiveantagonist [27] such as dicyclomine Expectedly at thehigher concentration of 3mgml FsCr further shifted theCRCs of Ach to the right but suppressed the maximalresponse possibly due to the Ca+ channel antagonist-likemechanism which has also been reported earlier [7]Hence FsCr has been proved to exhibit multiple anti-spasmodic mechanisms ranging from PDEI-like effect at alower concentration followed by anticholinergic at a rel-atively higher concentration and Ca+ antagonist-like effectat a further higher concentration Contrary to the extractFlaxseed oil mediated antispasmodic effect predominatelythrough K+ channel opener (KCO) activity [6] Both PDEIand anticholinergic activity were evident in oil at a rela-tively higher concentration whereas CCB activity couldnot be observed However the possibility of some addi-tional mechanisms cannot be ruled out

With current findings Flaxseed as a whole mediatesantispasmodic action through multiple mechanisms in-cluding PDEI (subtype PDE-4) anticholinergic KCO andCa+2 antagonist-like activities All of these mechanisms areknown to have a protective role in IBD [10 11 13] and thusprovide an additional pharmacological basis for the effec-tiveness of Flaxseedrsquos protective effect in IBD

Besides therapeutic use of antispasmodics in IBD an-tibacterials also are used as an adjunct because reducing theaggressive microbial load has shown improvement in clinicaloutcomes in patients with IBD [38 39] (erefore in thecurrent study we targeted a few of the E coli strains im-plicated in IBD Flaxseed oil showed a bactericidal activityagainst all the three strains tested but FsCr was bactericidalagainst EPEC (is difference in efficacy and potency in-dicates that Flaxseed oil may have a different antibacterialmechanism or additional chemical constituent(s) with an-tibacterial activity

(e bactericidal effect of both FsCr and Flaxseed oilagainst EPEC is a very important finding because EPECrsquosinvasive level is more or less similar to AIEC strain (LF82)[13] Moreover EPEC has also been shown to sustainiodoacetamide-induced UC-like colitis in rats [40] HenceFlaxseed may reduce the invasive ability of Ecoli in IBDpatients and possibly its consequent manifestation thatincludes upregulation of NF-kβ [41] modulation of IL-1βIL-6 TNF-α COX-2 and apoptosis hence reducing in-flammatory cascades (is effect coupled with the earlier

Evidence-Based Complementary and Alternative Medicine 13

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

34 Antibacterial Activity Both Flaxseed extract (FsCr)and oil were tested in vitro for their effect against EPECETEC and EAEC Flaxseed oil was more potent and ef-ficacious against all the tested bacteria as compared toFsCr (e bactericidal effect of Flaxseed oil increaseddose-dependently at 9 and 14 μgml (Figures 6(a)ndash6(c))whereas the effect of FsCr was only evident against EPECat 100 μgml and no effect was observed against ETEC andEAEC (Figures 6(d)ndash6(f )) (e colony counts (cfu) areelaborated in Supplementary Table 3 Hence the minimaleffective dose for antibacterial activity against thesebacteria is almost 100 μgml for FsCr and 9 μgml forFlaxseed oil

Since FsCr did not exhibit bactericidal activity againstETEC and EAEC a possibility of bacteriostatic mechanismwas tested by increasing the incubation time from 2 to 16 hrs(to provide time for bacteria to grow) Doses of 5 and125mgml of FsCr were used for assay time of 16 hrswhereas doses of 005 and 01mg (50 and 100 microgml) of FsCrwere used when assay time was 2 hrs (e analysis is shownin Supplementary Figures 2ndash4 (A-B)

4 Discussion

Inflammatory bowel disease (IBD) is a manifestation ofmultiple dysregulations acting simultaneously therefore it

Isoprenaline alone

+FsCr (003mgml)+FsCr (001mgml)

Isoprenaline (μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1 3

(a)

Isoprenaline alone+Flaxseed oil (1mgml)+Flaxseed oil (3mgml)

Isoprenaline (microg)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0003 03 1 3 5

(b)

Isoprenaline (μM)

Isoprenaline alone+Papaverine (1μM)+Papaverine (3μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(c)

Isoprenaline (μM)

Isoprenaline alone

+Verapamil (01μM)+Verapamil (003μM)

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

001 01 03 1003

(d)

Figure 2 Concentration-response curves (CRCs) of isoprenaline

8 Evidence-Based Complementary and Alternative Medicine

Cilostazol control+FsCr (003mgml) +Zaprinast (100μM)

+Rolipram (3μM)

Cilostazol (μM)

CCh-

indu

ced

cont

ract

ion

()

0003 03 30 3000

100

75

50

25

0

(a)

Zaprinast control+FsCr (003mgml)

+Rolipram (3μM)+Cilostazol (100μM)

Zaprinast (μM)00003 03 30 1000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(b)

Rolipram control+FsCr (003mgml)

+Cilostazol (100μM)+Zaprinast (100μM)

Rolipram (μM)0003 03 30 3000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(c)

Figure 3 ((a)ndash(c)) Effect of FsCr on the relaxant responses of cilostazol (a) zaprinast (b) and rolipram (c) in rabbit jejunum precontractedwith CCh (1 μM)

Table 4 EC50 values of different PDE-subtype inhibitors with and without FsCr pretreatmentPDE inhibitorCilostazol (microM) Control +FsCr +Rolipram +ZaprinastEC50 1563 091lowastlowastlowast 015lowastlowastlowast 283lowastlowastlowastCI 7632 to 3199 035 to 235 006 to 039 148 to 541Rolipram (microM) Control +FsCr +Cilostazol (microM) +ZaprinastEC50 1241 1034 2421lowastlowastlowast 161lowastlowastlowastCI 642 to 2398 549 to 1946 121 to 483 096 to 270Zaprinast (microM) Control +FsCr +Rolipram +CilostazolEC50 1463 359lowastlowastlowast 1928lowastlowastlowast 038lowastlowastCI 7445 to 2874 1381 to 937 1191 to 3121 015 to 096EC50 effective concentration to produce 50 response CI confidence interval FsCr aqueous-methanolic crude extract of Flaxseed (70 30) All con-centrations are in microM lowastlowastlowastplt 0001 lowastlowastplt 001 one-way ANOVA was done followed by Tukeyrsquos posttest to compare the significance of meansplusmn SEM ofcontrols with other groups All treatment groups are compared with Control

Evidence-Based Complementary and Alternative Medicine 9

0

25

50

75

100

FsCr (mgml)

CCh (1μM)

+1mgml

+3mgml

+5mgml

cAM

P (p

mol

ml)

Sham

lowast

lowastlowastlowast

(a)

0

50

100

150

200

Papaverine

CCh (1μM)

+100μM

+300μM

+1000μMcA

MP

(pm

olm

l)

Sham

lowastlowastlowast

lowast

(b)

Figure 4 Effect of FsCr (a) and papaverine (b) on the cyclic nucleotide content of rabbit jejunum

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

FsCr (01mgmL)Control FsCr (03mgmL)

FsCr (3mgmL)

(a)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash45 ndash35 ndash25ndash85Log [ACh] M

Fs oil (1mgmL)Control Fs oil (3mgmL)

Fs oil (5mgmL)

(b)

Figure 5 Continued

10 Evidence-Based Complementary and Alternative Medicine

needs to be catered from different ends for an effectivedisease control Multitargeted approach has gained a lot ofinterest in IBD therapeutics [1] therefore we explored thevarious pharmacological targets of Flaxseed in order torationalize its medicinal use in IBD

Our preliminary studies have shown that Flaxseed ex-tract (FsCr) was effective against 6 AA-induced colitis inmice due to its antioxidant and anti-inflammatory activities[4] However this does not represent the potential of wholeFlaxseed as the extract so prepared mainly constitutespolar compounds of Flaxseed [5] Hence we studied dif-ferent mechanisms of both Flaxseed extract (known to

contain polar constituents) and oil (known to containnonpolar constituents) and then compared their effects onvarious parameters implicated in IBD

(e first step was to identify the effectiveness of Flaxseedoil against IBD and compare it with Flaxseed extract (FsCr)It was evident with parameters such as mortality rate diseaseactivity index (DAI) and microscopic scoring that Flaxseedoil was as effective in ameliorating the severity of IBD inmice model as FsCr however there were some differencesOne of the differences was that FsCr was more potent thanFlaxseed oil in producing these effects Flaxseed oil was moreefficacious in reducing ulcer size as compared to FsCr

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

Dicyclomine (003μM)Dicyclomine (01μM)

Control

(c)

Atropine (0003μM)Control Atropine (001μM)

Atropine (003μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

(d)

ControlVerapamil (003μM)Verapamil (01μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55ndash85Log [ACh] M

(e)

Figure 5 Concentration-response curves of Ach in the absence and presence of the increasing concentrations of (a) FsCr FS oil (b)dicyclomine (c) atropine (d) and verapamil (e) in isolated rabbit jejunum preparations

Evidence-Based Complementary and Alternative Medicine 11

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Flaxseed oil

Num

ber o

f EPE

C cf

u

Original inoculumUntreated (EPEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(a)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Original inoculumUntreated EPEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

lowast

(b)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Flaxseed oil

Original inoculumUntreated (ETEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(c)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f ETE

C cf

u

Original inoculumUntreated ETEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(d)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Flaxseed oil

Original inoculumUntreated (EAEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(e)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Original inoculumUntreated EAEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(f )

Figure 6 Bar charts show colony counts of sim106 EPEC (a d) ETEC (b e) and EAEC (c f ) incubated with and without Flaxseed oil (andashc)and Flaxseed extract (FsCr) (dndashf)

12 Evidence-Based Complementary and Alternative Medicine

reduction in neutrophil and lymphocytic infiltration wasslightly better with FsCr than Flaxseed oil FsCr was alsomore potent and efficacious in promoting mucin productionthan Flaxseed oil Despite these advancements the mortalityrate of Flaxseed oil was less as compared to FsCr Differencesin inflammatory and mucosal protective parameters in theextract and oil indicate that both the polar and nonpolarfractions of Flaxseed have the potential to mediate mucosalprotective effect in the IBDmodel of mice andmay provide asynergistic effect when given as a whole seed(is seems trueas Flaxseed and its oil showed clinical improvement in ul-cerative colitis patients in one of the latest reported studies[31] On the contrary our current findings are in contrast toan earlier report where 10 Flaxseed diet aggravated theseverity of dextran sodium sulfate- (DSS-) induced colitismodel of mice [32] One of the possibilities for this could bethat Flaxseed meal has a laxative effect [33] which is knownto aggravate colitis Nevertheless improvement in clinicalstudies in UC patients shows that this may not be the case orthis aspect may vary depending on the severity of the diseaseIn the clinical study patients who were not on corticoste-roids andor immune modulators were opted for whichmeans that the recruited UC patients have limited diseaseseverity More animal and clinical studies are needed toclarify this aspect

(e other part of our study aimed to identify themechanisms of Flaxseed in terms of its antispasmodic effectInterestingly not only did we find the antispasmodicmechanisms but we also got some lead for its anti-in-flammatory effect We found that both FsCr and Flaxseedoil caused complete inhibition of CCh-induced contractioncompared with other spasmogens which is indicative ofPDE inhibitory activity andor anticholinergic activity[26 34] Upon further experimentation it was evident thatFsCr not only exhibited a stronger PDEI but also increasedthe tissue cAMP levels Hence we further aimed to identifywhich subtype of PDEI receptor was inhibited It was foundthat FsCr mediated the similar inhibitory pathway for PDEenzyme as rolipram a standard PDE4 inhibitor (is wasidentified because FsCr and rolipram given simultaneouslycould not produce synergistic inhibitory effect against CCh-induced contraction whereas other PDEI when coad-ministered with FsCr potentiated the effect indicating thatperhaps rolipram and FsCr worked through the samesubtype of PDE enzyme (is interpretation is furthersupported by previous studies where the effect of one of thesubtypes of PDE inhibitors has been potentiated againstCCh-induced contraction when pretreated with the othersubtypes of PDE inhibitor [23 24 30] (is finding has avery important implication because blocking PDE-4 enzymehas been proposed as one of the novel approaches to targetIBD PDE-4 enzymes are present on inflammatory cells andtheir inhibition prevents mucosal damage and subsequentinflammatory cell infiltration by increasing cAMP [35] Ourfindings are also relatable because subsequently we were ableto show the increased level of cAMP in the FsCr-treatedtissues which is the end result of PDE-4 subtype inhibitionInterestingly in the HPLC fingerprints of FsCr (unpub-lished) one of the peaks matched quercetin a flavonoid that

has lately been identified as a potential PDE-4 inhibitor [36]and has been shown to ameliorate the severity of acetic acidcolitis in rats [37]

FsCr was the most potent against CCh-induced con-traction resembling papaverine It was however different inthat it showed some selectivity against high K+ likedicyclomine whereas papaverine showed similar inhibi-tion against both spontaneous and high K+-induced con-tractions (is raised the possibility of an additionalanticholinergic along with Ca+2 antagonist-like activitysimilar to dicyclomine component at a higher dose In-terestingly when tested for its anticholinergic activityFsCr at 03 and 1mgml concentrations caused therightward shift of the CRCs of Ach without suppression ofthe maximum response a classic pattern of a competitiveantagonist [27] such as dicyclomine Expectedly at thehigher concentration of 3mgml FsCr further shifted theCRCs of Ach to the right but suppressed the maximalresponse possibly due to the Ca+ channel antagonist-likemechanism which has also been reported earlier [7]Hence FsCr has been proved to exhibit multiple anti-spasmodic mechanisms ranging from PDEI-like effect at alower concentration followed by anticholinergic at a rel-atively higher concentration and Ca+ antagonist-like effectat a further higher concentration Contrary to the extractFlaxseed oil mediated antispasmodic effect predominatelythrough K+ channel opener (KCO) activity [6] Both PDEIand anticholinergic activity were evident in oil at a rela-tively higher concentration whereas CCB activity couldnot be observed However the possibility of some addi-tional mechanisms cannot be ruled out

With current findings Flaxseed as a whole mediatesantispasmodic action through multiple mechanisms in-cluding PDEI (subtype PDE-4) anticholinergic KCO andCa+2 antagonist-like activities All of these mechanisms areknown to have a protective role in IBD [10 11 13] and thusprovide an additional pharmacological basis for the effec-tiveness of Flaxseedrsquos protective effect in IBD

Besides therapeutic use of antispasmodics in IBD an-tibacterials also are used as an adjunct because reducing theaggressive microbial load has shown improvement in clinicaloutcomes in patients with IBD [38 39] (erefore in thecurrent study we targeted a few of the E coli strains im-plicated in IBD Flaxseed oil showed a bactericidal activityagainst all the three strains tested but FsCr was bactericidalagainst EPEC (is difference in efficacy and potency in-dicates that Flaxseed oil may have a different antibacterialmechanism or additional chemical constituent(s) with an-tibacterial activity

(e bactericidal effect of both FsCr and Flaxseed oilagainst EPEC is a very important finding because EPECrsquosinvasive level is more or less similar to AIEC strain (LF82)[13] Moreover EPEC has also been shown to sustainiodoacetamide-induced UC-like colitis in rats [40] HenceFlaxseed may reduce the invasive ability of Ecoli in IBDpatients and possibly its consequent manifestation thatincludes upregulation of NF-kβ [41] modulation of IL-1βIL-6 TNF-α COX-2 and apoptosis hence reducing in-flammatory cascades (is effect coupled with the earlier

Evidence-Based Complementary and Alternative Medicine 13

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

Cilostazol control+FsCr (003mgml) +Zaprinast (100μM)

+Rolipram (3μM)

Cilostazol (μM)

CCh-

indu

ced

cont

ract

ion

()

0003 03 30 3000

100

75

50

25

0

(a)

Zaprinast control+FsCr (003mgml)

+Rolipram (3μM)+Cilostazol (100μM)

Zaprinast (μM)00003 03 30 1000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(b)

Rolipram control+FsCr (003mgml)

+Cilostazol (100μM)+Zaprinast (100μM)

Rolipram (μM)0003 03 30 3000

CCh-

indu

ced

cont

ract

ion

()

100

75

50

25

0

(c)

Figure 3 ((a)ndash(c)) Effect of FsCr on the relaxant responses of cilostazol (a) zaprinast (b) and rolipram (c) in rabbit jejunum precontractedwith CCh (1 μM)

Table 4 EC50 values of different PDE-subtype inhibitors with and without FsCr pretreatmentPDE inhibitorCilostazol (microM) Control +FsCr +Rolipram +ZaprinastEC50 1563 091lowastlowastlowast 015lowastlowastlowast 283lowastlowastlowastCI 7632 to 3199 035 to 235 006 to 039 148 to 541Rolipram (microM) Control +FsCr +Cilostazol (microM) +ZaprinastEC50 1241 1034 2421lowastlowastlowast 161lowastlowastlowastCI 642 to 2398 549 to 1946 121 to 483 096 to 270Zaprinast (microM) Control +FsCr +Rolipram +CilostazolEC50 1463 359lowastlowastlowast 1928lowastlowastlowast 038lowastlowastCI 7445 to 2874 1381 to 937 1191 to 3121 015 to 096EC50 effective concentration to produce 50 response CI confidence interval FsCr aqueous-methanolic crude extract of Flaxseed (70 30) All con-centrations are in microM lowastlowastlowastplt 0001 lowastlowastplt 001 one-way ANOVA was done followed by Tukeyrsquos posttest to compare the significance of meansplusmn SEM ofcontrols with other groups All treatment groups are compared with Control

Evidence-Based Complementary and Alternative Medicine 9

0

25

50

75

100

FsCr (mgml)

CCh (1μM)

+1mgml

+3mgml

+5mgml

cAM

P (p

mol

ml)

Sham

lowast

lowastlowastlowast

(a)

0

50

100

150

200

Papaverine

CCh (1μM)

+100μM

+300μM

+1000μMcA

MP

(pm

olm

l)

Sham

lowastlowastlowast

lowast

(b)

Figure 4 Effect of FsCr (a) and papaverine (b) on the cyclic nucleotide content of rabbit jejunum

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

FsCr (01mgmL)Control FsCr (03mgmL)

FsCr (3mgmL)

(a)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash45 ndash35 ndash25ndash85Log [ACh] M

Fs oil (1mgmL)Control Fs oil (3mgmL)

Fs oil (5mgmL)

(b)

Figure 5 Continued

10 Evidence-Based Complementary and Alternative Medicine

needs to be catered from different ends for an effectivedisease control Multitargeted approach has gained a lot ofinterest in IBD therapeutics [1] therefore we explored thevarious pharmacological targets of Flaxseed in order torationalize its medicinal use in IBD

Our preliminary studies have shown that Flaxseed ex-tract (FsCr) was effective against 6 AA-induced colitis inmice due to its antioxidant and anti-inflammatory activities[4] However this does not represent the potential of wholeFlaxseed as the extract so prepared mainly constitutespolar compounds of Flaxseed [5] Hence we studied dif-ferent mechanisms of both Flaxseed extract (known to

contain polar constituents) and oil (known to containnonpolar constituents) and then compared their effects onvarious parameters implicated in IBD

(e first step was to identify the effectiveness of Flaxseedoil against IBD and compare it with Flaxseed extract (FsCr)It was evident with parameters such as mortality rate diseaseactivity index (DAI) and microscopic scoring that Flaxseedoil was as effective in ameliorating the severity of IBD inmice model as FsCr however there were some differencesOne of the differences was that FsCr was more potent thanFlaxseed oil in producing these effects Flaxseed oil was moreefficacious in reducing ulcer size as compared to FsCr

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

Dicyclomine (003μM)Dicyclomine (01μM)

Control

(c)

Atropine (0003μM)Control Atropine (001μM)

Atropine (003μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

(d)

ControlVerapamil (003μM)Verapamil (01μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55ndash85Log [ACh] M

(e)

Figure 5 Concentration-response curves of Ach in the absence and presence of the increasing concentrations of (a) FsCr FS oil (b)dicyclomine (c) atropine (d) and verapamil (e) in isolated rabbit jejunum preparations

Evidence-Based Complementary and Alternative Medicine 11

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Flaxseed oil

Num

ber o

f EPE

C cf

u

Original inoculumUntreated (EPEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(a)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Original inoculumUntreated EPEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

lowast

(b)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Flaxseed oil

Original inoculumUntreated (ETEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(c)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f ETE

C cf

u

Original inoculumUntreated ETEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(d)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Flaxseed oil

Original inoculumUntreated (EAEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(e)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Original inoculumUntreated EAEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(f )

Figure 6 Bar charts show colony counts of sim106 EPEC (a d) ETEC (b e) and EAEC (c f ) incubated with and without Flaxseed oil (andashc)and Flaxseed extract (FsCr) (dndashf)

12 Evidence-Based Complementary and Alternative Medicine

reduction in neutrophil and lymphocytic infiltration wasslightly better with FsCr than Flaxseed oil FsCr was alsomore potent and efficacious in promoting mucin productionthan Flaxseed oil Despite these advancements the mortalityrate of Flaxseed oil was less as compared to FsCr Differencesin inflammatory and mucosal protective parameters in theextract and oil indicate that both the polar and nonpolarfractions of Flaxseed have the potential to mediate mucosalprotective effect in the IBDmodel of mice andmay provide asynergistic effect when given as a whole seed(is seems trueas Flaxseed and its oil showed clinical improvement in ul-cerative colitis patients in one of the latest reported studies[31] On the contrary our current findings are in contrast toan earlier report where 10 Flaxseed diet aggravated theseverity of dextran sodium sulfate- (DSS-) induced colitismodel of mice [32] One of the possibilities for this could bethat Flaxseed meal has a laxative effect [33] which is knownto aggravate colitis Nevertheless improvement in clinicalstudies in UC patients shows that this may not be the case orthis aspect may vary depending on the severity of the diseaseIn the clinical study patients who were not on corticoste-roids andor immune modulators were opted for whichmeans that the recruited UC patients have limited diseaseseverity More animal and clinical studies are needed toclarify this aspect

(e other part of our study aimed to identify themechanisms of Flaxseed in terms of its antispasmodic effectInterestingly not only did we find the antispasmodicmechanisms but we also got some lead for its anti-in-flammatory effect We found that both FsCr and Flaxseedoil caused complete inhibition of CCh-induced contractioncompared with other spasmogens which is indicative ofPDE inhibitory activity andor anticholinergic activity[26 34] Upon further experimentation it was evident thatFsCr not only exhibited a stronger PDEI but also increasedthe tissue cAMP levels Hence we further aimed to identifywhich subtype of PDEI receptor was inhibited It was foundthat FsCr mediated the similar inhibitory pathway for PDEenzyme as rolipram a standard PDE4 inhibitor (is wasidentified because FsCr and rolipram given simultaneouslycould not produce synergistic inhibitory effect against CCh-induced contraction whereas other PDEI when coad-ministered with FsCr potentiated the effect indicating thatperhaps rolipram and FsCr worked through the samesubtype of PDE enzyme (is interpretation is furthersupported by previous studies where the effect of one of thesubtypes of PDE inhibitors has been potentiated againstCCh-induced contraction when pretreated with the othersubtypes of PDE inhibitor [23 24 30] (is finding has avery important implication because blocking PDE-4 enzymehas been proposed as one of the novel approaches to targetIBD PDE-4 enzymes are present on inflammatory cells andtheir inhibition prevents mucosal damage and subsequentinflammatory cell infiltration by increasing cAMP [35] Ourfindings are also relatable because subsequently we were ableto show the increased level of cAMP in the FsCr-treatedtissues which is the end result of PDE-4 subtype inhibitionInterestingly in the HPLC fingerprints of FsCr (unpub-lished) one of the peaks matched quercetin a flavonoid that

has lately been identified as a potential PDE-4 inhibitor [36]and has been shown to ameliorate the severity of acetic acidcolitis in rats [37]

FsCr was the most potent against CCh-induced con-traction resembling papaverine It was however different inthat it showed some selectivity against high K+ likedicyclomine whereas papaverine showed similar inhibi-tion against both spontaneous and high K+-induced con-tractions (is raised the possibility of an additionalanticholinergic along with Ca+2 antagonist-like activitysimilar to dicyclomine component at a higher dose In-terestingly when tested for its anticholinergic activityFsCr at 03 and 1mgml concentrations caused therightward shift of the CRCs of Ach without suppression ofthe maximum response a classic pattern of a competitiveantagonist [27] such as dicyclomine Expectedly at thehigher concentration of 3mgml FsCr further shifted theCRCs of Ach to the right but suppressed the maximalresponse possibly due to the Ca+ channel antagonist-likemechanism which has also been reported earlier [7]Hence FsCr has been proved to exhibit multiple anti-spasmodic mechanisms ranging from PDEI-like effect at alower concentration followed by anticholinergic at a rel-atively higher concentration and Ca+ antagonist-like effectat a further higher concentration Contrary to the extractFlaxseed oil mediated antispasmodic effect predominatelythrough K+ channel opener (KCO) activity [6] Both PDEIand anticholinergic activity were evident in oil at a rela-tively higher concentration whereas CCB activity couldnot be observed However the possibility of some addi-tional mechanisms cannot be ruled out

With current findings Flaxseed as a whole mediatesantispasmodic action through multiple mechanisms in-cluding PDEI (subtype PDE-4) anticholinergic KCO andCa+2 antagonist-like activities All of these mechanisms areknown to have a protective role in IBD [10 11 13] and thusprovide an additional pharmacological basis for the effec-tiveness of Flaxseedrsquos protective effect in IBD

Besides therapeutic use of antispasmodics in IBD an-tibacterials also are used as an adjunct because reducing theaggressive microbial load has shown improvement in clinicaloutcomes in patients with IBD [38 39] (erefore in thecurrent study we targeted a few of the E coli strains im-plicated in IBD Flaxseed oil showed a bactericidal activityagainst all the three strains tested but FsCr was bactericidalagainst EPEC (is difference in efficacy and potency in-dicates that Flaxseed oil may have a different antibacterialmechanism or additional chemical constituent(s) with an-tibacterial activity

(e bactericidal effect of both FsCr and Flaxseed oilagainst EPEC is a very important finding because EPECrsquosinvasive level is more or less similar to AIEC strain (LF82)[13] Moreover EPEC has also been shown to sustainiodoacetamide-induced UC-like colitis in rats [40] HenceFlaxseed may reduce the invasive ability of Ecoli in IBDpatients and possibly its consequent manifestation thatincludes upregulation of NF-kβ [41] modulation of IL-1βIL-6 TNF-α COX-2 and apoptosis hence reducing in-flammatory cascades (is effect coupled with the earlier

Evidence-Based Complementary and Alternative Medicine 13

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

0

25

50

75

100

FsCr (mgml)

CCh (1μM)

+1mgml

+3mgml

+5mgml

cAM

P (p

mol

ml)

Sham

lowast

lowastlowastlowast

(a)

0

50

100

150

200

Papaverine

CCh (1μM)

+100μM

+300μM

+1000μMcA

MP

(pm

olm

l)

Sham

lowastlowastlowast

lowast

(b)

Figure 4 Effect of FsCr (a) and papaverine (b) on the cyclic nucleotide content of rabbit jejunum

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

FsCr (01mgmL)Control FsCr (03mgmL)

FsCr (3mgmL)

(a)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash45 ndash35 ndash25ndash85Log [ACh] M

Fs oil (1mgmL)Control Fs oil (3mgmL)

Fs oil (5mgmL)

(b)

Figure 5 Continued

10 Evidence-Based Complementary and Alternative Medicine

needs to be catered from different ends for an effectivedisease control Multitargeted approach has gained a lot ofinterest in IBD therapeutics [1] therefore we explored thevarious pharmacological targets of Flaxseed in order torationalize its medicinal use in IBD

Our preliminary studies have shown that Flaxseed ex-tract (FsCr) was effective against 6 AA-induced colitis inmice due to its antioxidant and anti-inflammatory activities[4] However this does not represent the potential of wholeFlaxseed as the extract so prepared mainly constitutespolar compounds of Flaxseed [5] Hence we studied dif-ferent mechanisms of both Flaxseed extract (known to

contain polar constituents) and oil (known to containnonpolar constituents) and then compared their effects onvarious parameters implicated in IBD

(e first step was to identify the effectiveness of Flaxseedoil against IBD and compare it with Flaxseed extract (FsCr)It was evident with parameters such as mortality rate diseaseactivity index (DAI) and microscopic scoring that Flaxseedoil was as effective in ameliorating the severity of IBD inmice model as FsCr however there were some differencesOne of the differences was that FsCr was more potent thanFlaxseed oil in producing these effects Flaxseed oil was moreefficacious in reducing ulcer size as compared to FsCr

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

Dicyclomine (003μM)Dicyclomine (01μM)

Control

(c)

Atropine (0003μM)Control Atropine (001μM)

Atropine (003μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

(d)

ControlVerapamil (003μM)Verapamil (01μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55ndash85Log [ACh] M

(e)

Figure 5 Concentration-response curves of Ach in the absence and presence of the increasing concentrations of (a) FsCr FS oil (b)dicyclomine (c) atropine (d) and verapamil (e) in isolated rabbit jejunum preparations

Evidence-Based Complementary and Alternative Medicine 11

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Flaxseed oil

Num

ber o

f EPE

C cf

u

Original inoculumUntreated (EPEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(a)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Original inoculumUntreated EPEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

lowast

(b)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Flaxseed oil

Original inoculumUntreated (ETEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(c)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f ETE

C cf

u

Original inoculumUntreated ETEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(d)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Flaxseed oil

Original inoculumUntreated (EAEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(e)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Original inoculumUntreated EAEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(f )

Figure 6 Bar charts show colony counts of sim106 EPEC (a d) ETEC (b e) and EAEC (c f ) incubated with and without Flaxseed oil (andashc)and Flaxseed extract (FsCr) (dndashf)

12 Evidence-Based Complementary and Alternative Medicine

reduction in neutrophil and lymphocytic infiltration wasslightly better with FsCr than Flaxseed oil FsCr was alsomore potent and efficacious in promoting mucin productionthan Flaxseed oil Despite these advancements the mortalityrate of Flaxseed oil was less as compared to FsCr Differencesin inflammatory and mucosal protective parameters in theextract and oil indicate that both the polar and nonpolarfractions of Flaxseed have the potential to mediate mucosalprotective effect in the IBDmodel of mice andmay provide asynergistic effect when given as a whole seed(is seems trueas Flaxseed and its oil showed clinical improvement in ul-cerative colitis patients in one of the latest reported studies[31] On the contrary our current findings are in contrast toan earlier report where 10 Flaxseed diet aggravated theseverity of dextran sodium sulfate- (DSS-) induced colitismodel of mice [32] One of the possibilities for this could bethat Flaxseed meal has a laxative effect [33] which is knownto aggravate colitis Nevertheless improvement in clinicalstudies in UC patients shows that this may not be the case orthis aspect may vary depending on the severity of the diseaseIn the clinical study patients who were not on corticoste-roids andor immune modulators were opted for whichmeans that the recruited UC patients have limited diseaseseverity More animal and clinical studies are needed toclarify this aspect

(e other part of our study aimed to identify themechanisms of Flaxseed in terms of its antispasmodic effectInterestingly not only did we find the antispasmodicmechanisms but we also got some lead for its anti-in-flammatory effect We found that both FsCr and Flaxseedoil caused complete inhibition of CCh-induced contractioncompared with other spasmogens which is indicative ofPDE inhibitory activity andor anticholinergic activity[26 34] Upon further experimentation it was evident thatFsCr not only exhibited a stronger PDEI but also increasedthe tissue cAMP levels Hence we further aimed to identifywhich subtype of PDEI receptor was inhibited It was foundthat FsCr mediated the similar inhibitory pathway for PDEenzyme as rolipram a standard PDE4 inhibitor (is wasidentified because FsCr and rolipram given simultaneouslycould not produce synergistic inhibitory effect against CCh-induced contraction whereas other PDEI when coad-ministered with FsCr potentiated the effect indicating thatperhaps rolipram and FsCr worked through the samesubtype of PDE enzyme (is interpretation is furthersupported by previous studies where the effect of one of thesubtypes of PDE inhibitors has been potentiated againstCCh-induced contraction when pretreated with the othersubtypes of PDE inhibitor [23 24 30] (is finding has avery important implication because blocking PDE-4 enzymehas been proposed as one of the novel approaches to targetIBD PDE-4 enzymes are present on inflammatory cells andtheir inhibition prevents mucosal damage and subsequentinflammatory cell infiltration by increasing cAMP [35] Ourfindings are also relatable because subsequently we were ableto show the increased level of cAMP in the FsCr-treatedtissues which is the end result of PDE-4 subtype inhibitionInterestingly in the HPLC fingerprints of FsCr (unpub-lished) one of the peaks matched quercetin a flavonoid that

has lately been identified as a potential PDE-4 inhibitor [36]and has been shown to ameliorate the severity of acetic acidcolitis in rats [37]

FsCr was the most potent against CCh-induced con-traction resembling papaverine It was however different inthat it showed some selectivity against high K+ likedicyclomine whereas papaverine showed similar inhibi-tion against both spontaneous and high K+-induced con-tractions (is raised the possibility of an additionalanticholinergic along with Ca+2 antagonist-like activitysimilar to dicyclomine component at a higher dose In-terestingly when tested for its anticholinergic activityFsCr at 03 and 1mgml concentrations caused therightward shift of the CRCs of Ach without suppression ofthe maximum response a classic pattern of a competitiveantagonist [27] such as dicyclomine Expectedly at thehigher concentration of 3mgml FsCr further shifted theCRCs of Ach to the right but suppressed the maximalresponse possibly due to the Ca+ channel antagonist-likemechanism which has also been reported earlier [7]Hence FsCr has been proved to exhibit multiple anti-spasmodic mechanisms ranging from PDEI-like effect at alower concentration followed by anticholinergic at a rel-atively higher concentration and Ca+ antagonist-like effectat a further higher concentration Contrary to the extractFlaxseed oil mediated antispasmodic effect predominatelythrough K+ channel opener (KCO) activity [6] Both PDEIand anticholinergic activity were evident in oil at a rela-tively higher concentration whereas CCB activity couldnot be observed However the possibility of some addi-tional mechanisms cannot be ruled out

With current findings Flaxseed as a whole mediatesantispasmodic action through multiple mechanisms in-cluding PDEI (subtype PDE-4) anticholinergic KCO andCa+2 antagonist-like activities All of these mechanisms areknown to have a protective role in IBD [10 11 13] and thusprovide an additional pharmacological basis for the effec-tiveness of Flaxseedrsquos protective effect in IBD

Besides therapeutic use of antispasmodics in IBD an-tibacterials also are used as an adjunct because reducing theaggressive microbial load has shown improvement in clinicaloutcomes in patients with IBD [38 39] (erefore in thecurrent study we targeted a few of the E coli strains im-plicated in IBD Flaxseed oil showed a bactericidal activityagainst all the three strains tested but FsCr was bactericidalagainst EPEC (is difference in efficacy and potency in-dicates that Flaxseed oil may have a different antibacterialmechanism or additional chemical constituent(s) with an-tibacterial activity

(e bactericidal effect of both FsCr and Flaxseed oilagainst EPEC is a very important finding because EPECrsquosinvasive level is more or less similar to AIEC strain (LF82)[13] Moreover EPEC has also been shown to sustainiodoacetamide-induced UC-like colitis in rats [40] HenceFlaxseed may reduce the invasive ability of Ecoli in IBDpatients and possibly its consequent manifestation thatincludes upregulation of NF-kβ [41] modulation of IL-1βIL-6 TNF-α COX-2 and apoptosis hence reducing in-flammatory cascades (is effect coupled with the earlier

Evidence-Based Complementary and Alternative Medicine 13

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

needs to be catered from different ends for an effectivedisease control Multitargeted approach has gained a lot ofinterest in IBD therapeutics [1] therefore we explored thevarious pharmacological targets of Flaxseed in order torationalize its medicinal use in IBD

Our preliminary studies have shown that Flaxseed ex-tract (FsCr) was effective against 6 AA-induced colitis inmice due to its antioxidant and anti-inflammatory activities[4] However this does not represent the potential of wholeFlaxseed as the extract so prepared mainly constitutespolar compounds of Flaxseed [5] Hence we studied dif-ferent mechanisms of both Flaxseed extract (known to

contain polar constituents) and oil (known to containnonpolar constituents) and then compared their effects onvarious parameters implicated in IBD

(e first step was to identify the effectiveness of Flaxseedoil against IBD and compare it with Flaxseed extract (FsCr)It was evident with parameters such as mortality rate diseaseactivity index (DAI) and microscopic scoring that Flaxseedoil was as effective in ameliorating the severity of IBD inmice model as FsCr however there were some differencesOne of the differences was that FsCr was more potent thanFlaxseed oil in producing these effects Flaxseed oil was moreefficacious in reducing ulcer size as compared to FsCr

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

Dicyclomine (003μM)Dicyclomine (01μM)

Control

(c)

Atropine (0003μM)Control Atropine (001μM)

Atropine (003μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55 ndash5ndash85Log [ACh] M

(d)

ControlVerapamil (003μM)Verapamil (01μM)

o

f con

trol

0

25

50

75

100

ndash75 ndash65 ndash55ndash85Log [ACh] M

(e)

Figure 5 Concentration-response curves of Ach in the absence and presence of the increasing concentrations of (a) FsCr FS oil (b)dicyclomine (c) atropine (d) and verapamil (e) in isolated rabbit jejunum preparations

Evidence-Based Complementary and Alternative Medicine 11

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Flaxseed oil

Num

ber o

f EPE

C cf

u

Original inoculumUntreated (EPEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(a)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Original inoculumUntreated EPEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

lowast

(b)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Flaxseed oil

Original inoculumUntreated (ETEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(c)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f ETE

C cf

u

Original inoculumUntreated ETEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(d)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Flaxseed oil

Original inoculumUntreated (EAEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(e)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Original inoculumUntreated EAEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(f )

Figure 6 Bar charts show colony counts of sim106 EPEC (a d) ETEC (b e) and EAEC (c f ) incubated with and without Flaxseed oil (andashc)and Flaxseed extract (FsCr) (dndashf)

12 Evidence-Based Complementary and Alternative Medicine

reduction in neutrophil and lymphocytic infiltration wasslightly better with FsCr than Flaxseed oil FsCr was alsomore potent and efficacious in promoting mucin productionthan Flaxseed oil Despite these advancements the mortalityrate of Flaxseed oil was less as compared to FsCr Differencesin inflammatory and mucosal protective parameters in theextract and oil indicate that both the polar and nonpolarfractions of Flaxseed have the potential to mediate mucosalprotective effect in the IBDmodel of mice andmay provide asynergistic effect when given as a whole seed(is seems trueas Flaxseed and its oil showed clinical improvement in ul-cerative colitis patients in one of the latest reported studies[31] On the contrary our current findings are in contrast toan earlier report where 10 Flaxseed diet aggravated theseverity of dextran sodium sulfate- (DSS-) induced colitismodel of mice [32] One of the possibilities for this could bethat Flaxseed meal has a laxative effect [33] which is knownto aggravate colitis Nevertheless improvement in clinicalstudies in UC patients shows that this may not be the case orthis aspect may vary depending on the severity of the diseaseIn the clinical study patients who were not on corticoste-roids andor immune modulators were opted for whichmeans that the recruited UC patients have limited diseaseseverity More animal and clinical studies are needed toclarify this aspect

(e other part of our study aimed to identify themechanisms of Flaxseed in terms of its antispasmodic effectInterestingly not only did we find the antispasmodicmechanisms but we also got some lead for its anti-in-flammatory effect We found that both FsCr and Flaxseedoil caused complete inhibition of CCh-induced contractioncompared with other spasmogens which is indicative ofPDE inhibitory activity andor anticholinergic activity[26 34] Upon further experimentation it was evident thatFsCr not only exhibited a stronger PDEI but also increasedthe tissue cAMP levels Hence we further aimed to identifywhich subtype of PDEI receptor was inhibited It was foundthat FsCr mediated the similar inhibitory pathway for PDEenzyme as rolipram a standard PDE4 inhibitor (is wasidentified because FsCr and rolipram given simultaneouslycould not produce synergistic inhibitory effect against CCh-induced contraction whereas other PDEI when coad-ministered with FsCr potentiated the effect indicating thatperhaps rolipram and FsCr worked through the samesubtype of PDE enzyme (is interpretation is furthersupported by previous studies where the effect of one of thesubtypes of PDE inhibitors has been potentiated againstCCh-induced contraction when pretreated with the othersubtypes of PDE inhibitor [23 24 30] (is finding has avery important implication because blocking PDE-4 enzymehas been proposed as one of the novel approaches to targetIBD PDE-4 enzymes are present on inflammatory cells andtheir inhibition prevents mucosal damage and subsequentinflammatory cell infiltration by increasing cAMP [35] Ourfindings are also relatable because subsequently we were ableto show the increased level of cAMP in the FsCr-treatedtissues which is the end result of PDE-4 subtype inhibitionInterestingly in the HPLC fingerprints of FsCr (unpub-lished) one of the peaks matched quercetin a flavonoid that

has lately been identified as a potential PDE-4 inhibitor [36]and has been shown to ameliorate the severity of acetic acidcolitis in rats [37]

FsCr was the most potent against CCh-induced con-traction resembling papaverine It was however different inthat it showed some selectivity against high K+ likedicyclomine whereas papaverine showed similar inhibi-tion against both spontaneous and high K+-induced con-tractions (is raised the possibility of an additionalanticholinergic along with Ca+2 antagonist-like activitysimilar to dicyclomine component at a higher dose In-terestingly when tested for its anticholinergic activityFsCr at 03 and 1mgml concentrations caused therightward shift of the CRCs of Ach without suppression ofthe maximum response a classic pattern of a competitiveantagonist [27] such as dicyclomine Expectedly at thehigher concentration of 3mgml FsCr further shifted theCRCs of Ach to the right but suppressed the maximalresponse possibly due to the Ca+ channel antagonist-likemechanism which has also been reported earlier [7]Hence FsCr has been proved to exhibit multiple anti-spasmodic mechanisms ranging from PDEI-like effect at alower concentration followed by anticholinergic at a rel-atively higher concentration and Ca+ antagonist-like effectat a further higher concentration Contrary to the extractFlaxseed oil mediated antispasmodic effect predominatelythrough K+ channel opener (KCO) activity [6] Both PDEIand anticholinergic activity were evident in oil at a rela-tively higher concentration whereas CCB activity couldnot be observed However the possibility of some addi-tional mechanisms cannot be ruled out

With current findings Flaxseed as a whole mediatesantispasmodic action through multiple mechanisms in-cluding PDEI (subtype PDE-4) anticholinergic KCO andCa+2 antagonist-like activities All of these mechanisms areknown to have a protective role in IBD [10 11 13] and thusprovide an additional pharmacological basis for the effec-tiveness of Flaxseedrsquos protective effect in IBD

Besides therapeutic use of antispasmodics in IBD an-tibacterials also are used as an adjunct because reducing theaggressive microbial load has shown improvement in clinicaloutcomes in patients with IBD [38 39] (erefore in thecurrent study we targeted a few of the E coli strains im-plicated in IBD Flaxseed oil showed a bactericidal activityagainst all the three strains tested but FsCr was bactericidalagainst EPEC (is difference in efficacy and potency in-dicates that Flaxseed oil may have a different antibacterialmechanism or additional chemical constituent(s) with an-tibacterial activity

(e bactericidal effect of both FsCr and Flaxseed oilagainst EPEC is a very important finding because EPECrsquosinvasive level is more or less similar to AIEC strain (LF82)[13] Moreover EPEC has also been shown to sustainiodoacetamide-induced UC-like colitis in rats [40] HenceFlaxseed may reduce the invasive ability of Ecoli in IBDpatients and possibly its consequent manifestation thatincludes upregulation of NF-kβ [41] modulation of IL-1βIL-6 TNF-α COX-2 and apoptosis hence reducing in-flammatory cascades (is effect coupled with the earlier

Evidence-Based Complementary and Alternative Medicine 13

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Flaxseed oil

Num

ber o

f EPE

C cf

u

Original inoculumUntreated (EPEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(a)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Original inoculumUntreated EPEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

lowast

(b)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EPE

C cf

u

Flaxseed oil

Original inoculumUntreated (ETEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(c)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f ETE

C cf

u

Original inoculumUntreated ETEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(d)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Flaxseed oil

Original inoculumUntreated (EAEC)

9μgml (18μl)14μgml (28μl)

OI

Con

trol

lowastlowastlowast

(e)

10 times 102

10 times 103

10 times 104

10 times 105

10 times 106

10 times 107

Num

ber o

f EAE

C cf

u

Original inoculumUntreated EAEC

50μgml (10μl)100μgml (20μl)

FsCrOI

Con

trol

(f )

Figure 6 Bar charts show colony counts of sim106 EPEC (a d) ETEC (b e) and EAEC (c f ) incubated with and without Flaxseed oil (andashc)and Flaxseed extract (FsCr) (dndashf)

12 Evidence-Based Complementary and Alternative Medicine

reduction in neutrophil and lymphocytic infiltration wasslightly better with FsCr than Flaxseed oil FsCr was alsomore potent and efficacious in promoting mucin productionthan Flaxseed oil Despite these advancements the mortalityrate of Flaxseed oil was less as compared to FsCr Differencesin inflammatory and mucosal protective parameters in theextract and oil indicate that both the polar and nonpolarfractions of Flaxseed have the potential to mediate mucosalprotective effect in the IBDmodel of mice andmay provide asynergistic effect when given as a whole seed(is seems trueas Flaxseed and its oil showed clinical improvement in ul-cerative colitis patients in one of the latest reported studies[31] On the contrary our current findings are in contrast toan earlier report where 10 Flaxseed diet aggravated theseverity of dextran sodium sulfate- (DSS-) induced colitismodel of mice [32] One of the possibilities for this could bethat Flaxseed meal has a laxative effect [33] which is knownto aggravate colitis Nevertheless improvement in clinicalstudies in UC patients shows that this may not be the case orthis aspect may vary depending on the severity of the diseaseIn the clinical study patients who were not on corticoste-roids andor immune modulators were opted for whichmeans that the recruited UC patients have limited diseaseseverity More animal and clinical studies are needed toclarify this aspect

(e other part of our study aimed to identify themechanisms of Flaxseed in terms of its antispasmodic effectInterestingly not only did we find the antispasmodicmechanisms but we also got some lead for its anti-in-flammatory effect We found that both FsCr and Flaxseedoil caused complete inhibition of CCh-induced contractioncompared with other spasmogens which is indicative ofPDE inhibitory activity andor anticholinergic activity[26 34] Upon further experimentation it was evident thatFsCr not only exhibited a stronger PDEI but also increasedthe tissue cAMP levels Hence we further aimed to identifywhich subtype of PDEI receptor was inhibited It was foundthat FsCr mediated the similar inhibitory pathway for PDEenzyme as rolipram a standard PDE4 inhibitor (is wasidentified because FsCr and rolipram given simultaneouslycould not produce synergistic inhibitory effect against CCh-induced contraction whereas other PDEI when coad-ministered with FsCr potentiated the effect indicating thatperhaps rolipram and FsCr worked through the samesubtype of PDE enzyme (is interpretation is furthersupported by previous studies where the effect of one of thesubtypes of PDE inhibitors has been potentiated againstCCh-induced contraction when pretreated with the othersubtypes of PDE inhibitor [23 24 30] (is finding has avery important implication because blocking PDE-4 enzymehas been proposed as one of the novel approaches to targetIBD PDE-4 enzymes are present on inflammatory cells andtheir inhibition prevents mucosal damage and subsequentinflammatory cell infiltration by increasing cAMP [35] Ourfindings are also relatable because subsequently we were ableto show the increased level of cAMP in the FsCr-treatedtissues which is the end result of PDE-4 subtype inhibitionInterestingly in the HPLC fingerprints of FsCr (unpub-lished) one of the peaks matched quercetin a flavonoid that

has lately been identified as a potential PDE-4 inhibitor [36]and has been shown to ameliorate the severity of acetic acidcolitis in rats [37]

FsCr was the most potent against CCh-induced con-traction resembling papaverine It was however different inthat it showed some selectivity against high K+ likedicyclomine whereas papaverine showed similar inhibi-tion against both spontaneous and high K+-induced con-tractions (is raised the possibility of an additionalanticholinergic along with Ca+2 antagonist-like activitysimilar to dicyclomine component at a higher dose In-terestingly when tested for its anticholinergic activityFsCr at 03 and 1mgml concentrations caused therightward shift of the CRCs of Ach without suppression ofthe maximum response a classic pattern of a competitiveantagonist [27] such as dicyclomine Expectedly at thehigher concentration of 3mgml FsCr further shifted theCRCs of Ach to the right but suppressed the maximalresponse possibly due to the Ca+ channel antagonist-likemechanism which has also been reported earlier [7]Hence FsCr has been proved to exhibit multiple anti-spasmodic mechanisms ranging from PDEI-like effect at alower concentration followed by anticholinergic at a rel-atively higher concentration and Ca+ antagonist-like effectat a further higher concentration Contrary to the extractFlaxseed oil mediated antispasmodic effect predominatelythrough K+ channel opener (KCO) activity [6] Both PDEIand anticholinergic activity were evident in oil at a rela-tively higher concentration whereas CCB activity couldnot be observed However the possibility of some addi-tional mechanisms cannot be ruled out

With current findings Flaxseed as a whole mediatesantispasmodic action through multiple mechanisms in-cluding PDEI (subtype PDE-4) anticholinergic KCO andCa+2 antagonist-like activities All of these mechanisms areknown to have a protective role in IBD [10 11 13] and thusprovide an additional pharmacological basis for the effec-tiveness of Flaxseedrsquos protective effect in IBD

Besides therapeutic use of antispasmodics in IBD an-tibacterials also are used as an adjunct because reducing theaggressive microbial load has shown improvement in clinicaloutcomes in patients with IBD [38 39] (erefore in thecurrent study we targeted a few of the E coli strains im-plicated in IBD Flaxseed oil showed a bactericidal activityagainst all the three strains tested but FsCr was bactericidalagainst EPEC (is difference in efficacy and potency in-dicates that Flaxseed oil may have a different antibacterialmechanism or additional chemical constituent(s) with an-tibacterial activity

(e bactericidal effect of both FsCr and Flaxseed oilagainst EPEC is a very important finding because EPECrsquosinvasive level is more or less similar to AIEC strain (LF82)[13] Moreover EPEC has also been shown to sustainiodoacetamide-induced UC-like colitis in rats [40] HenceFlaxseed may reduce the invasive ability of Ecoli in IBDpatients and possibly its consequent manifestation thatincludes upregulation of NF-kβ [41] modulation of IL-1βIL-6 TNF-α COX-2 and apoptosis hence reducing in-flammatory cascades (is effect coupled with the earlier

Evidence-Based Complementary and Alternative Medicine 13

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

reduction in neutrophil and lymphocytic infiltration wasslightly better with FsCr than Flaxseed oil FsCr was alsomore potent and efficacious in promoting mucin productionthan Flaxseed oil Despite these advancements the mortalityrate of Flaxseed oil was less as compared to FsCr Differencesin inflammatory and mucosal protective parameters in theextract and oil indicate that both the polar and nonpolarfractions of Flaxseed have the potential to mediate mucosalprotective effect in the IBDmodel of mice andmay provide asynergistic effect when given as a whole seed(is seems trueas Flaxseed and its oil showed clinical improvement in ul-cerative colitis patients in one of the latest reported studies[31] On the contrary our current findings are in contrast toan earlier report where 10 Flaxseed diet aggravated theseverity of dextran sodium sulfate- (DSS-) induced colitismodel of mice [32] One of the possibilities for this could bethat Flaxseed meal has a laxative effect [33] which is knownto aggravate colitis Nevertheless improvement in clinicalstudies in UC patients shows that this may not be the case orthis aspect may vary depending on the severity of the diseaseIn the clinical study patients who were not on corticoste-roids andor immune modulators were opted for whichmeans that the recruited UC patients have limited diseaseseverity More animal and clinical studies are needed toclarify this aspect

(e other part of our study aimed to identify themechanisms of Flaxseed in terms of its antispasmodic effectInterestingly not only did we find the antispasmodicmechanisms but we also got some lead for its anti-in-flammatory effect We found that both FsCr and Flaxseedoil caused complete inhibition of CCh-induced contractioncompared with other spasmogens which is indicative ofPDE inhibitory activity andor anticholinergic activity[26 34] Upon further experimentation it was evident thatFsCr not only exhibited a stronger PDEI but also increasedthe tissue cAMP levels Hence we further aimed to identifywhich subtype of PDEI receptor was inhibited It was foundthat FsCr mediated the similar inhibitory pathway for PDEenzyme as rolipram a standard PDE4 inhibitor (is wasidentified because FsCr and rolipram given simultaneouslycould not produce synergistic inhibitory effect against CCh-induced contraction whereas other PDEI when coad-ministered with FsCr potentiated the effect indicating thatperhaps rolipram and FsCr worked through the samesubtype of PDE enzyme (is interpretation is furthersupported by previous studies where the effect of one of thesubtypes of PDE inhibitors has been potentiated againstCCh-induced contraction when pretreated with the othersubtypes of PDE inhibitor [23 24 30] (is finding has avery important implication because blocking PDE-4 enzymehas been proposed as one of the novel approaches to targetIBD PDE-4 enzymes are present on inflammatory cells andtheir inhibition prevents mucosal damage and subsequentinflammatory cell infiltration by increasing cAMP [35] Ourfindings are also relatable because subsequently we were ableto show the increased level of cAMP in the FsCr-treatedtissues which is the end result of PDE-4 subtype inhibitionInterestingly in the HPLC fingerprints of FsCr (unpub-lished) one of the peaks matched quercetin a flavonoid that

has lately been identified as a potential PDE-4 inhibitor [36]and has been shown to ameliorate the severity of acetic acidcolitis in rats [37]

FsCr was the most potent against CCh-induced con-traction resembling papaverine It was however different inthat it showed some selectivity against high K+ likedicyclomine whereas papaverine showed similar inhibi-tion against both spontaneous and high K+-induced con-tractions (is raised the possibility of an additionalanticholinergic along with Ca+2 antagonist-like activitysimilar to dicyclomine component at a higher dose In-terestingly when tested for its anticholinergic activityFsCr at 03 and 1mgml concentrations caused therightward shift of the CRCs of Ach without suppression ofthe maximum response a classic pattern of a competitiveantagonist [27] such as dicyclomine Expectedly at thehigher concentration of 3mgml FsCr further shifted theCRCs of Ach to the right but suppressed the maximalresponse possibly due to the Ca+ channel antagonist-likemechanism which has also been reported earlier [7]Hence FsCr has been proved to exhibit multiple anti-spasmodic mechanisms ranging from PDEI-like effect at alower concentration followed by anticholinergic at a rel-atively higher concentration and Ca+ antagonist-like effectat a further higher concentration Contrary to the extractFlaxseed oil mediated antispasmodic effect predominatelythrough K+ channel opener (KCO) activity [6] Both PDEIand anticholinergic activity were evident in oil at a rela-tively higher concentration whereas CCB activity couldnot be observed However the possibility of some addi-tional mechanisms cannot be ruled out

With current findings Flaxseed as a whole mediatesantispasmodic action through multiple mechanisms in-cluding PDEI (subtype PDE-4) anticholinergic KCO andCa+2 antagonist-like activities All of these mechanisms areknown to have a protective role in IBD [10 11 13] and thusprovide an additional pharmacological basis for the effec-tiveness of Flaxseedrsquos protective effect in IBD

Besides therapeutic use of antispasmodics in IBD an-tibacterials also are used as an adjunct because reducing theaggressive microbial load has shown improvement in clinicaloutcomes in patients with IBD [38 39] (erefore in thecurrent study we targeted a few of the E coli strains im-plicated in IBD Flaxseed oil showed a bactericidal activityagainst all the three strains tested but FsCr was bactericidalagainst EPEC (is difference in efficacy and potency in-dicates that Flaxseed oil may have a different antibacterialmechanism or additional chemical constituent(s) with an-tibacterial activity

(e bactericidal effect of both FsCr and Flaxseed oilagainst EPEC is a very important finding because EPECrsquosinvasive level is more or less similar to AIEC strain (LF82)[13] Moreover EPEC has also been shown to sustainiodoacetamide-induced UC-like colitis in rats [40] HenceFlaxseed may reduce the invasive ability of Ecoli in IBDpatients and possibly its consequent manifestation thatincludes upregulation of NF-kβ [41] modulation of IL-1βIL-6 TNF-α COX-2 and apoptosis hence reducing in-flammatory cascades (is effect coupled with the earlier

Evidence-Based Complementary and Alternative Medicine 13

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

reported antibacterial activity of FsCr against Salmonellatyphi [7] which is implicated in UC [39] and other Gram-positive and Gram-negative organisms [7 42] providesanother pharmacological basis for medicinal use of Flaxseedin IBD (is effectiveness of FsCr could be correlated withthe resemblance of HPLC peaks of FsCr with two syntheticcompounds that we have identified in one of our unpub-lished reports (ose compounds are ofloxacin and met-ronidazole both of which have broad-spectrum coverageagainst aerobic and anaerobic pathogens [43] However thepresence of some novel compounds is speculated based on100 kill of VRE which is resistant to conventional anti-biotics On the other hand Flaxseed oil used in the currentstudy exhibited a peak at later retention times (supple-mentary Figure 1) indicating the presence of polar con-stituents when analyzed through HPLC (is peak maycorrespond to alpha linolenic acid (ALA) as identified inprevious reports [44] that has also exhibited effectiveness inthe colitis model of mice [45] Future studies will be directedtoward the confirmation of these active constituents andtheir contribution to ameliorating effects in IBD

(is study is the first report on the effect of Flaxseed onbacteria implicated in IBD Earlier antibacterial effect ofFlaxseed oil has been shown against Ecoli [42] which is anonpathogenic strain and hence cannot be compared (ecurrent study shows that Flaxseed has bactericidal effectagainst pathogens implicated in IBD which may contributeto managing immune dysregulation from a different endthan cytokine modulation

5 Conclusion

Based on the current findings we understand that bothFlaxseed oil and extract are effective against IBD model ofmice with possibly different mucosal protective mecha-nisms Both the extract and the oil exhibit antispasmodicactivity with partly similar mechanisms FsCr has pre-dominant PDE inhibitor-like effect and weaker Ca++ an-tagonist effect whereas Flaxseed oil has predominant KCO-like effect and weak PDEI effect Both the extract and the oilare effective against microbes implicated in IBD with astronger antibacterial activity in Flaxseed oil (is coupledwith previously reported cytokine modulatory and antiox-idant effects of FsCr indicates that the whole seed mustencompass the potential of both the extract and the oil andtherefore shall give an augmented response in IBD HenceFlaxseed may become a choice of remedy that helps to caterthe basic pathophysiological tenets of IBD which involveimmune dysregulation and barrier defects as well as mi-crobial dysregulation

Abbreviations

PercentdegC Degree centigrademicrog MicrogramμM MicromolarμL MicroliterAA Acetic acid

Ach Acetylcholine chlorideCa+2 CalciumcAMP Cyclic adenosine monophosphateCCh CarbacholCD Crohnrsquos diseasecfu Colony forming unit(s)COX-2 Cyclooxygenase 2CRCs Concentration-response curvesDAI Disease activity indexDMSO Di methyl sulfoxideEAEC Enteroaggregative Escherichia coliECACU Ethics Committee for Animal Care and UseEPEC Enteropathogenic Escherichia coliETEC Enterotoxigenic Escherichia coliFsCr Aqueous-methanolic crude extract of Linum

usitatissimum seedsGm GramGI GastrointestinalH and E Hematoxylin and eosinK+ PotassiumKCO Potassium channel openerHr(s) Hour(s)IBD Inflammatory bowel diseaseIL-1β Interleukin-1 betaIL-6 Interleukin-6ip IntraperitoneallyIR IntrarectalKg KilogramLB Luria-Bertani brothMeOH MethanolicmL MillilitermM MillimolarNF-kβ Nuclear factor kappa-betaOD Optical densityOI Original inoculumPAS-AB

Periodic acid-Schiff-Alcian blue

PDE-4 Phosphodiesterase-4 subtypePDEI Phosphodiesterase inhibitorsSec SecondSEM Standard error of meanTNF-α Tumour necrosis factor-alphaUC Ulcerative colitisvv Volumevolumeww Weight by weight

Data Availability

(e data and materials supporting this study are availablefrom the corresponding author upon request

Conflicts of Interest

(e authors declare no conflicts of interest

Authorsrsquo Contributions

Amber Hanif Palla conceived the idea and executed theanimal-based work on IBD models and ex vivo tissue

14 Evidence-Based Complementary and Alternative Medicine

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

assembly set-up as well as in vitro assays Dr Najeeb-ur-Rehman along with Amber Hanif Palla supervised andconducted all the tissue assembly based experiments cAMPassays were designed and guided by Dr Samrah Bashir DrAnwar-ul Hassan Gilani supervised and intellectually con-cluded the whole project along with Amber Hanif Palla aswell as arranging for project funding All authors providedintellectual input in their respective domains they alsocritically reviewed the manuscript and modified it as pertheir expertise

Acknowledgments

(e authors would like to acknowledge Dr Kulsoom GhiasChair of the Department of Biological and BiomedicalSciences (BBS) and Dr Hassan Salman Siddiqi AssistantProfessor from the Department of BBS Aga Khan Uni-versity Hospital for their assistance in this project DrKhurram Minhas from Histopathology Department of AgaKhan University for assistance in conducting histopatho-logical analysis and Department of Pediatrics Aga KhanUniversity for providing bacterial strains (e authorswould also like to thank Miss Verdah Effendi and MissNoor Jehan Sarfaraz (anks are due to Dr Naveed AhmedKhan for teaching the authors this invaluable antibacterialtechnique and coordinating with the Department of Pe-diatrics for the arrangement of the bacterial strains (iswork was partially funded by HEC (Grant 106-2401-BM6-161)

Supplementary Materials

Supplementary Table 1 Multiple scoring criteria to assessmicroscopic damage in the colon of mice SupplementaryTable 2 Microscopic scoring of H and E stained and PAS-AB stained colon sections of mice after treatment withFsCr and Flaxseed oil Supplementary Table 3 Colonycount of EPEC ETEC and EAEC after treatment withFsCr and Flaxseed oil Supplementary Figure 1 HPLCfingerprint analysis of Flaxseed oil Supplementary Figure 2(A-B) Bar charts show effect on colony counts of sim106enteropathogenic E coli (EPEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 3 (A-B) Bar charts show effect on colony counts ofsim106 enterotoxigenic E coli (ETEC) incubated with andwithout FsCr for 16 hrs (A) and 2 hrs (B) Doses of 1 and25mg of FsCr were used for assay time of 16 hrs whereasdoses of 005 and 01mg (50 and 100 microgml) of FsCr wereused when assay time was 2 hrs (B) Supplementary Fig-ure 4 (A-B) Bar charts show the effect on colony counts ofsim106 enteroaggregative E coli (EAEC) incubated with andwithout Flaxseed extract (FsCr) for 16 hrs (A) and 2 hrs(B) Doses of 1 and 25mg of FsCr were used for assay timeof 16 hrs whereas doses of 005 and 01mg (50 and 100 microgml) of FsCr were used when assay time was 2 hrs (B)(Supplementary Materials)

References

[1] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152pp 427ndash434 2007

[2] R Rahimi M R Shams-Ardekani and M Abdollahi ldquoAreview of the efficacy of traditional Iranian medicine forinflammatory bowel diseaserdquo World Journal of Gastroenter-ology vol 16 no 36 p 4504 2010

[3] J A Duke M J Bogenschutz-Godwin M J du Cellier andP A Duke Catalog of Herbs (AaeuroldquoZ) Flax Handbook ofMedicinal Herbs CRC Press Boca Raton FL USA 2002

[4] A H Palla N T Iqbal K Minhas and A-H GilanildquoFlaxseed extract exhibits mucosal protective effect in aceticacid induced colitis in mice by modulating cytokines anti-oxidant and antiinflammatory mechanismsrdquo InternationalImmunopharmacology vol 38 pp 153ndash166 2016

[5] M-C Lin M-J Tsai and K-C Wen ldquoSupercritical fluidextraction of flavonoids from scutellariae radixrdquo Journal ofChromatography A vol 830 no 2 pp 387ndash395 1999

[6] A H Palla and A-H Gilani ldquoDual effectiveness of flaxseed inconstipation and diarrhea possible mechanismrdquo Journal ofEthnopharmacology vol 169 pp 60ndash68 2015

[7] A H Palla N A Khan S Bashir N ur-Rehman J Iqbal andA-H Gilani ldquoPharmacological basis for the medicinal use ofLinum usitatissimum (Flaxseed) in infectious and non-in-fectious diarrheardquo Journal of Ethnopharmacology vol 160pp 61ndash68 2015

[8] A J Farre M Colombo M Fort and B Gutierrez ldquoDif-ferential effects of various Ca2+ antagonistsrdquo General Phar-macology e Vascular System vol 22 no 1 pp 177ndash1811991

[9] A H Gilani N Aziz I M Khurram Z A Rao and N K Alildquo(e presence of cholinomimetic and calcium channel an-tagonist constituents in Piper betle Linnrdquo Phytotherapy Re-search vol 14 no 6 pp 436ndash442 2000

[10] R AwadM Dibildox and F Ortiz ldquoIrritable bowel syndrometreatment using pinaverium bromide as a calcium channelblocker A randomized double-blind placebo-controlledtrialrdquo Acta Gastroenterologica Latinoamericana vol 25 no 3pp 137ndash144 1995

[11] A Hosseini-Tabatabaei and M Abdollahi ldquoPotassiumchannel openers and improvement of toxic stress do theyhave role in the management of inflammatory bowel diseaserdquoInflammation amp Allergy-Drug Targets vol 7 no 3 pp 129ndash135 2008

[12] M Rescigno ldquo(e pathogenic role of intestinal flora in IBDand colon cancerrdquo Current Drug Targets vol 9 no 5pp 395ndash403 2008

[13] A da Silva Santos F Gomes Romeiro L Yukie Sassaki andJ Rodrigues ldquoEscherichia coli from Crohnrsquos disease patientdisplays virulence features of enteroinvasive (EIEC) enter-ohemorragic (EHEC) and enteroaggregative (EAEC) path-otypesrdquo Gut Pathogens vol 7 no 1 p 2 2015

[14] G Kaithwas and D K Majumdar ldquo(erapeutic effect ofLinum usitatissimum (flaxseedlinseed) fixed oil on acute andchronic arthritic models in albino ratsrdquo Inflammopharma-cology vol 18 no 3 pp 127ndash136 2010

[15] N R Council Guide for the Care and Use of LaboratoryAnimals National Academies Press Washington DC USA2010

[16] W C Evans Trease and Evans Pharmacognosy ElsevierHealth Sciences Amsterdam Netherlands 2009

Evidence-Based Complementary and Alternative Medicine 15

[17] B R MacPherson and C J Pfeiffer ldquoExperimental productionof diffuse colitis in ratsrdquo Digestion vol 17 no 2 pp 135ndash1501978

[18] S Wirtz C Neufert B Weigmann and M NeurathldquoChemically induced mouse models of intestinal inflamma-tionrdquo Nature Protocols vol 2 no 3 pp 541ndash546 2005

[19] M F Neurath I Fuss B L Kelsall E Stuber andW StroberldquoAntibodies to interleukin 12 abrogate established experi-mental colitis in micerdquoe Journal of Experimental Medicinevol 182 no 5 pp 1281ndash1290 1995

[20] J Vilaseca A Salas F Guarner R Rodriguez M Martinezand J R Malagelada ldquoDietary fish oil reduces progression ofchronic inflammatory lesions in a rat model of granulomatouscolitisrdquo Gut vol 31 no 5 pp 539ndash544 1990

[21] A Dorofeyev I Vasilenko O Rassokhina and R KondratiukldquoMucosal barrier in ulcerative colitis and Crohnrsquos diseaserdquoGastroenterology Research and Practice vol 2013 Article ID431231 9 pages 2013

[22] A H Gilani A-U Khan T Ali and S Ajmal ldquoMechanismsunderlying the antispasmodic and bronchodilatory propertiesof Terminalia bellerica fruitrdquo Journal of Ethnopharmacologyvol 116 no 3 pp 528ndash538 2008

[23] Y Sato T Akao J-X He et al ldquoGlycycoumarin from Gly-cyrrhizae Radix acts as a potent antispasmodic through inhi-bition of phosphodiesterase 3rdquo Journal of Ethnopharmacologyvol 105 no 3 pp 409ndash414 2006

[24] Y Sato J-X He H Nagai T Tani and T Akao ldquoIso-liquiritigenin one of the antispasmodic principles of Gly-cyrrhiza ularensis roots acts in the lower part of intestinerdquoBiological amp Pharmaceutical Bulletin vol 30 no 1 pp 145ndash149 2007

[25] J Van Rossum ldquoCumulative dose-response curves IITechnique for the making of dose-response curves in isolatedorgans and the evaluation of drug parametersrdquo Archivesinternationales de pharmacodynamie et de therapie vol 143p 299 1963

[26] T Kaneda Y Takeuchi H Matsui K Shimizu N Urakawaand S Nakajyo ldquoInhibitory mechanism of papaverine oncarbachol-induced contraction in bovine tracheardquo Journal ofPharmacological Sciences vol 98 no 3 pp 275ndash282 2005

[27] O Arunlakshana and H O Schild ldquoSome quantitative uses ofdrug antagonistsrdquo British Journal of Pharmacology andChemotherapy vol 14 no 1 pp 48ndash58 1959

[28] M H Mehmood N Aziz M N Ghayur and A-H GilanildquoPharmacological basis for the medicinal use of psyllium husk(Ispaghula) in constipation and diarrheardquo Digestive Diseasesand Sciences vol 56 no 5 pp 1460ndash1471 2011

[29] N A Khan K Osman and G J Goldsworthy ldquoLysates ofLocusta migratoria brain exhibit potent broad-spectrum an-tibacterial activityrdquo Journal of Antimicrobial Chemotherapyvol 62 no 3 pp 634-635 2008

[30] A L Harris M J Connell E W Ferguson et al ldquoRole of lowKm cyclic AMP phosphodiesterase inhibition in trachealrelaxation and bronchodilation in the Guinea pigrdquo eJournal of Pharmacology and Experimental erapeuticsvol 251 no 1 pp 199ndash206 1989

[31] N Morshedzadeh S Shahrokh H A Aghdaei et al ldquoEffectsof flaxseed and flaxseed oil supplement on serum levels ofinflammatory markers metabolic parameters and severity ofdisease in patients with ulcerative colitisrdquo Complementaryerapies in Medicine vol 46 pp 36ndash43 2019

[32] L Zarepoor J T Lu C Zhang et al ldquoDietary flaxseed intakeexacerbates acute colonic mucosal injury and inflammationinduced by dextran sodium sulfaterdquo American Journal of

Physiology-Gastrointestinal and Liver Physiology vol 306no 12 pp G1042ndashG1055 2014

[33] J Xu X Zhou C Chen et al ldquoLaxative effects of partiallydefatted flaxseed meal on normal and experimental consti-pated micerdquo BMC Complementary and Alternative Medicinevol 12 no 1 p 14 2012

[34] V Boswell-Smith D Spina and C P Page ldquoPhosphodies-terase inhibitorsrdquo British Journal of Pharmacology vol 147no 1 pp S252ndashS7 2006

[35] M Tsujij S Kawano S Tsuji et al ldquoColonic mucosal he-modynamics and tissue oxygenation in patients with ulcer-ative colitis investigation by organ reflectancespectrophotometryrdquo Journal of Gastroenterology vol 30no 2 pp 183ndash188 1995

[36] A L-F Chan H-L Huang H-C Chien C-M ChenC-N Lin and W-C Ko ldquoInhibitory effects of quercetinderivatives on phosphodiesterase isozymes and high-affinity[3 H]-rolipram binding in Guinea pig tissuesrdquo InvestigationalNew Drugs vol 26 no 5 pp 417ndash424 2008

[37] D Dodda R Chhajed and J Mishra ldquoProtective effect ofquercetin against acetic acid induced inflammatory boweldisease (IBD) like symptoms in rats possible morphologicaland biochemical alterationsrdquo Pharmacological Reportsvol 66 no 1 pp 169ndash173 2014

[38] R Rahimi S Nikfar and M Abdollahi ldquoMeta-analysistechnique confirms the effectiveness of anti-TNF-alpha in themanagement of active ulcerative colitis when administered incombination with corticosteroidsrdquo Medical Science MonitorInternational Medical Journal of Experimental and ClinicalResearch vol 13 no 7 2007

[39] M Campieri and P Gionchetti ldquoBacteria as the cause ofulcerative colitisrdquo Gut vol 48 no 1 pp 132ndash135 2001

[40] I H H Abdallah J Freund J Reimund et al ldquoEntero-pathogenic e coli sustains iodoacetamide-induced ulcerativecolitis-like colitis in rats modulation of IL-1β IL-6 TNF-αCOX-2 and apoptosisirdquo Journal of Biological Regulators andHomeostatic Agents vol 26 no 3 pp 515ndash526 2012

[41] G A Hecht ldquoEarly enterocyte responses to enteropathogenicE colirdquo Journal of Pediatric Gastroenterology and Nutritionvol 40 no 1 p S32 2005

[42] G Kaithwas A Mukerjee P Kumar and D K MajumdarldquoLinum usitatissimum (linseedflaxseed) fixed oil antimi-crobial activity and efficacy in bovine mastitisrdquo Inflammo-pharmacology vol 19 no 1 pp 45ndash52 2011

[43] C D Freeman N E Klutman and K C Lamp ldquoMetroni-dazolerdquo Drugs vol 54 no 5 pp 679ndash708 1997

[44] S K Gebauer T L Psota W S Harris and P M Kris-Etherton ldquonminus3 Fatty acid dietary recommendations and foodsources to achieve essentiality and cardiovascular benefitsrdquoe American Journal of Clinical Nutrition vol 83 no 6pp 1526ndash1535 2006

[45] R Reifen A Karlinsky A H Stark Z Berkovich andA Nyska ldquoα-Linolenic acid (ALA) is an anti-inflammatoryagent in inflammatory bowel diseaserdquo e Journal of Nutri-tional Biochemistry vol 26 no 12 pp 1632ndash1640 2015

16 Evidence-Based Complementary and Alternative Medicine