Journal of Ethnopharmacology 160 (2015) 61–68

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Research paper

Pharmacological basis for the medicinal use of Linum usitatissimum (Flaxseed) in infectious and non-infectious diarrhea Amber Hanif Palla a, Naveed Ahmed Khan a, Samra Bashir a, Najeeb ur-Rehman a,b, Junaid Iqbal a, Anwarul-Hassan Gilani a,b,n a b

Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi 74800, Pakistan Department of Pharmacy, College of Health Sciences, Mekelle University, PO Box 1871, Mekelle, Ethiopia

art ic l e i nf o

a b s t r a c t

Article history: Received 8 July 2014 Received in revised form 8 October 2014 Accepted 19 November 2014 Available online 26 November 2014

Ethnopharmacological relevance: Linum usitatissimum, commonly known as Flaxseed has traditionally been used for the management of diarrhea and gastrointestinal infections. This study was planned to assess pharmacological basis for the medicinal use of Flaxseed in infectious and non-infectious diarrhea. Materials and methods: The crude aqueous-methanolic extract of Flaxseed was studied using the in vivo castor oil-induced diarrhea, gut motility and enteropooling assays. Mechanistic basis was further elucidated by testing the inhibitory effect on spontaneously contracting isolated rabbit jejunum preparations, suspended in a 10 ml tissue bath containing Tyrode' solution, maintained at 37 1C and aerated with carbogen. Antibacterial efficacy of the Flaxseed extract was tested against different enteric and non-enteric pathogenic bacteria using in vitro antibacterial assays. Results: Flaxseed extract reduced the diarrheal score in mice, by 39%, 63.90% and 68.34% at the respective doses of 100, 300 and 500 mg/kg. Intestinal secretions were reduced by 24.12%, 28.09% and 38.80%, whereas the intestinal motility was reduced by 31.66%, 46.98% and 56.20% at respective doses of 100, 300 and 500 mg/kg. When tested on isolated rabbit jejunum preparations, Flaxseed extract produced a dosedependent inhibition of both spontaneous and high K þ (80 mM)-induced contractions, and shifted the concentration–response curves of Ca þ þ to the right with suppression of the maximum response, similar to that caused by verapamil. Flaxseed extract was found to possess bactericidal activity at the tested concentrations of 12.5 mg/ml, against vancomycin-resistant Enterococcus faecalis (100%), Escherichia coli K1 (88.88%), methicillin-resistant Staphylococcus aureus (98.76%), Bacillus cereus (92.64%), Pseudomonas aeruginosa (76.83%) and Salmonella typhi (26.91 73.35%). The concentration of 10 mg/ml showed bactericidal effects against all the aforementioned pathogens except Escherichia coli K1, whereas for Pseudomonas aeruginosa and Salmonella typhi, it was bacteriostatic at this concentration. Conclusions: Our results indicate that Linum usitatissimum (Flaxseed) extract exhibits antidiarrheal and antispasmodic activities by virtue of its antimotility and antisecretory effects which are mediated possibly through inhibition of Ca þ þ channels, though additional mechanism(s) cannot be ruled out. Flaxseed extract proved effective against both enteric and non-enteric pathogens causing diarrhea, thus ensuring wide coverage and rationalizing its medicinal use in both the infectious and non-infectious diarrhea. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Antidiarrheal Antispasmodic Flaxseed Ca þ þ antagonist Bactericidal Antisecretory

1. Introduction Diarrhea is characterized by an increased frequency of loose or liquid stools often accompanied with abdominal cramps. Diarrheal diseases affect 3–5 billion people causing approximately 5 million n Corresponding author at: Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi 74800, Pakistan. Tel.: þ 92 21 34864571. E-mail address: [email protected] (A.-H. Gilani).

http://dx.doi.org/10.1016/j.jep.2014.11.030 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

deaths annually. Pakistan stands 5th amongst the countries where there are reports of under-5 deaths due to diarrheal diseases (Boschi-Pinto et al., 2008). Diarrhea can either be a manifestation of any chronic disease or infection (WHO, 2013). In case of chronic diarrheal diseases, antidiarrheal management is needed for longer periods which could be for months or in some cases even lifetime. This management includes antimotility and/or bulk-forming agents that are associated with side effects such as distention, bloating, nausea, vomiting and constipation, particularly in long term use (Grahame-Smith and Aronson, 2002).

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Infectious diarrhea could be due to bacterial, viral and parasitic invasion. In bacterial diarrheal diseases, Escherichia coli is the most common cause in developing countries (Boschi-Pinto et al., 2008). Bacillus cereus, Campylobacter jejunii, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexinerii and Vibrio cholerae are some other enteric pathogens also implicated in diarrhea. Besides these, hospital acquired drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus faecalis (VRE) and antibiotic associated Clostridium difficile are also implicated in diarrhea, which are resistant to multiple antibiotics (Bartlett, 2006; Aman and Carl, 2008; Thakkar and Agrawal, 2010). Though in past, antibiotics have been used effectively for the management of bacterial diarrheas (Farthing et al., 2013), but because of the emergence of antibiotic resistance among different pathogenic bacteria, currently very few antibiotics are effective against different enteric bacterial pathogens (Smith and Coast, 2002). Based on this emerging resistance against antibiotics, it has been predicted that we will be pushed into post-antibiotic era very soon, where once again, like pre-antibiotic era, the common bacterial infections would lead into deadly outcomes (Alanis, 2005). Consistent with this, recently South Asian countries have been highlighted because of the discovery of carbapenem-resistance gene in enteric bacteria such as Escherichia coli and Klebsiella pneumoniae (Trivedi and Sabnis, 2009; Kumarasamy et al., 2010; Oberoi et al., 2013). Hence, there is a need for an alternative approach that could reduce this emerging resistance pattern but at the same time caters the etiology. Hence, a logical approach to avoid either long term side-effects of antidiarrheals in case of chronic diarrheas and to combat emerging resistanc(e pattern in case of infectious diarrhea is to revert towards nature. Natural products are known to contain multiple chemicals acting at multiple targets, to offer “effect enhancing and/or side-effect neutralizing” combinations (Gilani and Atta-ur-Rahman, 2005) and may also help to cope with challenge of resistance (Elfawal et al., 2012). Amongst the natural products, Flaxseed (Linum usitatissimum; Family: Linaceae), locally known as “Alsi”, has traditionally been used as a remedy for diarrhea (Gruenwald et al., 2000; Duke et al., 2002). It is making its mark as a functional food and is one of the richest plant sources of essential fatty acids. It is known to contain oil (40–50%) and meal, comprising of protein (23–34%), ash (4%), viscous fiber/mucilage (5%), and lignan precursors (9–30 mg/g of defatted meal) (Tarpila et al., 2005), in addition to other medicinally active constituents. To date, no study has been carried out to identify the pharmacological basis for the medicinal use of Flaxseed in infectious and non-infectious diarrhea. Earlier, a preliminary report on Flaxseed oil elaborated the mechanism of anti-inflammatory activity by employing castor oil-induced diarrhea as an indicator of prostaglandin mechanism (Kaithwas and Majumdar, 2010a). In another study, based on some preliminary in vitro experiments, the same group reported that the antiulcer and antisecretory potential of Flaxseed oil is due to anticholinergic or antihistaminergic activities respectively (Kaithwas and Majumdar, 2010b). Hence, present study on the crude extract of Flaxseed was undertaken to rationalize its medicinal use in diarrhea and to explore mechanistic basis for this effect by using the in vivo, ex vivo as well as in vitro assays. The study also assessed the antibacterial activity of Flaxseed extract on some of the enteric bacterial pathogens.

antibacterial assays were also purchased from the Sigma chemicals. Chemicals used for making physiological salt solutions including potassium chloride, calcium chloride, glucose, magnesium chloride, magnesium sulfate, sodium bicarbonate, sodium dihydrogen phosphate, EGTA and sodium chloride were obtained from Merck, Darmstadt, Germany. DMSO and Tween-80 used for solubilization were purchased from Merck, Darmstadt, Germany and Scharlau chemicals, Barcelona, Spain, respectively. Acacia powder, hydrolyzed starch, and vegetable charcoal used for charcoal meal transit were purchased from BDH Laboratory Supplies, Poole, England. All chemicals used were of the available analytical grade. 2.2. Animals BALB-c mice (20–25 g) and rabbits (1–1.5 kg) of local breed, of either sex, were used for this study. Animals were housed at the animal house of The Aga Khan University, maintained at 23–25 1C. They were kept in plastic cages (47  34  18 cm3) with sawdust (changed at every 48 h) and fasted for 24 h before the experiment, whereas they were given tap water and standard diet routinely. Animals had free access to water but food was withdrawn 24 h prior to experiments. Rabbits starved for 24 h were sacrificed by cervical dislocation and mice were also sacrificed by cervical dislocation, as routinely done in our laboratory. The study protocol (005-Ani-BBS-13) was also approved by ECACU (Ethics Committee for Animal Care and Use) of The Aga Khan University. 2.3. Bacterial isolates Bacterial strains used in this study include methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli 018:K1:H7, strain RS218 (Escherichia coli K1), Salmonella typhi (ATCC 14028), Bacillus cereus, vancomycin-resistant Enterococcus faecalis (VRE), and Pseudomonas aeruginosa. All bacteria are clinical isolates available upon request. Bacterial strains were routinely grown aerobically at 37 1C in Luria-Bertani broth (LB) for 16 h. 2.4. Preparation of the crude extract Flaxseed was purchased from an authentic herb supplier in the local market of Karachi, Pakistan. A sample of the plant material was deposited to the herbarium of the Natural Products Research Unit at the Department of Biological and Biomedical Sciences of The Aga Khan University, with a voucher number LU-SE-0812-106. The seeds were made free of dirt and other adulterants and were ground to coarse powder by an electrically driven mill. Approximately 1 kg ground seeds were soaked in the aqueous-methanol (30:70 v/v) at room temperature for 3 days with occasional shaking (Ghayur and Gilani, 2005; Rehman et al., 2012). It was filtered through a double layered muslin cloth and subsequently through a filter paper. The residue was re-soaked in the fresh solvent and the process was repeated twice to get maximum yield of crude extract from the plant material. The combined filtrate was concentrated in a rotary evaporator at 40 1C under reduced pressure ( 760 mmHg) to a thick, semi-solid mass, labeled as the crude extract of Flaxseed, with approximated yield of 8% and was stored at  20 1C until used. 2.5. Working stocks

2. Materials and methods 2.1. Standard drugs The reference drugs, acetylcholine chloride, potassium chloride verapamil hydrochloride, carbamyl choline chloride and loperamide were purchased from the Sigma Chemicals Co, St. Louis, MO, USA. Nutrient agar and Luria-Bertani broth powders used for

For the in vivo and ex vivo experiments, on the day of experiment, the extract was solubilized in 10% DMSO–5% Tween-80 and subsequent dilutions were made in distilled water. For the in vitro antibacterial experiments, 1 ml of Flaxseed extract ( 2.08 g) was solubilized in 50% methanol and then centrifuged at 14,000g for 60 min. The supernatant was collected and microfiltered whereas the remaining pellet was weighed and subtracted from the amount

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of extract taken before centrifuge (2.08 g). The final concentration of Flaxseed extract was 180 μg/μL.

2.6. In vivo experiments 2.6.1. Castor oil-induced diarrhea Antidiarrheal effect of Flaxseed extract was tested on castor oilinduced diarrhea in mice by slight modification of the method previously used (Wang et al., 2006; Bashir et al., 2011). Mice fasted for 24 h before the experiment, were divided into 6 groups of 5 mice each, housed in individual cages. All the drugs were administered to the mice orally using feeding needles. The first group labeled as negative control received saline in vehicle (10 ml/kg), whereas the second group (diseased) also received control solvent to be later followed by castor oil treatment. The third group (positive control) received loperamide (10 mg/kg), whereas, the fourth, fifth and sixth (treatment) groups received increasing concentrations of 100, 300 and 500 mg/kg of Flaxseed extract respectively. 1 h after the treatment, each animal, except those in negative control group, received 10 ml/kg of castor oil. After 4 h, the cages were inspected for the frequency of the typical diarrheal droppings as well as the consistency of stool which was scored according to Wirtz et al. (2005) as follows: score 0: no diarrhea; score 1: soft but formed stool; score 2: very soft stool; score 3: diarrhea. For each group, total score was calculated as sum of individual scores. Individual score was calculated by multiplying the respective score with the number of diarrheal droppings. Percent reduction i.e. how much the tested treatments reduced the control (castor oil-induced) diarrheal score was calculated by using equation as follows:

Percent reduction ¼

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on marker meal transit. BALB-c mice fasted for 18 h were classified into groups in the similar way as previously described in enteropooling assay and dosed orally (except positive control) through a feeding needle. 1 h after the administration of vehicle, extract, and control drug loperamide (10 mg/kg, i.p.), 10 ml/kg of castor oil was administered orally to all groups except the negative control group. 1 h later, 0.3 ml charcoal meal (10% activated charcoal suspension in 5% gum acacia and 5% starch) was administered orally as a marker meal. 30 min later each animal was sacrificed and the peristaltic index was measured by comparing the distance traveled by the charcoal meal from pylorus to the cecum of each animal to the total length of the small intestine of the respective animal and expressed as percentage (Teke et al., 2007) as follows: PI ¼ LM=LSI  100 where PI¼peristaltic index; LM¼length of charcoal meal; LSI¼length of small intestine. The percent inhibition relative to the control was also calculated as follows: Percent inhibition ¼ ½ðcontrol–testÞ=control  100 2.6.4. Acute toxicity study A total of 20 mice (20–25 g) of either sex were equally divided into four groups. The test was performed using increasing doses of the crude extract of the Flaxseed (300, 500 and 1000 mg/kg) given orally, in 10 ml/kg control vehicle to different animals serving as the test groups. Another group of mice was administered control vehicle (10 ml/kg) orally as the negative control. The animals were allowed food and water ad libitum and kept under regular observation for 6 h to observe their piloerection, changes in exploratory behavior and blindness, while lethality was monitored up to 24 h.

½Mean castor oil diarrheal score ðcontrolÞ–mean score of treatment group  100 mean castor oil diarrheal score ðcontrolÞ

2.6.2. Enteropooling assay To study the mechanism of antidiarrheal activity, intestinal fluid accumulation also called as enteropooling assay was used, that would tell whether the extract produced its anti-diarrheal effect by reducing secretions or not (Capasso et al., 1994, 2002; Gilani et al., 2005a). BALB-c mice fasted for 18 h were divided into 6 groups and dosed orally (except positive control) through a feeding needle. The first group labeled as negative control group received saline (10 ml/kg) dissolved in vehicle, whereas the second group (diseased) also received control solvent to be later followed by castor oil treatment. The third group received the control drug atropine (10 mg/kg, intraperitoneally), as described by Mehmood et al. (2011). The fourth, fifth and sixth (treatment) groups received 100, 300 and 500 mg/kg of the Flaxseed extract orally. 1 h after the control and test material administration, each animal received 10 ml/kg of castor oil except the negative control group, to induce secretions. 30 min later, the mice were sacrificed by cervical dislocation. Subsequently, the entire intestine was removed and weighed with care making sure there was no leakage of fluid. The results are expressed as follows: P i =P m  1000 where Pi is the weight of the intestine and Pm is the weight of the animal in grams. 2.6.3. Castor oil-induced gut motility To further elaborate whether Flaxseed extract has antidiarrheal activity by its effect on gut motility, the method of Aye-Than et al. (1989) and modified by Bakare et al. (2011) was followed in which motility was induced by using castor oil and then effect was checked

2.7. Ex vivo experiments 2.7.1. Isolated rabbit jejunum Experiments on isolated rabbit jejunal preparations were carried out as described previously (Gilani and Coblin, 1987; Jabeen et al., 2009). Briefly, intestinal segments of 2 cm length were suspended in a 10 ml tissue bath containing Tyrode's solution, aerated with carbogen and maintained at 37 1C. Composition of Tyrode's solution (mM) was KCl (2.68), NaCl (136.9), MgCl2 (1.05), NaHCO3 (11.90), NaH2PO4 (0.42), CaCl2 (1.8), and glucose (5.55). A preload of 1 g was applied to each tissue segment and spontaneous contractions were recorded by using isotonic transducers (50-6360, Harvard Apparatus, Holliston, MA, USA) coupled with Power Lab data acquisition system (model ML-845, AD Instruments, Sydney, Australia) coupled with computer using chart software (version 5.3). Each tissue was allowed to equilibrate for at least 30 min before the addition of any drug. The rabbit jejunum exhibits spontaneous rhythmic contractions under these experimental conditions, allowing testing the relaxant (spasmolytic) activity without the use of an agonist (Gilani et al., 2000). To assess the mechanism behind the spasmolytic activity, high K þ (80 mM) was used to depolarize the preparations as described by Farre et al. (1991). This method allows determining whether the spasmolytic activity of the test substance is through Ca þ þ channel blockade or some other mechanism(s). Contraction of smooth muscle induced by high K þ (430 mM) is known to be mediated via influx of Ca þ þ from extracellular fluid and the substance which inhibits this contraction is considered to act through blockade of Ca þ þ channels (Bolton, 1979).

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Hence, high K þ (80 mM) was added to the tissue bath and once the sustained contraction achieved, test material was then added in a cumulative fashion to obtain concentration-dependent inhibitory responses (Van Rossum, 1963). The relaxation of intestinal preparations, pre-contracted with high K þ (80 mM), was expressed as a percent of the control response mediated by K þ . To further confirm the Ca þ þ antagonist activity of the test substance, the tissue was allowed to stabilize in normal Tyrode's solution, which was then replaced with Ca þ þ free Tyrode's solution containing EGTA (0.1 mM) for 30 min in order to remove Ca þ þ from the tissues. This solution was further replaced with K þ -rich and Ca þ þ free Tyrode's solution, having the following composition (mM): KCl (50), NaCl (91.04), MgCl2 (1.05), NaHCO3 (11.90), NaH2PO4 (0.42), glucose (5.55), and EGTA (0.1). Following an incubation period of 30 min, control concentration–response curves (CRCs) of Ca þ þ were obtained. When the control CRCs of Ca þ þ were found super-imposable (usually after two cycles), the tissue was pretreated with the test material for 60 min to test the possible Ca þ þ channel blocking effect. The CRCs of Ca þ þ were reconstructed in the presence of different concentrations of Flaxseed extract. The same procedure was repeated for verapamil, a standard Ca þ þ channel blocker (Fleckenstein, 1977).

since it decreased the diarrheal score by 39% (score: 21.672.85), 63.9% (score: 1372.13) and 68.34% (score: 11.371.35) at 100, 300 and 500 mg/kg doses, respectively compared to castor oil. This effect was dose-dependent at 100 mg/kg and 300 mg/kg (po0.01 and po0.001 respectively). The next higher dose of 500 mg/kg did not show further change in the anti-diarrheal effect (Fig. 1).

3.1.2. Effect on enteropooling Castor oil caused significant increase in intestinal secretions from 77.3571.52 g to 14975.90 g. Flaxseed extract reduced the castor oil-induced intestinal secretions in mice by 24.12% (11375.12 g), 28.09% (10772.49 g) and 38.8% (9173.06 g) at respective doses of 100, 300 and 500 mg/kg. The doses of 100 and 300 mg/kg produced a dose-dependent inhibitory effect (po0.01 and po0.001, respectively), whereas the next higher dose (500 mg/kg) did not cause further increase in the effect (Fig. 2).

2.8. In vitro experiments 2.8.1. Bactericidal activity of Flaxseed extract Antibacterial assays were performed as previously described (Khan et al., 2008). Briefly, 10 ml of inoculum (od¼0.22) corresponding to approximately 106 colony forming units (c.f.u.) suspended with various concentrations of Flaxseed extract (1.0 and 2.5 mg in 200 ml assay volume; 5 and 12.5 mg/ml) was incubated aerobically at 37 1C for 16 h. After this incubation, bacterial cultures were 10-fold serially diluted and plated on nutrient agar plates and incubated further at 37 1C overnight. Bacteria incubated with LB alone served as negative control. For 100% kill, bacteria were incubated with appropriate antibiotics comprising of 100 mg/ml gentamycin for Bacillus cereus, Escherichia coli K1, Pseudomonas aeruginosa, and Salmonella typhi and 100 mg/ml vancomycin for MRSA. Next day, colonies were counted. Data were presented in terms of c.f.u. compared to inoculated bacterial growth. The percentage bactericidal effects were determined as follows: 100  ½ðc:f:u: in extract=original inoculumÞ  100:

Fig. 1. Antidiarrheal effect of increasing doses of crude extract of Flaxseed along with loperamide (10 mg/kg), given orally as a positive control, on castor oil-induced diarrhea in mice. Score 0: no diarrhea, score 1: soft but formed stool, score 2: very soft stool and score 3: very lose stool. Each experiment was repeated thrice. Results shown are mean 7 SEM of 5 animals for each experimental group. n, nn, and nnn indicate p o 0.05, p o 0.01 and p o0.001 vs. castor oil respectively. Fs.Cr: Flaxseed extract; vehicle: 5% DMSO–10% Tween-80 and distilled water.

2.9. Statistical analysis All the data expressed are mean7standard error of the mean (S.E.M). The statistical parameter applied in the castor oil-induced diarrhea, antisecretory and gut motility assay was One-way analysis of variance (ANOVA) followed by Tukey's post-test and/or unpaired t-test (two-tailed), while in case of antibacterial analysis, paired or unpaired t-test (two-tailed) was used. Concentration response curves were analyzed by nonlinear-regression using Graph-Pad program (Graph-Pad, San Diego, California, USA). All other graphing, calculations and statistical analysis were also carried out by using Graph-PAD software (Graph-Pad, San Diego, California, USA).

3. Results 3.1. In vivo activities 3.1.1. Effect on castor oil-induced diarrhea After administration of castor oil, the mean diarrheal score of castor oil group was 35.770.81 as compared to 0 score in vehicle control group. Flaxseed extract showed antidiarrheal activity in mice

Fig. 2. Antisecretory effect of increasing doses of crude extract of Flaxseed along with atropine (10 mg/kg, i.p.), as a positive control, on castor oil-stimulated fluid accumulation in small intestine of mice. Intestinal fluid accumulation is expressed as (Pi/Pm)  1000 where Pi is the weight of the small intestine and Pm is the weight of the mouse. Results shown are mean7 SEM of three experiments with 5 animals in each experimental group. n, nn, and nn indicate p o0.05, po 0.01 and po 0.001 respectively vs. control vehicle. Fs.Cr: Flaxseed extract; vehicle: 5% DMSO–10% Tween-80 and distilled water.

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3.1.3. Effect on gut motility (charcoal meal transit) Castor oil increased the intestinal motility as represented by the charcoal meal transit from 45.6871.96 cm to 68.4373.95 cm (po0.01). Flaxseed extract reduced the intestinal motility in mice by 31.66% (46.7673.46 cm; po0.01), 46.98% (36.2873.73 cm; po0.001) and 56.2% (29.9774.30 cm; po0.001) at the respective doses of 100, 300 and 500 mg/kg. The efficacy was dose-dependent up to 300 mg/kg, whereas no further change was observed at next higher dose of 500 mg/kg (Fig. 3a).

verapamil (Fig. 4b). Further, CRCs of Ca þ þ were constructed in the absence and presence of test material for confirmation whether or not the inhibitory response was mediated through Ca þ þ channel blockade. The results showed that pre-treatment of the tissue with 0.1 mg/ml and 0.3 mg/ml shifted the Ca þ þ CRCs to the right with suppression of maximal response (Fig. 4c) in a dose-dependent fashion similar to that caused by verapamil (Fig. 4d).

3.2. Ex vivo activities

3.3.1. Bactericidal effects To determine the bactericidal activity of Flaxseed extract against Escherichia coli K1, MRSA, VRE, Bacillus cereus, Pseudomonas aeruginosa, and Salmonella typhi, 106 c.f.u. were incubated with different concentrations of Flaxseed extract. Flaxseed extract exhibited bactericidal activity against all the bacteria tested at 1 and 2.5 mg/200 μl of assay volume (10 and 12.5 mg/ml), except Salmonella typhi and Pseudomonas aeruginosa, in whom Flaxseed extract showed bacteriostatic effect at 1 mg/200 μl of assay volume whereas it was ineffective for Escherichia coli K1 at this concentration (Fig. 5a–f). In case of VRE, 1 and 2.5 mg/200 μl of assay volume Flaxseed extract produced 99.73% and 100% bactericidal activity respectively (Fig. 5a). When tested against Escherichia coli K1, 2.5 mg/200 μl of assay volume of Flaxseed extract produced 66.8671.80% bactericidal activity; however there was no effect at the concentration of 1 mg/200 μl of assay volume (Fig. 5b). Flaxseed extract at the concentration of 1 and 2.5 mg/200 μl of assay volume produced 88.88% and 98.6070.07% bactericidal activity against MRSA, respectively (Fig. 5c). For Pseudomonas aeruginosa, 2.5 mg/200 μl of assay volume of Flaxseed extract produced 76.8376.20% bactericidal activity (Fig. 5d). In case of Bacillus cereus, Flaxseed extract showed 84.2672.59% and 92.6473.00% bactericidal activity, at the concentration of 1 and 2.5 mg/200 μl of assay volume respectively (Fig. 5e). However, for Salmonella typhi, 2.5 mg/200 μl of assay volume of the Flaxseed extract showed moderate bactericidal

3.2.1. Effect on rabbit jejunum Flaxseed extract caused relaxation of both spontaneous and high K þ (80 mM)-induced contractions (Fig. 4a), similar to that caused by

Fig. 3. Antimotility effect of increasing doses of crude extract of Flaxseed along with loperamide, 10 mg/kg given i.p., as a positive control, on castor oil-induced charcoal meal transit from pylorus to cecum in mice. Charcoal meal transit is expressed as LM/LSI  100 where LM is the length of charcoal meal traveled and LSI is the length of small intestine. Results shown are mean7 SEM of three experiments with 5 animals in each experimental group. n, nn, and nnn indicate p o 0.05, p o 0.01 and po 0.001 respectively. Fs.Cr: Flaxseed extract; vehicle: 5% DMSO–10% Tween-80 and distilled water.

3.3. In vitro activities

Fig. 4. Inhibitory effect of Flaxseed extract (a and c) and verapamil (b and d) on spontaneous (a) and high K þ (80 mM)-induced contractions (b) and Ca þ þ concentration response curves in the presence and absence of Flaxseed extract (c) and verapamil (d) in isolated rabbit jejunal preparations. Data shown are mean 7 SEM of 5 experiments. Fs.Cr: Flaxseed extract.

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Fig. 5. Bactericidal activity of Flaxseed extract against gut pathogens. Flaxseed extract at the concentration of 1 and 2.5 mg in 200 μl assay volume (10 and 12.5 mg/ml) was incubated with  106 c.f.u. of VRE (a) Escherichia coli K1 (b), MRSA (c), Pseudomonas aeruginosa (d), Bacillus cereus (e) and Salmonella typhi (f) at 37 1C for 24 h in 200 ml. Escherichia coli K1, Bacillus cereus, Pseudomonas aeruginosa and Salmonella typhi were incubated with 100 mg/ml gentamicin, while MRSA were incubated with 100 mg/ml vancomycin, to serve as a positive control. For negative control, bacteria were incubated with methanol in LB alone. After incubation, bacterial colonies were counted and percentage bactericidal effects were determined as described in Section 2. The data are presented as the mean 7 standard error of three independent experiments performed in duplicate. p-Values were calculated by comparing original inoculum and different extract concentrations using un-paired t-test. n, nn and nnn indicate p o0.05, po 0.01 and p o0.001 vs. OI. Fs.Cr: Flaxseed extract; OI: original inoculum.

activity (26.9073.40%). Surprisingly 1 mg/200 μl of assay volume of Flaxseed extract did not allow the growth of Pseudomonas aeruginosa and Salmonella typhi beyond the original inoculum, hence suggestive of bacteriostatic activity (Fig. 5d and f). 3.4. Acute toxicity test The extract was well tolerated by the animals up to the tested oral dose of 1000 mg/kg. No sign of acute toxicity like restlessness, seizures and piloerection were noticed over the period of observation (6 h) and there were no deaths recorded.

4. Discussion Flaxseed is used in traditional medicine for the treatment of diarrhea and gastrointestinal infections (Gruenwald et al., 2000; Duke et al., 2002). This investigation was carried out to evaluate pharmacological basis for the medicinal use of this plant in diarrhea due to non-infectious and infectious causes. We observed that pretreatment of mice with Flaxseed extract caused a dosedependent reduction in the incidence and severity of diarrheal stool production. The usual indicator of this type of diarrhea is to see all or none response (Gilani et al., 2005a). In this study we categorized diarrhea quantitatively on the basis of both the frequency and consistency (Wirtz et al., 2005), as diarrhea is the consequence of both increase in frequency and decrease in consistency

of the stool. We found that Flaxseed extract was effective in reducing both the frequency and increasing the consistency of the diarrheal stool. One possibility for antidiarrheal activity could be by affecting gut motility. This aspect was confirmed when we tested Flaxseed extract in the accelerated gut motility model in mice that was induced by castor oil. Castor oil increases intestinal fluid contents and caused diarrhea indirectly through ricinoleic acid formation, which changes the electrolyte and water transport, and subsequently resulting in strong and generates enormous contractions in the transverse and distal colon (Iwao and Terada, 1962). Earlier, we have observed that plants also mediate their spasmolytic effect through Ca þ þ channel blockade (Gilani et al., 2005b, 2005c, 2007). To evaluate whether Flaxseed also mediates the decrease in gut motility by the same mechanism, we performed isolated tissue experiments on rabbit jejunum. Ca þ þ channel blocking like activity was evident as the extract inhibited the high K þ (80 mM)-induced contractions and also produced a rightward shift in the Ca þ þ CRCs along with suppression of the maximal response, which is the characteristic of the standard Ca þ þ channel blocker, like verapamil (Fleckenstein, 1977) that is known to inhibit gut motility non-specifically. In one of the earlier reports, Flaxseed oil was shown to possess anticholinergic activity in guinea-pig ileum, which has been presented through a bar graph showing a decrease in cholinergic activity at increasing doses of Flaxseed oil (Kaithwas and Majumdar, 2010b). Since Ca þ þ channel blockers are known to have non-specific spasmolytic effect, they are expected to

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block the effect of all agonists including Ach, histamine and 5-HT (Gilani et al., 2008). Hence, if any substance causes the decrease in cholinergic or histaminergic activities, it may not necessarily mean that those substances are absolute anticholinergic or antihistaminergic. This is because, a typical characteristic of competitive antagonists (anticholinergic or antihistaminergic) is a parallel displacement in Ach or histamine CRCs without the suppression of maximum response (Arunlakshana and Schild, 1959), which was not the case reported in that study (Kaithwas and Majumdar, 2010b). To further elucidate antidiarrheal mechanism, enteropooling assay was done to determine the effect of Flaxseed extract on intestinal secretions and it was found effective in reducing castor oil-induced intestinal secretions in mice. Earlier antisecretory activity of Flaxseed oil in relation to its antiulcer activity has been attributed to antihistaminergic and anticholinergic activities (Kaithwas and Majumdar, 2010b), which may not be the correct interpretation of the results due to the aforementioned reason. It is possible that the antihistaminergic and anticholinergic activities that have been suggested for Flaxseed oil were probably due to the Ca þ þ channel blocking effect, which is also supported by our present findings of Flaxseed extract in rabbit jejunum. Thus to conclude, the mechanistic basis of Flaxseed extract in diarrhea can be due to its effect on intestinal secretions coupled with decreased propulsion of material in the intestinal tract due to gut motility inhibitory effect possibly mediated through Ca þ þ antagonist effect; consequently it may promote reabsorption of materials in the intestine causing decreased loss of fluid and electrolytes. Besides functional disorders, bacterial infection is one of the major causes of diarrhea (NDDIC, 2011) and antibiotics have been found effective in managing them as well as in avoiding complications (Farthing et al., 2013), but with the current increasing trend of microbial resistance against currently available antibiotics (Kumarasamy et al., 2010), a safer alternative for antimicrobial agents seemed imperative. Previously our group reported the discovery of anti-microbial activity from the plant (Khan et al., 2011), animal (Khan et al., 2008) and microbial sources (Iqbal et al., 2014). Here, we are reporting antibacterial activity of Flaxseed extract against Escherichia coli K1, MRSA, VRE, Pseudomonas aeruginosa, Bacillus cereus and Salmonella typhi, which are known to cause both enteric and non-enteric infections (NDDIC, 2011). The effectiveness of Flaxseed extract against Gram positive and negative bacteria, including the drug-resistant pathogens, suggests the presence of constituents that could serve as a source of new and better antimicrobial compounds. Earlier, antibacterial activity of Flaxseed extract have been reported for Salmonella typhi, Bacillus cereus, Pseudomonas aeruginosa and Escherichia coli K-12 (Seher et al., 2006; Bakht et al., 2011). Besides these, Flaxseed oil has also been shown to have antibacterial activity against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli K-12, Bacillus cereus and Pseudomonas aeruginosa (Kaithwas et al., 2011). This effect could not be correlated with Flaxseed extract because oil has different constituents as compared to extract and their penetration in the bacteria will also be different (Nazzaro et al., 2013). Our antibacterial assay has an edge on earlier reports where antibacterial activity was measured qualitatively or semi-quantitatively. In our assays, we have noted, both bactericidal and bacteriostatic activity in a quantitative manner. Though in aforementioned studies, antibacterial activity was reported against Escherichia coli K 12, Staphylococcus aureus and Enterococcus faecalis, but in our study, instead of non-pathogenic Escherichia coli K 12, we have employed pathogenic Escherichia coli K1 and multi drug-resistant strains of Staphylococcus aureus and Enterococcus faecalis. Future studies may be directed to test the Flaxseed extract on other bacterial pathogens including Vibrio cholera, Campylobacter jejunii, Shigella flexinerii, Proteus mirabilis as well as viral and parasitic pathogens causing enteric infections. Additionally, the same in vitro studies

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will also be carried out in vivo and subsequently into clinical studies. We also plan to acquire several commensal bacteria and undertake a detailed study to determine the effects and their mode of action in future studies. One aspect that we need to address related to Flaxseed is that literature suggests its use in both diarrhea and constipation (Duke et al., 2002). When we tested Flaxseed extract for its laxative effect in normal mice, no significant increase in frequency and weight of fecal material was observed (data not shown). Earlier, the laxative effect has been reported for partially defatted Flaxseed meal which was attributed to its insoluble fiber content (Xu et al., 2012) that decreases transit times within stomach and small intestine (Brownlee, 2011). Since we have used the crude aqueous-methanolic extract, the insoluble fiber content is excluded from the material, though presence of laxative or gut stimulant component(s) cannot be ruled out. To assess the laxative activity of the Flaxseed, we tested the mucus part, which showed moderate degree of spasmogenic effect (data not shown); detailed study on possible mechanism of action is in progress and will be presented as a separate study. Interestingly, in our study, Flaxseed extract did not halt the stool frequency completely when diarrhea was induced, which could be considered a merit, in terms of avoiding the side-effect of constipation, as seen with antidiarrheal drugs like loperamide (Grahame-Smith and Aronson, 2002). It is speculated that possible presence of spasmogenic component may have presented this type of antidiarrheal activity which is particularly important in case of infectious diarrhea, where completely abolishing the stool frequency increases the chances of dissemination of infection. Thus, Flaxseed as a whole appears to have both the spasmogenic and spasmolytic components, like some of the popular herbs, such as Psyllium husk (Ispaghol), (Mehmood et al., 2011) Ginger (Ghayur and Gilani, 2005), which have been shown in our laboratory to possess dual efficacy in constipation and diarrhea. However, depending on the form and the way it is used, its effect will vary like in case of Fumaria indica (Gilani et al., 2005b).

5. Conclusion The results of the present study suggest that Flaxseed extract possesses antidiarrheal activity due to its effect on consistency and frequency of loose stool possibly due to inhibition of gut motility and secretions in the mice. The predominant antispasmodic mechanism involved seems to be Ca þ þ -channels blocking like activity. This coupled with antibacterial effect against number of enteric and non-enteric pathogens gives ample evidence to suggest that Flaxseed extract could be used in both the infectious and non-infectious diarrhea.

Acknowledgments This study was financed in part by the Higher Education Commission, Government of Pakistan (Grant no. Bm6-161), as an indigenous PhD scholarship awarded to Miss Amber Hanif Palla and partially supported by the Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi, Pakistan We would also like to acknowledge Mehwish Sagheer who provided technical help for performing in vitro antibacterial assays. References Alanis, A.J., 2005. Resistance to antibiotics: are we in the post-antibiotic era? Archives of Medical Research 36, 697–705. Aman, S., Carl, U., 2008. Enterocolitis caused by methicillin-resistant Staphylococcus aureus. Infectious Disease Clinical Practice 16 (4), 222–223.

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Arunlakshana, O., Schild, H.O., 1959. Some quantitative uses of drug antagonists. British Journal of Pharmacological Chemotherapy 14 (1), 48–58. Aye, -Than, Kulkarni, H., Hmone, W., Tha, S., 1989. Anti-diarrhoeal efficacy of some Burmese indigenous drug formulations in experimental diarrhoeal test models. Pharmaceutical Biology 27 (4), 195–200. Bakare, R., Magbagbeola, O., Akinwande, A., Okunowo, O., Green, M., 2011. Antidiarrheal activity of aqueous leaf extract of Momordica charantia in rats. Journal of Pharmacognosy and Phytotherapy 3 (1), 1–7. Bakht, J., Ali, H., Khan, M., Khan, A., Saeed, M., 2011. Antimicrobial activities of different solvents extracted samples of Linum usitatissimum by disc diffusion method. African Journal of Biotechnology 10 (85), 19825–19835. Bartlett, J.G., 2006. Narrative review: the new epidemic of Clostridium difficileassociated enteric disease. Annals of Internal Medicine 145, 758. Bashir, S., Memon, R., Gilani, A.H., 2011. Antispasmodic and antidiarrheal activities of Valeriana hardwickii Wall. rhizome are putatively mediated through Ca þ þ channel blockade. Evidence Based Complementary and Alternative Medicine 2011, 1–6. Bolton, T., 1979. Mechanisms of action of transmitters and other substances on smooth muscle. Physiological Reviews 59 (3), 606–718. Boschi-Pinto, C., Velbeit, L., Shibuya, K., 2008. Estimating child mortality due to diarrhea in developing countries. Bulletin of the World Health Organization 86 (9), 710–717. Brownlee, I.A., 2011. The physiological roles of dietary fibre. Food Hydrocolloids 25, 238–250. Capasso, F., Mascolo, N., Izzo, A.A., Gaginella, T.S., 1994. Dissociation of castor oilinduced diarrhea and intestinal mucosal injury in rat: effect of NG nitro-Larginine methylester. British Journal of Pharmacology 113, 1127–1130. Capasso, R., Izzo, A.A., Borelli, F., Russo, A., Sautebin, L., Pinto, A., et al., 2002. Effect of piperine, the active ingredient of black pepper on intestinal secretion in mice. Life Sciences 71, 2311–2317. Duke, J.A., Bogenschutz-Godwin, M.J., duCellier, M.J., Duke, P.A., 2002. Catalog of herbs (A–Z): flax, Handbook of Medicinal Herbs, second ed. CRC press, Boca Raton, Florida, pp. 305–306. Elfawal, M.A., Towler, M.J., Reich, N.G., Golenbock, D., Weathers, P.J., Rich, S.M., 2012. Dried whole plant Artemisia annua as an antimalarial therapy. PLoS One 7 (12), e52746. http://dx.doi.org/10.1371/journal.pone (0052746. Epub 2012 Dec 20). Farre, J., Colombo, M., Fort, M., Gutierrez, B., 1991. Differential effects of various Ca þ þ antagonists. General Pharmacology 22 (1), 177–181. Farthing, M., Salam, M.A., Lindberg, G., Dite, P., Khalif, I., Salazar-Lindo, E., WGO, 2013. Acute diarrhea in adults and children: a global perspective. Journal of Clinical Gastroenterology 47 (1), 12–20. http://dx.doi.org/10.1097/MCG.0b013e31826df662. Fleckenstein, A., 1977. Specific pharmacology of Ca þ þ in myocardium, cardiac pacemakers, and vascular smooth muscle. Annual Review of Pharmacology and Toxicology 17, 149–166. Ghayur, M.N., Gilani, A.H., 2005. Pharmacological basis for the medicinal use of Ginger in gastrointestinal disorders. Digestive Diseases and Sciences 50, 1889–1897. Gilani, A.H., Coblin, L.B., 1987. Interaction of himbacine with carbachol at muscarinic receptors of heart and smooth muscle. Archives internationales de pharmacodynamie et de thérapie 290, 46–53. Gilani, A.H., Aziz, N., Khurram, I.M., Rao, Z.A., Ali, N.K., 2000. The presence of cholinomimetic and Ca þ þ channel antagonist constituents in Piper betle Linn 14, 436–442Phytotherapy Research 14, 436–442. Gilani, A.H., Atta-ur-Rahman, 2005. Trends in ethnopharmacology. Journal of Ethnopharmacology 100 (1–2), 43–49. Gilani, A.H., Ghayur, M.N., Khalid, A., Zaheer-ul-Haq, Q., Choudhary, M.I., Rahman, A., 2005a. Presence of antispasmodic, antidiarrheal, antisecretory, Ca þ þ antagonist and acetylcholinesterase inhibitory steroidal alkaloids in Sarcocca saligna. Planta Medica 71 (2), 1–6. Gilani, A.H., Bashir, S., Janbaz, K.H., Khan, A., 2005b. Pharmacological basis for the use of Fumaria indica in constipation and diarrhea. Journal of Ethnopharmacology 96, 585–589. Gilani, A.H., Bashir, S., Janbaz, K.H., Shah, A.J., 2005c. Presence of cholinergic and Ca þ þ channel blocking activities explains the traditional use of Hibiscus rosasinensis in constipation and diarrhea. Journal of Ethnopharmacology 102, 289–294. Gilani, A.H., Bashir, S., Khan, A., 2007. Pharmacological basis for the use of Borago officinalis in gastrointestinal, respiratory and cardiovascular disorders. Journal of Ethnopharmacology 114, 393–399. Gilani, A.H., Khan, A.U., Ali, T., Ajmal, S., 2008. Mechanisms underlying the antispasmodic and bronchodilatory properties of Terminalia bellerica fruit. Journal of Ethnopharmacology 116 (3), 528–538. Grahame-Smith, D.G., Aronson, J.K., 2002. Pharmacopoeia. In: Grahame-Smith, D., Aronson, J., Grahame-Smith, D., Aronson, J. (Eds.), Oxford Textbook of Clinical

Pharmacology and Drug Therapy, 3rd ed. Oxford University Press, Bournemouth, pp. 469–551. Gruenwald, J., Brendler, T., Jaenicke, C., 2000. PDR for Herbal Medicines, second ed. Medical Economics Company, Montvale, NJ. Iqbal, J., Siddiqui, R., Khan, N.A., 2014. Acanthamoeba and bacteria produce antimicrobials to target their counterpart. Parasites & Vectors 7 (56), http: //dx.doi.org/10.1186/1756-3305-7-56. Iwao, I., Terada, Y., 1962. On the mechanism of diarrhea due to castor oil. Jpn J Pharmacol 12, 137–145. Jabeen, Q., Bashir, S., Lyoussi, B., Gilani, A.H., 2009. Coriander fruit exhibits gut modulatory, blood pressure lowering and diuretic activities. Journal of Ethnopharmacology 122, 123–130. Kaithwas, G., Majumdar, D.K., 2010a. Therapeutic effect of Linum usitatissimum (Flaxseed/linseed) fixed oil on acute and chronic arthritic models in albino rats. Inflammopharmacology 18, 127–136. Kaithwas, G., Majumdar, D.K., 2010b. Evaluation of antiulcer and antisecretory potential of Linum usitatissimum fixed oil and possible mechanism of action. Inflammopharmacology 18 (3), 137–145. Kaithwas, G., Mukerjee, A., Kumar, P., Majumdar, D.K., 2011. Linum usitatissimum (linseed/flaxseed) fixed oil: antimicrobial activity and efficacy in bovine mastitis. Inflammopharmacology 19, 45–52. Khan, H.U., Ali, I., Khan, A.U., Naz, R., Gilani, A.H., 2011. Antibacterial, antifungal, antispasmodic and Ca þ þ antagonist effects of Caesalpinia bonducella. Natural Product Research 25 (4), 444–449. http://dx.doi.org/10.1080/14786419.2010.529445. Khan, N.A., Osman, K., Goldsworthy, G.J., 2008. Lysates of Locusta migratoria brain exhibit potent broad-spectrum antibacterial activity. Journal of Antimicrobial Chemotherapy 62 (3), 634–635. Kumarasamy, K., Toleman, M., Walsh, T., Bagaria, J., Butt, F., Balakrishnan, R., et al., 2010. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infectious Diseases 10 (9), 597–602. Mehmood, M.H., Aziz, N., Ghayur, M.N., Gilani, A.H., 2011. Pharmacological basis for the medicinal use of Psyllium Husk (Ispaghol) in constipation and diarrhea. Digestive Diseases and Sciences 56, 1460–1471. Nazzaro, F., Fratianni, F., De Martino, L., Coppola, R., De Feo, V., 2013. Effect of essential oils on pathogenic bacteria. Pharmaceuticals 6, 1451–1474. http://dx. doi.org/10.3390/ph6121451. NDDIC, 2011. National Digestive Diseases Information Clearinghouse. Retrieved from Diarrhea: 〈http://digestive.niddk.nih.gov/ddiseases/pubs/diarrhea/#cause〉 (accessed 18.03.14.). Oberoi, L., Singh, N., Sharma, P., Aggarwal, A., 2013. ESBL, MBL and Ampc beta Lactamases producing superbugs—havoc in the intensive care units of Punjab India. Journal of Clinical Diagnostic Research 7 (1), 70–73. Rehman, N.R., Bashir, S., Al-Rehaily, A.J., Gilani, A.H., 2012. Mechanisms underlying the antidiarrheal, antispasmodic and bronchodilator activities of Fumaria parviflora and involve-ment of tissue and species specificity. Journal of Ethnopharmacology 144 (1), 128–137. Seher, G., Turgut-Balik, N., Nazmi, G., 2006. Antimicrobial activities of some fatty acids of turmeric, ginger root and linseed used in the treatment of infectious diseases. World Journal of Agricultural Sciences 40 (2), 439–442. Smith, R.D., Coast, J., 2002. Antimicrobial resistance: a global response. Bulletin of the World Health Organization 80, 126–133. Tarpila, A., Wennberg, T., Tarpila, S., 2005. Flaxseed as a functional food. Current Topics in Nutraceutical Research 3 (3), 167–188. Teke, G.N., Kuiate, J.R., Gatsing, D., 2007. Antidiarrheal and antimicrobial activities of Emilia Coccinea (Sims) G. Don extracts. Journal of Ethnopharmacology 112, 278–283. Thakkar, S., Agrawal, R., 2010. A case of Staphylococcus aureus enterocolitis: a rare entity. Gastroenterology and Hepatology 6 (2), 115–117. Trivedi, T.H., Sabnis, R.G., 2009. Superbugs in ICU: is there any hope for solution? Journal of the Association of Physicians of India 57, 623–625. Van Rossum, J., 1963. Cumulative dose–response curves. II. Techniques for the making of dose–response curves in isolated organs and the evaluation of drug parameters. Archives Internationales de Pharmacodynamie 143, 299–330. Wang, H., Tan, C., Bai, X., Du, Y., Lin, B., 2006. Pharmacological studies of anti-diarrhoeal activity of Gentianopsis paludosa. Journal of Ethnopharmacology 105 (1–2), 114–117. WHO, 2013. Diarrheoal Disease: Factsheet no. 330. 〈http://www.who.int/mediacen tre/factsheets/fs330/en/index.html〉 (accessed 03.03.14.). Wirtz, S., Neufert, C., Weigmann, B., Neurath, M., 2005. Chemically induced mouse models of intestinal inflammation. Nature Protocols 2 (3), 541–546 (PMID: 17406617). Xu, J., Zhou, X., Chen, C., Deng, Q., Huang, Q., Yang, J., et al., 2012. Laxative effects of partially defatted flaxseed meal on normal and experimental constipated mice. BMC Complementary and Alternative Medicine 12 (14), 1–5.