THE AGA KHAN UNIVERSITY Faculty of Health Sciences Department Of

THE AGA KHAN UNIVERSITY Faculty of Health Sciences

Department of Biological and Biomedical Sciences

PHARMACOLOGICAL BASIS FOR MEDICINAL USE OF

FLAXSEED (LINUM USITATISIMUM) IN INFLAMMATORY

BOWEL DISEASE

AMBER HANIF PALLA (M.Pharm)

A thesis submitted in partial fulfilment of the requirements for the degree of degree of

Doctorate of Philosophy (Health Sciences) in the discipline of Pharmacology

Karachi, Pakistan

2016

THE AGA KHAN UNIVERSITY Faculty of Health Sciences

Department of Biological and Biomedical Sciences

A thesis submitted in partial fulfilment of the requirements for the degree of Doctorate of Philosophy (Health Sciences) in the discipline of Pharmacology

PHARMACOLOGICAL BASIS FOR MEDICINAL USE OF FLAXSEED (LINUM USITATISIMUM) IN INFLAMMATORY BOWEL DISEASE

Members of the Thesis Defence Panel appointed to examine the thesis of AMBER HANIF PALLA find it satisfactory and recommended that it be accepted

Dedication

Dedicated to

My Abbu, Mr. Hanif Palla and Ammi, Farida Palla,

To my lovely daughters, Rabia & Aleena.

My Husband, Aamir

My siblings, Fayyaz, Fahad and Anam

& last but not the least

My Late.Uncle Yusuf, who would have been very proud of me

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Abstract

Inflammatory bowel disease (IBD) comprising of Ulcerative colitis (UC) and

Crohn’s disease (CD) is a multifactorial disease with major maneuvering targets being immune dysregulation, microbial homeostasis, and barrier dysfunctioning. Natural products which comprise of multiple bioactive constituents could control multiple targets of a disease simultaneously. Flaxseed has traditionally been used in IBD but its effectiveness has not yet been pharmacologically validated. Hence, it was aimed to explore the pharmacological basis for medicinal use of Flaxseed in IBD. For this purpose, methanolic-aqueous (70:30) extract of Flaxseed (Fs.Cr), was used to test the different parameters of IBD. This was followed by screening of Flaxseed oil for its potential in IBD.

Fs.Cr was at first assessed for its effect against acetic acid (AA)-induced colitis model of mice. Colitis was induced under anaesthesia by intrarectal administration of

0.1 ml of 6% AA, and the parameters were assessed after 24 hrs of induction. Fs.Cr reduced the disease activity index (DAI) dose-dependently at 150 mg/kg and 300 mg/kg and macroscopic damage with the highest ulcer index (21.88) for untreated groups, whereas, pre-treatment of animals with Fs.Cr, significantly reduced this ulcer index to

11.68, 7.83 and 6.01 at 150, 300 and 500 mg/kg doses respectively, as compared to untreated group. Hemotoxylin and Eosin stained sections of colon in Fs.Cr treated groups, showed reduction in Neurath scores, where untreated mice had a score of

3.48±0.65 (max score = 4) and Fs.Cr pretreated mice showed a reduction in score to

2.33±0.57 and 1.56±0.5 at 150 mg/kg and 300 mg/kg respectively. Vilaseca score parameters (including ulceration, inflammation and depth of lesion) with untreated groups score being 2.08±0.275 reduced significantly to 1.51±0.2 and 1.08±0.14 at 150 mg/kg and 300 mg/kg doses, respectively. Periodic acid Schiff-alcian blue (PAS-Ab) stains showed an increase in mucin levels and restoration of goblet cells in the treated groups with a score of 1.50±0.25, 2.100±0.25 and 2.3±0.5 with increasing doses of 150 mg/kg, 300 mg/kg and 500 mg/kg respectively, as compared to the untreated mice

IV groups who had the lowest scores (1.2±0.37 out of 3). Anti-inflammatory potential of

Fs.Cr was evidently supported by the reduced myeloperoxidase activity with untreated animals exhibiting activity at 400±59.5 mU/ml, which reduced to 150.85±65 mU/ml and 107±13.89mU/ml at at 150 mg/kg and 300 mg/kg doses, respectively. Its effect against oxidative stress was evident by reduced malondialdehyde activity, where untreated mice exhibited 77.25±4 nmol/mg activity, whereas this reduced to 49.66±4.88 nmol/mg and 26.36±2.14 nmol/mg at respective doses of 150 mg/kg and 300 mg/kg.

Enhanced antioxidant activity (catalase, superoxide dismutase, glutathione peroxidase and total glutathione) in Fs.Cr groups was significantly higher than both the untreated control as well as sham control, evident at 300 mg/kg and 500 mg/kg doses. Anti- inflammatory effect of Fs.Cr was characterized by its effect on modulation of cytokines.

Colons of untreated mice showed elevated levels of IFN- (a pro-inflammatory marker), as compared to sham controls at all the time points (1238±195.2 pg/ml, 859±100.9 pg/ml and 943.2±177.8 pg/ml). Compared to this, the colons of Fs.Cr pretreated mice showed reduced IFN-levels to 605±62.17 pg/ml and 492.7±37.96 pg/ml at 6 hrs for

150 and 300 mg/kg doses with similar reducing trend evident at 12 hrs and 24 hrs. TNF-

levels were also elevated in untreated mice colons’ at all time points 194.1±27.36 pg/ml, 110.3±3.39 pg/ml and 142.4±4.93 pg/ml), whereas Fs.Cr pre-treated groups showed a reduction in these levels, with effect evident at 300 mg/kg and 500 mg/kg at all the tested time points. Kinetics of IL-17 levels was studied in AA-induced colitis model for the first time. Untreated groups showed increased IL-17 levels at 6 hrs

(233.2±18.41 pg/ml), and sharply decreased levels at 12 hrs (166.1±32.26 pg/ml) and

24 hrs (114.2±16.29 pg/ml). On the contrary, Fs.Cr-treated tissues showed insignificant decline of IL-17 at 6 hrs, but exhibited a dose-dependent increase at 24 hrs with

175.9±31.1 pg/ml at 150 mg/kg dose, 209±29.85 pg/ml for 300 mg/kg dose and

223.8±13.69 pg/ml for 500 mg/kg dose. Hence, it was concluded that Fs.Cr ameliorated the severity of AA-induced colitis model of mice by mucosal protective, anti- inflammatory and antioxidant mechanisms with modulation of multiple cytokines.

Diarrhea and spasm are the main manifestation of IBD and hence, Fs.Cr was also assessed for its effectiveness in diarrhoea and spasm along with possible mechanisms. Fs.Cr showed antidiarrheal activity in castor oil-induced diarrhea in mice with mean diarrhoeal score being 35.7±0.81 for untreated mice, whereas, Fs.Cr significantly reduced the diarrhoeal score to 21.6±2.85, 13±2.13 and 11.3±1.35 at 150,

300 and 500 mg/kg doses. Reduced intestinal secretions in mice were evident in Fs.Cr treated groups as compared to untreated groups whose intestinal secretions were increased from 77.35±1.52g (sham control) to 149±5.90 g (untreated), whereas, Fs.Cr pretreated groups showed secretions significantly being reduced to 24.12% (113±5.12 g), 28.09% (107±2.49 g) and 38.8% (91±3.06 g) at respective doses of 150, 300 and

500 mg/kg. Gut motility was increased in untreated controls by administration of castor oil that increased the charcoal meal transit from 45.68±1.96 cm to 68.43±3.95 cm, whereas, Fs.Cr reduced the intestinal motility in mice by 31.66% (46.76±3.46 cm),

46.98% (36.28±3.73 cm; and 56.2% (29.97±4.30 cm); at the respective doses of 150,

300 and 500 mg/kg. The main antispasmodic mechanism of Fs.Cr was mediated by phosphodiesterase enzyme inhibitory activity (PDEI), followed by Ca++ antagonist effect, as Fs.Cr dose-dependently inhibited spontaneous-induced contractions, carbachol (CCh)-induced contractions (at 3 mg/ml) and high K+-induced contractions

(at 5 mg/ml), in isolated rabbit jejunum with higher potency against CCh, similar to that of papaverine, while verapamil was more potent against high K+. This was also supported by potentiation of isoprenaline concentration-response curves (CRCs) by pretreatment with low doses of Fs.Cr (0.01 and 0.03 mg/ml). Fs.Cr mediated PDE inhibitory activity via PDE-4 subtype, as pre-treatment of Fs.Cr did not potentiate

Rolipram (PDE-4 inhibitor) inhibitory activity against CCh-induced contractions, whereas it potentiated the inhibitory effect of Cilostazol (PDE-3 inhibitor) and zaprinast

(PDE-5 inhibitor). Subsequent dose dependent increase in cAMP levels after pre- treatment with 1, 3 and 5 mg/ml of Fs.Cr, similar to papaverine (1 M and 3 M doses), further supported its PDEI effect. Possible presence of Ca++ antagonist like activity was

VI studied because of Fs.Cr’s inhibitory effect against high K+-induced contractions at high doses. Fs.Cr at 0.1 mg/ml and 0.3 mg/ml caused a rightward shift in the Ca++ CRCs with suppression of maximal response, indicating its Ca++ antagonist effect. Hence, Fs.Cr possessed antispasmodic effect mediated through a dual inhibition of PDE enzyme and

Ca++ influx.

Microbes being the persistent source of immune trigger and inflammation, Fs.Cr was therefore, also studied for its antimicrobial potential in the in vitro assay. Fs.Cr at the dose of 50 µg/ml showed cidal effect against enteropathogenic E. coli (EPEC),

Pseudomonas aeruginosa and Bacillus cereus, whereas, a static effect was observed against enterotoxigenic E.coli (ETEC) and enteroaggregative E. coli (EAEC). The cidal activity against methicillin-resistant Staphylococcus aureus was observed at 12.5 mg/ml and at 5 mg/ml against vancomycin-resistant Enterococcus faecalis, along with remarkable 100% kill at 12.5 mg/ml indicating presence of some novel compounds.

Interestingly, the HPLC peaks of Fs.Cr resembled with retention times of some synthetic antimicrobial compounds

Further to this, Flaxseed oil which is an available edible source was studied for its potential in IBD. Flaxseed oil ameliorated the severity of AA-induced colitis as evident by improved DAI, mortality rate and microscopic damage. The comparison of microscopic damage caused by test materials revealed that Flaxseed oil exhibited a stronger effect on neutrophil infiltration as compared to Fs.Cr, whereas, Fs.Cr produced more pronounced increase on mucin levels indicating possibility for employing different mechanisms to mediate the protective effect.

When studied for its potential against diarrhoea and spasm, Flaxseed oil showed a combination of laxative effect at low doses and antidiarrheal effect at high doses. The gut stimulant effect was prominently cholinergic and at lesser level histaminergic. The antispasmodic activity of Flaxseed oil was mediated by K+ channel opening (KCO) mechanism, followed by a weak PDEI activity as it inhibited CCh-induced contraction.

Flaxseed oil was more potent and efficacious as bactericidal against enteropathogenic

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Eschericia.coli (EPEC), enterotoxigenic E.coli (ETEC) and enteroaggregative E.coli

(EAEC) which are implicated in IBD, as they killed these microbes at 9 µg/ml and 14

µg/ml, whereas, Fs.Cr was only cidal against EPEC at a concentration 10 times higher than that of Flaxseed oil (100 µg/ml), whereas, it had a static mechanism against ETEC and EAEC.

A different mechanism is speculated for Flaxseed oil and Fs.Cr; this is further advocated by difference in HPLC fingerprints of Fs.Cr and Flaxseed oil.

Based on the above mentioned findings, it is concluded that Flaxseed extract and oil have potential for targeting multiple etiological aspects of IBD through multiple pathways; thus, indicating that Flaxseed as a whole has the potential to be used as alternate remedy with a known safety profile being edible in nature.

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List of Abbreviations

# Number % Percent

4-AP 4-Aminopyridine 5-ASA 5-aminosalicylic acid 6-MP 6-Mercaptopurine  Alpha AA Acetic acid Ach Acetylcholine ALA Alpha-linoleic acid

APC Antigen presenting cells AZA Azathioprine  Beta B.cereus Bacillus cereus Ca++ Calcium ion

CaCl2 Calcium chloride cAMP Cyclic adenosine monophosphate CAT Catalase

CCB Calcium channel blockade CCh Carbachol CD Crohn’s disease CI Confidence interval

CO2 Carbon dioxide Cr Crude CRCs Concentration-response curves CSA Cyclosporine DAI Disease activity index DC Dendritic cells DECs Diarrhoeaogenic E. coli EAEC Enteroaggregative Eschericia coli

EC50 Median effective concentration E.coli K1 Eschericia coli K1 edn Edition EDTA Ethylenediamine tetra acetic acid EAEC Enteroaggregative E.coli

EPEC Enteropathogenic E.coli

ETEC Enterotoxigenic E. coli

T cells Gamma-delta T cells

GATA Trans-acting T-cell-specific transcription factor-3 GI Gastrointestinal

GPx Glutathione peroxidase

GSH Glutathione

GST Glutahtione S transferases

IBD Inflammatory Bowel Disease ICAM1 Intercellular Adhesion Molecule 1 iDC Immature dendritic cells IEC Intestinal epithelial cells IFN- Interferon gamma ILC Innate lymphoid cells i.p Intraperitoneal I.R. Intra-rectal

K+ Potassium ion

KATP ATP-dependent potassium Channel Kg Kilogram

KH2PO4 Potassium dihydrogen phosphate LB Leuria Broth LA Linolenic acid

µ Micro µM Micromolar M Molar MDA Malondialdehyde

Mg Milligram

MgCl2 Magnesium chloride

MgSO4 Magnesium sulfate MHC Major histocompatibility complex Min Minute Ml Milliliter mm Hg Millimeter of mercury mM Millimolar MPO Myeloperoxidase

MTX Methotrexate MYD88 Myeloid differentiation primary response gene 88 NF- Nuclear factor kappa-light-chain-enhancer of activated B cells NK Natural killer

NO Nitric oxide

OA Oleic acid

OI Original inoculum P.O. Per oral PRR Pattern recognition receptors

4-AP 4-Aminopyridine PDE Phosphodiesterase PDEIs Phophodiesterase Inhibitors PTGER4 prostaglandin receptor EP4

ROR- Retinoic acid receptor gamma

ROS Reigure 2.12

active oxygen species

SASP Sulfasalazine S. flexinerii Shigella flexinerii SOD Superoxide dismutase

SSG-Red Glutathione reductase S.typhi Salmonella typhi

Teff Effector memory T cell

Tem T memory cells

TF Transcription factor

TGF- Tumour growth factor-beta

Th Helper T cells TLR Toll like receptor

TNF- Tumour necrosis factor-alpha Tregs Regulatory T cells

VCAM-1 Vascular cell adhesion molecule 1

VRE Vancomycin-resistant Enterococcus faecailis WHO World Health Organization SEM Standard error mean Syn Synonym UC Ulcerative colitis vs. Versus

Acknowledgement

This thesis is one more of Allah’s countless blessings on me; my first and foremost thanks to Allah, the Almighty. He has always been my support system and whenever I gave up, He took over, and got me through. There have been times when I was striving to look for answers, where it seemed that there’s no way to move forward; and out of nowhere things surfaced in a way, which were beyond intellect and evidences. Sometimes I felt that I am merely doing stuff and that there’s a Divine helping hand behind me. My sincere gratitude to Prophet Hazrat Muhammad (Salallahu alayhee wasallam), for his Nazr-e-karam, which has made it all possible. Indeed “Yeh sub unka karam hai, kay baat ab tak bani hui hai”.

This thesis is materialized due to my supervisor, Dr. Anwar-ul-Hassan Gilani,

HEC Distinguished National Professor, whose vision and support has brought me till this point. He had been a mentor, a father figure, and the one who believed in me. The most amazing part is that he never refuted any idea by saying “No, you can’t do it”.

Infact, he always indicated pros and cons of the decision and then would leave it up on me to decide and then let me discover the path, guiding along. It was his far-sightedness and willingness to encourage new ideas that helped me in shaping up the thesis in multiple directions. He has been a strength and support for me, at levels that go beyond academics; that support enabled me to cope some of the most stressful years of life.

Special thanks to Dean, Farhat Abbas who facilitated the research process and

Dr. Naveed Ahmed Khan who guided me for the antimicrobial assays, facilitated research means and arranged for bacterial strains from Department of Pediatrics. I also am grateful to Dr. Najeeha Talat Iqbal who helped me with cytokines assay and their complicated data handling and Dr. Samrah Bashir, who helped me with various technical and intellectual aspects of research, unconditinally.

This thesis is the output of efforts and sacrifices from a lot of people; my Ammi and Abbu, who patiently beared with me, and took my responsibility in ways that couldn’t be elaborated. They took care of me and my two daughters, with utmost love

XII and care. Had it not been for their support and love to my kids, this journey couldn’t be possible. I am specifically grateful to my father and an un-named friend of his, for arranging funds to manage part of the fees of AKU.

I owe this to my two lovely daughters, Rabia and Aleena, who have compromised with my routines, and supported me, and prayed for me by literally saying, “Allah Taala Ammi ko Paas keraadain”. They were my inspiration for completing the PhD; they were the ones who kept me going in the most stressful of times.

I am also thankful to my husband, who gave me the confidence to apply in PhD program at AKU, and his vision for me to achieve this degree. In the initial years, I might have ended up leaving the program, but he stood by me and sent me back to AKU.

My brothers and sister’s counselling, support and care, for me and my kids, further enabled me to sustain this journey.

The whole Natural Product Research group is especially acknowledged since we all have worked together for past so many years. I am thankful to all my friends and coaleagues, especially, Najeeb, whose help and support was always unconditional. He is the one who helped me both technically and intellectually in any capacity requested.

A special mention for Dr. Malik Hassan Mehmood, who helped me with method to induce colitis in the given resources, and my friends here, with whom a heart to heart talk eased out the difficult times.

I acknowledge the support rendered to me by Ali Moosa for the administrative and non-administrative help. A special mention for Mam Noor Jehan Sarfaraz, from

Department of Biological and Biomedical Sciences (BBS), who trained me in histological techniques and helped me in optimizing the mice sections. The lab technologists including Miss Naseema Mehboob Ali, Miss Saeeda Sheryaar, Miss

Ayesha Rashid, Miss Kausar Sabuhi, Miss Verdah Affendi, Mr. Adil Siddiqui, Mr.

Masnoon and Mr. Ghulam Haider, who rendered unconditional support and guidance, and tried to solve my problems in their best of capacities. Animal house staff including

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Tahir Chauhan, Asghar Ali and Mukhtar Ahmed offered their utmost support when required in managing animal based research. Special thanks to Dr. Zubair Ahmed, Dr.

Khurram Minhas, Mr Chander and Mr. Ali from histopathology department and Prof.

Bina Siddiqui from H.E.J Research Institute of Chemistry to perform HPLC analysis in her department.

I would like to gratefully acknowledge the financial support as, “Indigenous

PhD fellowship” awarded by Higher Education Comission.

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Declaration

I, Amber Hanif Palla solemnly declare that the research work presented in the thesis entitled “PHARMACOLOGICAL BASIS FOR MEDICINAL USE OF

FLAXSEED (LINUM USITATISIMUM) IN INFLAMMATORY BOWEL

DISEASE” is solely my research work with no significant contribution from any other person. Small contribution /help wherever taken has been duly acknowledged and that complete thesis has been written by me.

I understand the zero tolerance policy of the HEC and the Aga Khan University towards plagiarism. Therefore, I, as an author of the above titled thesis declare that no portion of my thesis has been plagiarized and any material used as reference is properly cited/referred.

I undertake that if I am found guilty of any form of plagiarism in the above titled thesis even after the award of PhD degree, the University reserves the rights to withdraw/revoke my PhD degree and that HEC and the University has the right to publish my name on the HEC/University website on which names of students are placed who submit plagiarized thesis.

Student/Author Signature:

Name: Amber Hanif Palla

Table of Contents Dedication ...... III Abstract ...... IV List of Abbreviations ...... IX Acknowledgement ...... XII Declaration ...... XV List of Tables ...... XXI List of Figures...... XXII CHAPTER 1 ...... 1 1. Introduction and Background ...... 1 1.1. Inflammatory Bowel Disease ...... 2 1.2. Epidemiology in Asia and Pakistan ...... 2 1.3. Pathophysiology ...... 5 1.3.1. Role of External environment ...... 5

1.3.2. Role of Genetics ...... 5

1.3.3. Role of Barrier defect ...... 6

1.3.4. Role of Immune dysregulation ...... 6

1.3.4.1. Role of neutrophils ...... 9

1.3.4.2. IL-17 release from innate immune cells ...... 11

1.3.5. Microbes ...... 11

1.4. Pharmacotherapy ...... 12 1.4.1. Advancements in IBD Pharmacotherapy ...... 17

1.4.2. Limitation of pharmacotherapy ...... 17

1.4.3. Natural products as a therapeutic option ...... 19

1.5. Potential of Flaxseed in IBD ...... 20 1.5.1. Literature search ...... 21

1.5.2. Selection of animal model of colitis ...... 24

1.5.3. Research Objectives ...... 25

1.5.4. Outline of Thesis ...... 26

Chapter 2 ...... 28 2. Flaxseed in colitis model of mice ...... 28

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2.1. Introduction ...... 29 2.2. Materials and Methods ...... 31 2.2.1. Reagents ...... 31

2.2.2. Preparation of crude extract of Flaxseed ...... 31

2.2.3. Working stocks ...... 32

2.2.4. Ethical statement ...... 33

2.2.5. Experimental animals and diet ...... 33

2.2.6. Study Design ...... 33

2.2.7. Phytochemical analysis ...... 36

2.2.8. Acute toxicity study ...... 36

2.2.9. Induction of acetic acid colitis ...... 37

2.2.10. Disease activity Index ...... 37

2.2.11. Macroscopic damage...... 39

2.2.12. Microscopic damage ...... 40

2.2.13. Myeloperoxidase assay ...... 40

2.2.14. Malondialdehyde assay ...... 41

2.2.15. Antioxidant assay ...... 43

2.2.16. Tissue Cytokine ELISA ...... 43

2.2.17. Statistical analysis ...... 44

2.3. Results: ...... 45 2.3.1. Phytochemical and HPLC analysis ...... 45

2.3.2. Acute toxicity test ...... 45

2.3.3. Effect on Disease activity index ...... 48

2.3.4. Effect on macroscopic damage ...... 50

2.3.5. Effect on microscopic damage ...... 54

2.3.6. Effect on MPO activity ...... 61

2.3.7. Effect on Malondialdehyde activity ...... 61

2.3.8. Effect on antioxidant activity ...... 62

2.3.9. Effect on Interferon- and tumour necrosis factor- levels ...... 66 XVII

2.3.10. Effect on IL-17 levels ...... 67

2.4. Discussion ...... 72 Chapter 3 ...... 78 3. Flaxseed in diarrhoea and spasms ...... 78 3.1. Introduction ...... 79 3.2. Materials and Methods ...... 81 3.2.1. Standard drugs...... 81

3.2.2. Animals ...... 81

3.2.3. Working stocks ...... 82

3.2.4. Castor oil-induced diarrhoea ...... 82

3.2.5. Enteropooling Assay ...... 83

3.2.6. Charcoal meal gut transit test ...... 84

3.2.7. Isolated rabbit-jejunum preparations ...... 84

3.2.8. PDE inhibitory (PDEI) activity ...... 85

3.2.11. Ca++ antagonist activity ...... 87

3.2.13. Statistical analysis ...... 88

3.3. Results ...... 89 3.3.1. Effect on castor oil-induced diarrhoea ...... 89

3.3.2. Effect on intestinal secretions ...... 89

3.3.3. Effect on gut motility ...... 90

3.3.4. Effect on isolated rabbit jejunum ...... 93

3.3.5. Effect on PDE enzyme ...... 93

3.3.8. Effect on Ca++channels ...... 94

3.4. Discussion ...... 100 CHAPTER 4 ...... 105 4. Flaxseed and microbes ...... 105 4.1. Introduction ...... 106 4.2. Materials and Methods ...... 107 4.2.1. Quantitative antimicrobial assay (16 hrs) ...... 109

4.2.2. Quantitative antimicrobial assay (2 hrs) ...... 110

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4.3. Results ...... 110 4.3.1. Effect on enteric pathogens ...... 110

4.3.2.1. Enteropathogenic Eschericia coli ...... 117

4.3.2.2. Enterotoxigenic E. coli ...... 117

4.3.2.3. Enteroaggregative Eschericia coli...... 118

4.4. Discussion ...... 122 Chapter 5 ...... 126 5. DIFFERENT FORMS OF FLAXSEED AND POTENTIAL FOR IBD ...... 126 5.1. Introduction ...... 127 5.2. Materials and Methods ...... 128 5.2.1. Preparation of Flaxseed oil and mucilage ...... 128

5.2.1. Chemicals ...... 128

5.2.2. Animals ...... 129

5.2.3. Working stocks ...... 129

5.2.4. Acute toxicity study ...... 130

5.2.5. HPLC fingerprints ...... 130

5.2.6. Spasmodic mechanisms in isolated tissues ...... 131

5.2.7. Laxative activity in mice ...... 132

5.2.8. Castor oil-induced diarrhoea in mice ...... 133

5.2.9. Enteropooling assay in mice ...... 134

5.2.10. Antimicrobial assay in vitro ...... 135

5.2.11. Acetic acid-induced colitis in mice ...... 136

5.2.9. Statistical analysis ...... 136

5.3. Results ...... 137 5.3.1. HPLC finger print analysis ...... 137

5.3.2. Spasmogenic effect of Flaxseed oil in isolated tissues ...... 139

5.3.3. Spasmolytic effect of Flaxseed oil in isolated tissues ...... 141

5.3.4. Laxative effect of Flaxseed oil in mice ...... 145

5.3.5. Antidiarrhoeal effect in mice ...... 147

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5.3.6. Antisecretory effect in mice ...... 149

5.3.7. In vitro antimicrobial activity of Flaxseed oil ...... 152

5.3.8. Flaxseed oil in acetic acid-induced colitis ...... 155

5.3.9. Spasmodic effect of Flaxseed mucilage ...... 159

5.4. Discussion ...... 162 6. General Discussion ...... 174 6.1. Conclusion: ...... 187

6.2. Limitations of the study ...... 188

6.3. Future plans ...... 189

7. Bibliography ...... 190 8. Vita ...... 207

List of Tables

Legend Page Table # # Distinctive and common features of ulcerative colitis and 1.1 4 Crohn’s Disease.

1.2 Studies of Flaxseed outcome on cytokines. 22

1.3 Effect of Flaxseed on oxidative parameters. 23

2.1 Disease activity index scoring criteria. 38 Multiple scoring criteria to assess microscopic damage in the 2.2 42 colon of mice. 2.3 Colon and spleen wet weight to length ratio. 51

2.4 Microscopic damage score of H and E and Periodic acid-Schiff- 56 alcian blue stained colon tissues. 2.5 Levels of cytokine at 6, 12 and 24 hrs with and without Flaxseed 68 treatment in acetic acid-induced colitis in mice. 5.1 Comparison of antidiarrhoeal score of Flaxseed extract vs 148 Flaxseed oil. 5.2 Comparison of antisecretory effect of Flaxseed oil vs Flaxseed 150 extract. Colony count of Enteropathogenic E.coli, Enterotoxigenic 5.3 E.coli and Enteroaggregative E.coli treated with and without 153 Flaxseed oil in antimicrobial assay.

Comparison of mortality rate and Disease activity index of 5.4 acetic acid induced colitis mice. 157

Microscopic scoring comparison of H & E stained, and Periodic 5.5 acid Schiff-Alcian blue stained, sections of mice colon induced 158 with colitis pretreated with either Flaxseed oil or Flaxseed extract.

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List of Figures

Legend Page Figure # # 1.1 Activated adaptive immune cells and their cytokine expression 10 1.2 Pharmacological targets of IBD and approved treatments 15 1.3 Step wise approach of use of pharmacological targets in IBD 16 2.1 Study design for dosing schedules in BALB/c mice 35 High performance liquid chromatography (HPLC) fingerprints 2.2 46 of Fs.Cr (A-E); High performance liquid chromatography fingerprints of 2.3 47 standards used to compare peaks of Fs.Cr The percent change in body weight (A) and disease activity 2.4 index (B) of BALB/c mice in control, untreated and treated 49 groups, 24 hrs after the induction of colitis. The percentage area affected (A) and length of ulceration and number of inflammation1 (B), scored according to Wallace 2.5 score1 showing macroscopic damage in colon sections of 52 control, untreated groups and Fs.Cr treated groups in BALB/c mice. Type of ulcer lesion (A), area covered by ulcer (B), and the sum of A and B represented as ulcer index (C), scored for assessing 2.6 macroscopic damage parameters in colon sections of control, 53 untreated groups and Fs.Cr treated groups in BALB/c mice, 24 hrs after induction of colitis. Representative microscopic images at 200x magnification of 2.7 57 colon sections, stained with H and E. Representative microscopic images at 400x magnification of 2.8 58 colon sections, stained with H and E. Representative microscopic images at 400x magnification of 2.9 colon sections, stained with Periodic acid Schiff-Alcian blue 59 (PAS-Ab) stains. Microscopic damage in colon sections of mice stained with H and E, (A) and PAS-Ab staining (B) scored by Vilaseca1 scoring 2.10 60 (ulceration, inflammation and depth of lesion) and Dorofeyev2 scoring for H and E and mucin levels respectively Effect on Myeloperoxidase (A), and Malondialdehyde (B) activities in homogenized colon tissues of BALB/c mice 2.11 64 obtained from sham control, acetic acid (untreated), positive control and Fs.Cr pre-treated groups. Changes in activity of catalase (A), glutathione peroxidase (B), superoxide dismutase (C) and total glutathione (D) in 2.12 65 homogenized colon tissues, obtained from controls, untreated and Fs.Cr pretreated groups. Effect on IFN- levels at 6 hrs (A), 12 hrs (B) and 24 hrs (C) in 2.13 homogenized colon tissues of BALB/c mice 69 obtained from controls, untreated and Fs.Cr pre-treated groups. Effect on TNF-α levels at 6 hrs (A), 12 hrs (B), and 24 hrs (C), 2.14 in homogenized colon tissues obtained from controls, untreated 70 and Fs.Cr pretreated groups.

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Effect on IL-17 levels at 6 hrs (A), 12 hrs (B), and 24 hrs (C), in 2.15 homogenized colon tissues of BALB/c mice obtained from 71 controls, untreated and Fs.Cr pretreated groups. Possible sources of innate IL-17 in acetic acid induced- colitis and 2.16 76 effect of Fs.Cr at 6, 12 and 24 hrs of BALB/c mice. 3.1 Diarrhea score in controls, untreated and Fs.Cr pre-treated 91 BALB/c mice with castor oil-induced diarrhea. Effect on castor oil-stimulated fluid accumulation in small 3.2 intestines (A) and on castor oil-induced charcoal meal transit in 92 intestine from pylorus to cecum (B) in controls, untreated and Fs.Cr treated BALB/c mice. Inhibitory effect of methanolic-aqueous crude extract of Flaxseed 3.3 (Fs.Cr) (A), papaverine (B), and verapamil (C), against 95 spontaneous, carbachol (CCh) and high K+-induced contractions in isolated rabbit-jejunal preparations of BALB/c mice Concentration-response curves (CRCs) of isoprenaline in the 3.4 absence and presence of the increasing concentrations of Fs.Cr 96 (A), papaverine (B), and verapamil (C), in isolated rabbit jejunal preparation 3.5 Effect of Fs.Cr (A), and papaverine (B), on the cyclic nucleotide 97 content of rabbit jejunum Effect of Fs.Cr on the relaxant responses of cilostazol (A), 3.6 rolipram (B), and zaprinast (C), with and without Fs.Cr in rabbit 98 jejunum precontracted with CCh (1 µM). CRCs of Ca++ in the absence and presence of the increasing 3.7 concentrations of Fs.Cr (A), verapamil (B), and papaverine (C), 99 in isolated rabbit jejunal preparations 4.1 Effect on ~106 c.f.u of S. typhi treated with and without Fs.Cr 113

6 4.2 Effect on colony count of ~10 c.f.u of Pseudomonas aeruginosa 114 (A), and Bacillus cereus (B), treated with and without Fs.Cr 6 4.3 Effect on colony count of ~10 c.f.u of Eschericia coli K1 treated 115 with and without Fs.Cr Effect on colony count of ~106 c.f.u of vancomycin-resistant 4.4 Enterococcus faecalis (A), and Methicillin-resistant 116 Staphylococcus aureus (B), treated with and without Fs.Cr 6 4.5 Effect on colony counts of ~10 Enteropathogenic Eschericia coli 119 incubated with and without Fs.Cr for 16 hrs (A), and for2 hrs (B). 6 4.6 Effect on colony counts of ~10 Enterotoxigenic Eschericia coli 120 incubated with and without Fs.Cr for 16 hrs (A), and 2 hrs (B). Effect on colony counts of ~106 Enteroaggregative Eschericia 4.7 coli incubated with and without Fs.Cr for 16 hrs (A), and 2 hrs 121 (B). High performance liquid chromatography (HPLC) fingerprints of 5.1 Flaxseed oil. Peaks identified in Figure indicate retention time (in 138 min) of respective compounds Spasmogenic effect of Flaxseed oil on spontaneous contractions 5.2 at low doses (0.01-1 mg/ml) in the absence and presence of 140 atropine and/or pyrilamine in isolated rabbit jejunal preparations Inhibitory effect of Flaxseed oil on: Low K+ (25 mM) and high + 5.3 K (80 mM)-induced sustained contractions (A), and on 143 inhibitory CRCs on low K+-induced contraction in the absence and presence of K+-channel antagonists (B).

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Concentration-response curves (CRC) of isoprenaline in the absence and presence of the increasing concentrations of Flaxseed 5.4 oil (A), Fs.Cr (B), and papaverine (C), in isolated rabbit jejunal 144 preparation, constructed against CCh (1 µM)-induced contractions. 5.5 Effect of Flaxseed oil on laxative score in absence and presence 146 of atropine pre-treatment (A), and on %age of wet feces (B). Effect on castor oil-induced diarrhoea (A), and on castor oil- 5.6 stimulated fluid accumulation in small intestines (B), of controls, 151 untreated and Flaxseed oil pre-treated BALB/c mice. Effect on colony counts of ~106 Enteropathogenic Eschericia 5.7 coli (A1-A2), Enterotoxigenic Eschericia coli (B1-B2), and 154 Enteroaggregative Eschericia coli (C1-C2), incubated with and without Flaxseed oil (A1-C1), and Fs.Cr (A2-C2). Spasmogenic effect of Flaxseed mucilage on spontaneous 5.8 contractions in the absence and presence of atropine and/or 160 pyrilamine in isolated rabbit jejunal preparations. 5.9 Effect of Flaxseed oil on laxative score in absence and presence 161 of atropine pre-treatment (A), and on %age of wet feces (B).

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CHAPTER 1

1. INTRODUCTION AND BACKGROUND

1.1. Inflammatory Bowel Disease

Inflammatory Bowel Disease (IBD) is the idiopathic intestinal inflammatory condition that involves colon and/or other part of intestine (Baumgart & Carding,

2007). It can be triggered as a result of exposure to microbes or any other environmental factor in a genetically susceptible host leading to persistent dysregulated immune response (Xavier & Podolsky, 2007). It is broadly categorized into Ulcerative colitis (UC) and Crohn’s disease (CD) sharing some distinctive and some similar features outlined in Table 1.1.

1.2. Epidemiology in Asia and Pakistan

IBD was thought to be associated as disease of the developed world with greater prevalence in Northern Europe, the United Kingdom, and North America; these rates are however, beginning to become steady in these places. On the contrary

Southern Europe, Asia, and most developing countries have shown a rising trend in incidence rates and prevalence in both UC and CD (Loftus, 2004), particularly in

Asian subcontinent and hence, IBD is now considered a global disease (Lakatos et al., 2004; Thia et al., 2008).

The growing modernization (Gismera et al.,2008) and westernization of the population has been considered the reason for this trend in Asian countries (Goh &

Xiao, 2009; Leong et al., 2004). This range of prevalence in Asian countries has increased roughly 11 times for UC, ranging from 4/100,000 to 44/100,000 people, however, the rising trend is relatively slower for CD patients with prevalence ranging 3.6/100,000 to 7.7/100,000 people (Lee et al., 2000; Ouyang et al., 2006;

Sood et al., 2009). The trend of UC prevalence seems to be more common in Asia as compared to for CD (Ahuja & Tandon, 2010). In India, prevalence of UC has been reported to be as high as 42.8–44.3/ 100 000 (Ouyang et al., 2005). This is in accordance with the other reports where racial differences in the prevalence of UC and CD showed higher incidence and prevalence of UC in Indians living in

Singapore as compared to Chinese and Malay populations (Tan and Goh, 2005; Lee et al., 2000).

Although true prevalence of IBD in Pakistan is still unknown, different studies have compared the incidence of UC in rectal bleed patients in different large referral centers and it has been reported to be 25-44% between 1989-2010 (Jafri,

1989; Khan et al., 2010). CD is also on a rise in Pakistan according to rough estimates from gastroenterologists of the country (Rogler et al., 2012).

Table 1.1: Distinctive and common features of Ulcerative colitis and Crohn’s Disease1,2

Features Ulcerative colitis Crohn’s Disease Can occur anywhere in the Location Involves portions of colon GI tract preferentially (rectum specifically) terminal ileum

Distribution Diffused Focal (segmental)-skip Limited to the mucosa of the lesions; All layers are colon; Superficial involved with sub mucosal inflammation layer affected to greatest; transmural inflammation; discontinuous skip lesions

T cells involved Predominant Helper type 2 Predominant Helper type 1 (Th2)-T cells; Th17 cells also (Th1); Th17 cells also act as involved as mediators. mediators.

Common symptoms Diarrhoea in all cases with Intermittent diarrhoea; Less varying intensity of blood bloody than UC. and mucus Abdominal pain, fecal Abdominal pain, fecal urgency and weight loss urgency and weight loss

Contradictory Constipation in left-sided Constipation in some cases symptoms colitis. of CD.

Histological Depletion of goblet cells Aggregation of macrophages characteristics mucin; diffused neutrophil frequently forming non- and lymphocytic infiltration; caseating granulomas. micro-abscesses in lamina Earliest mucosal lesions in propria (LP) due to edema. CD often appear over Lack granulomatous Peyer’s patches characteristics

Colonoscopic Diffused inflammation and/or Apparent cobblestones appearance ulceration Focal, lineal ulcers;

1(Cotran et al., 1999); 2 (Monteleone et al., 2011b).

1.3. Pathophysiology

There are four basic pathophysiological tenets of IBD that includes genetics, immune dysregulation, microbes and barrier defect (Kucharzik et al., 2006). The disease develops from multiple loci and their integration is what manifests disease and makes it multifactorial. External environmental factors cause epigenetic modifications, thereby triggering metabolic pathways and with altered composition of the gut flora initiates an inflammatory response (Scharl & Rogler, 2012).

1.3.1. Role of External environment

Many environmental factors have been attributed as a cause for triggering

IBD. These include use of refined sugar, perinatal events, childhood infections, microbes, stress, dysregulated enteric flora, tobacco, diet, appendectomy, certain drugs (antibiotics, oral contraceptive pills and likely non-steroidal ant-inflammatory drugs) and domestic hygiene (Baumgart & Carding, 2007; Blumberg, 2009) but the strength of association needs to be further validated (Koutroubakis et al., 1995).

Amongst them, non-smoking status has shown a correlation with UC and smoking has shown a correlation with CD (Tysk et al., 1988).

1.3.2. Role of Genetics

Genetic studies highlight that IBD is a polygenic disease, although these factors may be more important in CD than in UC (Orholm et al., 2000). Genes defective in IBD are the ones mostly involved in regulation of innate defenses as well as adaptive immune defense. Besides, some overlapping immunogenetic pathways also exist between CD and UC with immune dysregulation as a common problem (Shih et al., 2008). Latest evidence also suggests that certain environmental factors may cause/trigger epigenetic modifications and may possibly contribute in heredity (Scharl & Rogler, 2012). It’s not a single gene which causes the disease rather there are multiple defects which produce an additive effect or epistasis (Scharl

& Rogler, 2012). This multiple genes involvement can be correlated with failure to attain remission, which further emphasizes on need of multi-targeted approach for

IBD therapy.

1.3.3. Role of Barrier defect

The intestinal epithelial barrier is composed of intestinal epithelial cells

(IECs) and its paracellular spaces are sealed by junctional complexes. IECs also express different other mucosal monitors including mucin secreting goblet cells, trefoil peptides (Mashimo et al., 1996), prostaglandin receptor EP4 (PTGER4)

(Xavier and podolosky, 2007) and paneth cells (Peterson & Artis, 2014), all of which are involved in regulating the epithelial barrier function and maintain homeostasis.

Defect in these barriers result in leaky barrier which causes passive diffusion of ions and water from the circulation to the intestinal lumen as well as alteration in the motility (Andus & Gross, 1999), thereby causing leak flux diarrhoea (Hering &

Schulzke, 2009). Functional defect in IEC leads to dissemination of invasive bacteria through the epithelium (Benjamin et al., 2013) resulting in abundant release of mucous, serum protein, blood, and fluids into the bowel, further leading to exudative diarrhoea and triggering or perpetuation of the inflammation (Hering & Schulzke,

2009).

1.3.4. Role of Immune dysregulation

Development of IBD is thought to be mediated by an overt immune response initiated innate immune cells which is subsequently taken over once adaptive immune system is activated (Bamias et al., 2005). Recent advancement in evidence based studies confirms the role of defective innate immune system in IBD.

Professional and non-professional APCs are the innate immune cells that predominantly participate in intestinal immune regulation. Dendritic cells (DCs), macrophages (Mᵩ), B cells, gamma-delta (T cells, innate lymphoid cells (ILC) and natural killer cells (NK) cells are the predominant professional APCs. They have 6 pattern recognition receptors on their surface or cytosol and after recognizing the antigen they express major histocompatibility (MHC) class II proteins and also simultaneously release co-stimulatory molecules. These APCs are recognized by Th cells. Non-professional APCs are the ones which do not constitutively express MHC class II proteins and only express when stimulated by some cytokines; they include all nucleated cells such as epithelial cells and vascular endothelial cells in gut tissues.

These cells mostly express MHC class I proteins and therefore are presented to cytotoxic T cells (Xavier and Podolsky, 2007)

In IBD predisposed patients, the balance is disturbed at multiple levels of innate encounters. In normal individuals, DCs are conditioned to not trigger immune reaction upon encounter with commensals and only when there’s an imbalance in protective versus aggresopsive commensals, then DCs get activated in normal hosts, to maintain gut homeostasis. In IBD predisposed individuals, due to defective conditioning of DCs, they incorrectly recognize commensals as pathogens and therefore initiate an inflammatory response against them.

Another defect of innate immune system involves an imbalance between immature DCs (iDCs) and natural killer cells. In IBD predisposed patients, the ratio of iDCs and NK cells is disturbed with lesser iDCs and higher NK cells, thus favouring NK cells to cause DC maturation; the predominance of mature DCs result in maintenance of inflammatory state due to their longer survival. Naive T cells are differentiated by these matured DCs into effector T cells and NK T cells.

Effective downregulation of intestinal macrophages (Mᵩ) phenotype and function is another defect in innate immunity attributing to IBD pathogenesis. The reason why downregulation of Mᵩ attribute in pathogenesis is because these Mᵩ are actually the proinflammatory monocytes from the circulation that migrate to the site of immune reaction in gut lamina propria and unlike normal Mᵩ, they are converted into an “inflammation anergy state” (Smythies et al., 2005) with still exhibiting the ability to maintain bactericidal role through their phagocytic mechanisms. In IBD predisposed patients, these proinflammatory blood monocytes retain their

7 proinflammatory state because these Mᵩ are downregulated and hence, these Mᵩ retain their proinflammatory properties like normal Mᵩ (Grimm et al., 1995; Smith et al., 2009). Consequent inflammatory response of macrophages results in release of interleukin (IL)-6 and tumour necrosis factor (TNF)- which further aggravates the inflammatory process in IBD.

Defect in structure or function of intestinal epithelial cells (IEC) also lead to defective innate immune response. IECs, being the non-professional APCs, normally induce anergy in CD4+T cells; in IBD, in the presence of interferon gamma

(IFN-) and TNF-these IECs may be converted into functional APCs due to increased expression of both the histocompatibility molecule (Cruickshank et al.,

2004) and co-stimulatory molecules (Nakazawa et al., 2004). These APCs then activate effector T cell responses; ultimately, amplification and perpetuation of vicious circle of inflammation begins that subsequently results in tissue damage, causing rectal bleed, diarrhoea, abdominal pain and other associated symptoms of

IBD. T cells are alternately also activated by IECs via an alternate route of non- classical MHC molecules (Van De Wal et al., 2003).

Contrary to innate immune arm which is activated through bacterial/toll ligands, adaptive immune arm is simultaneously activated through innate immune response, but requires atleast 4-7 days to take its effect (Albert et al., 2002). The initiation of adaptive immune arm involves degradation of intracellular and extracellular pathogens in acidified vesicles of an APC. These APCs processes some part of antigens into peptides which are then linked to MHC class II proteins present on that APC and presented to CD4+ T cells. This presentation of peptides to T cells by DCs is a point where innate immunity is linked to adaptive immunity. In some cases B cells are activated causing secretion of antibody (Ig) to eliminate pathogen.

Consequently, cytokine chemokines and upregulation of membrane receptors on the cell surface of the T cell and APC directs the polarization of T cells into inflammatory lineage (Th1 or Th2 or Th17) or regulatory lineage (Tregs) (Bamias et al., 2005). The inflammatory lineages have been elaborated in Fig. 1.1. In normal

8 individuals, the proinflammatory activation and suppression of inflammation are in co-ordination by virtue of which they maintain gut homeostasis (Izcue et al., 2006).

In IBD predisposed patients, due to the defect in the regulatory mechanisms, their functioning is dysregulated due to which an overt proinflammatory response occur that downregulates ant-inflammatory mechanisms and cause a persistent inflammatory cascade. As a consequence, more inflammatory cells are migrated from surrounding vasculature towards the intestinal mucosa. The influx of aggressive metabolites and mediators lead to tissue damage. Furthermore, due to fibroblast growth as a consequence, collagen secretion is initiated which also leads to stricture formation (Keshavarzian et al., 2003; Baumgart and Carding, 2007).

1.3.4.1. Role of neutrophils

Neutrophils have an exceptional property of oxidative burst that results in release of toxic reactive oxidant species (ROS) and proteases and bioactive peptides

(Segal & Coade, 1978). When pathogen comes in contact with neutrophils, it fuses with its phagosomes (Segal et al., 1980) causing activation of the phagosomal membrane and in turn undergo oxidative burst releasing ROS and leading to a series of oxidative reactions forming superoxides (Segal & Coade, 1978). Neutrophil phagosomes also have a high proteolytic activity as they release proteases, antimicrobial peptides as well as bactericidal/permeability-increasing proteins

(Borregaard & Cowland, 1997; Ganz et al., 1985). Besides these, azurophilic granules in neutrophils also fuse with phagosome causing the release of myeloperoxidase (MPO). This MPO catalyzes the transformation of superoxide into hypochlorous acid, chlorines, chloramines, hydroxyl radicals, and single oxygen which are toxic molecules for microorganisms (Klebanoff, 2005). Under certain pathological conditions neutrophils reach intestinal epithelium and release monocyte chemoattractants. Thus shortly after neutrophils, monocytes are recruited that are converted into macrophages and then dominate the inflammatory mileu.

Figure 1.1; Activated adaptive immune cells and their cytokine expression. Antigen presenting cells (APCs) present the processed peptide via MHC class II to T cell receptor (TCR) present on T cells or via MHC class I to B cell receptor (BCR) to generate type of T or B cells respectively. T cells express different glycoprotein on their surfaces with CD4+ on Th cells and Treg cells, CD8+ on Tc cells and either of CD4+ or CD8+ on Tm cells. Depending on the adhesion molecules and the co-stimulatory molecules from APC, the type of T cells is polarized into Th1, Th2 or Th17 cells.

[APCs = antigen presenting cells; GATA = Trans-acting T-cell-specific transcription factor- 3; IFN- = interferon gamma; IL = interleukin; MHC = major histocompatibility complex; ROR- = retinoic acid receptor gamma; TGF- = tumour growth factor-beta Th = T helper;

Teff = Effector memory T cell; Tem = T memory cells; TF= transcription factor]. Information taken from (Bamias et al., 2005)

1.3.4.2. IL-17 release from innate immune cells

Earlier it was believed that IL-17 is the cytokine produced from Th-17 cells only and has a role in adaptive immune regulation. However, innate source of IL-17 is under studied. Some of the sources of IL-17 from innate immune cells include group 3 innate lymphoid cells (ILCs) that produce IL-17 and IL-22 and have dual role in protective and pathogenic effects in colitis (Spits et al., 2013; Sedda et al.,

2014). Another cell type involved in innate production of IL-17 is T cells

(intraepithelial lymphocytes) and release IL-17 from within a few hours after exposure with IL-23 (Oppmann et al., 2000; Smits et al., 2004). They have been reported to confer pathogenic (Do et al., 2016) as well as protective effect in different in vitro and in vivo models of IBD (Kuhl et al., 2007). Neutrophils are also the source of IL-17 in some animal models due to which they further attenuate the disease manifestation (Li et al., 2010; Katayama et al., 2013). Hence, IL-17 has an important role in innate immunity. However, their protective or pathogenic role depends on the source from which they are released and have role in both protection and pathogenicity in colitis (Fuss et al., 2011).

1.3.5. Microbes

In IBD there is an aberrant response against the commensal microbes in a genetically susceptible host because of misbalance between microbes and host defenses, although evidence also exists that this response by bacterial flora is attributed to disruption of the mucosal barrier or altered immune response (Rescigno,

2008). Following are the different forms in which microbes are involved in the pathogenesis of IBD. Microbial dysbiosis have been evident in IBD patients with an increased concentration of bacteria (Swidsinski et al., 2002) of proinflammatory phenotype (Matsuda et al., 2000). Pathogenic microbes are also implicated in the initiation of IBD in genetically susceptible hosts.

Amongst the pathogens, Adherent/invasive Escherichia coli (AIEC) is the pathogenic variants of E. coli which has been isolated frequently from intestinal 11 tissues of ileal CD patients (Boudeau et al., 1999; Smith et al., 2013). They possess specific properties of pathotypes of diarrhogenic E.coli (DEC) including enterohemorrhagic (EHEC), and enteroaggregative (EAEC) and enteroinvasive

(EIEC) (Santos et al., 2015). Consistent with the above mentioned facts, broad- spectrum antibiotics have shown improvement in clinical outcomes in patients with

IBD (Campieri & Gionchetti, 2001).

1.4. Pharmacotherapy

Current pharmacotherapeutic modalities against the pathological targets of

IBD are illustrated in Fig. 1.4. The stepwise approach has been designed to streamline the therapeutic options begins with the least toxic drugs. If these approaches do not provide relief, then drugs from a higher step are used. However, an aggressive disease stage requires the use of step 3 or 4 drugs earlier in the treatment.

The 1st step of the therapeutic paradigm is to use either sulfasalazine (SASP) or 5-aminosalicylic acid (5-ASA). However, SASP is reported to bear significant side effects which are attributed to sulfapyridine which include abdominal pain, headache, indigestion, nausea and vomiting. To avoid these side-effects, only 5-

ASA preparations were synthesized which were better tolerated as compared to

SASP (Feagan & MacDonald, 2012; Peppercorn, 1984).

If patients do not respond to 1st step, then the recommended 2nd step is to use corticosteroids. Although steroids have shown to induce remission in those patients who present the disease episode for the first time or with single exacerbation

(Mayberry et al., 2013) but they have not been very successful in maintaining remission. Besides, their long term use is associated with debilitating side effects which limits their use. Another concern with their use is either development of steroid resistance or steroid dependence. Despite of better results as compared to step 1 drugs, approximately 30% of patients using steroids still fail to attain clinical

12 remission. Subsequently, such patients become candidate for step 3 drugs, such as immunosuppressive and/or biological agents.

Immune modifiers are used as the 3rd step; they include azathioprine, 6- mercaptopurine, cyclosporin (CSA), tacrolimus, methotrexate and mycophenolate mofetil. Amongst these mentioned drugs, only CSA is recommended for acute UC cases, whereas, the rest of them cannot be used for acute UC cases. Current biologicals include anti-cytokine drugs that could be either used as a 3rd step rather than using immune modifiers and in case of aggressive disease may be used as 1st line of therapy for patients who present with aggressive diseased state and with periannal CD.

In case of inadequate response to standard therapies, biologicals are the next options. Amongst them anti-TNF- drugs are the most commonly used with infliximab, adalimumab and cetrozilumab being approved by Food and Drug

Administration (FDA) of USA, for treatment of CD (WGO, 2015). Limitation of current cytokine therapy is that it poses serious side effects including serious and minor infections, risk of lymphomas, and increased risk of opportunistic infections

(Ford & Peyrin-Biroulet, 2013). That’s the reason why despite being the most effective in the available treatment options, cytokine therapy is kept as a 3rd line of action when the escalating response is required (Rogler, 2015).

Supporting therapy is used to manage the symptoms as well as reduce the triggers of the disease. These include antibiotics to reduce the microbial overload that may have acted as a trigger, antidiarrhoeals and antispasmodics to relief from diarrhoea and abdominal pain.

Antibiotics most commonly used in practice are metronidazole and ciprofloxacin for CD (Sutherland et al., 1991; Arnold et al., 2002) and UC (Rahimi et al., 2006; Sartor, 2004). The limitation however is that with recurrent or prolonged antibiotics use, significant side effects are obvious causing noncompliance. Another complication is antibiotic associated colitis due to Clostridium difficile and development of antibiotic resistance (Nitzan et al., 2016). Another choice for

13 supportive therapy is the use of probiotics which although have no definite evidence but some studies support their effectiveness (Sood et al., 2009).

Although diarrhoea in IBD is a consequence of multiple causes, current antidiarrhoeal drugs only ameliorate the manifestation rather than treating cause.

They include diphenoxylate, loperamide and codeine phosphate. Diphenoxylate has both enteric and centrally-active opioid effect and affects gut motility. This drug can cross the blood–brain barrier due to which physical dependence is most common side effect. Euphoria and other psychoactive effects are also evident and that’s why manufacturer have added atropine to deter the abuse since in low doses it could augment the antidiarrhoeal effect of diphenoxylate, but at high doses cause unpleasant side effect like tachycardia, dry mouth and blurring of vision (Gutstein

& Akil, 2001). In contrast to diphenoxylate, loperamide has minimal central nervous system effects because of low oral absorption and inability to penetrate blood brain barrier. Accompanied constipation is a side effect for all these antimotility agents which can sometimes lead to complications like toxic megacolon which can be life threatening (Brown, 1979). It has been earlier reported for natural products that they contain components that exhibit dual mehcniasms, being effective against both constipation and diarrhoea, for example psyllium husk (Mehmood et al., 2011) and ginger (Ghayur and Gilani, 2005).

Antispasmodics are used to manage the symptoms and include anticholinergics (hyoscine butyl bromide and dicyclomine hydrochloride), antimuscarinics (mebeverine; musculotropic antispasmodic activity and calcium channel blockers (Pinaverium Bromide) (Talley, 2001). Amongst these, mebeverine, is selective for smooth intestinal muscle and is devoid of associated anticholinergics’ side-effects. Pinaverium has a high degree of selectivity for the intestinal smooth muscles specifically colon (Beech et al., 1990; Guslandi, 1994).

Figure 1.2; Pharmacological targets of IBD and approved treatments. The basic targets that are currently maneuvered in IBD include immune dysregulation, oxidative stress and microbial dysregulation, whereas, diarrhoea and spasm are the manifestation of above mentioned dysregulations and are managed with drugs.

[Abbreviations: 5-ASA = 5-aminosalicylic acid; CCB = Ca++ channel blockers; 6-MP = 6- Mercaptopurine; AZA = Azathioprine; MTX = Methotrexate; anti-TNF-anti tumour necrosis factor-

Figure 1.3; Step wise approach of use of pharmacological targets. Steps presented according to guidelines of World Gastroenterology Organization (WGO). Figure’s idea adopted from Pancione et al., (2008). The details of the steps are elaborated in section 1.3.

1.4.1. Advancements in IBD Pharmacotherapy

IBD is a rapidly growing field of research because with the current therapeutic paradigm, rate of relapses is significantly high. Hence, failure to attain remission or relapse led to discovery of other options. The current advancements include discovery of other biological options including anti-cytokine therapies, recombinant ant-inflammatory cytokine therapies, inhibitors of SMAD7 (Mothers against decapentaplegic homolog 7), kinase-inhibitors, non-selective anti-integrin antibodies, gut selective anti-adhesion molecules antibodies, anti-chemokines,

ICAM1 (Intercellular Adhesion Molecule 1) antisense oligonucleotide, delayed- release phosphatidylcholine as well as use of different helminth parasites to enhance regulatory mechanisms (Rogler, 2015).

1.4.2. Limitation of pharmacotherapy

The current pharmacotherapeutic options have a number of limitations which include dangerous adverse effect profile, poly-pharmacy and high cost leading to non-compliance and monitoring issues particularly for those patients who do not respond to conventional drugs and have to be prescribed second and third line drugs.

Current IBD treatments are associated with significant side-effects, such as weight gain, cushingoid appearance, osteopenia/osteoporosis, pancreatitis, and systemic immune suppression (Mackner et al., 2004). Amongst the recombinant cytokines, IFN-γ and TNF- are known to cause transient lymphopenias with monocytopenia and in some cases neutrophilia; flu-like symptoms have also been reported with the use of interferons. Depression, fatigue, irritability, loss of appetite and sleepiness are evoked by both IL-2 and IFN- The dangerous side-effect profile of these drugs indicate an overall need for more safe and effective treatments for therapies that could modulate the expression of cytokines but with a relatively safer profile and hence, phytotherapy seems to be a promising approach for addressing the cytokine modulation.

These side effects need to be given significant consideration because the duration of therapy is long for IBD medicines; in some cases, it could be for life time which adds in to the non-compliance. Also, the associated side-effects may be more drastic in Pakistani setting, than seen in the west because Pakistan, like other developing countries still have burden of tuberculosis and hepatitis which can be flared with the 2nd and 3rd line drugs used in IBD (Panaccione et al., 2008). Because of the burden of these diseases, additional monitoring is also imperative as these silent diseases could be reactivated as a result of immune suppression (Pai et al.,

2005).

Another considerable factor with current therapeutic strategy is expense of therapy for the patient in developing countries with an added expense of extensive monitoring (Pai et al., 2005). The estimated mean annual cost in US on IBD was $

8,265 for CD and $5,066 for UC (Kappelman et al., 2008) which is comparable to per capita income of Pakistan (US $1,027 in 2008). For an average Pakistani, this is clearly not an affordable cost.

Despite of these limitations, patients who opt for therapy, proportions of them fail to enter in remission phase remains high possibly due to multiple triggers.

The rates of maintaining remission are fairly low for 5-ASA as the induction of clinical remission in case of left-sided colitis and extensive UC, was observed in only 20% of patients treated with oral 5-ASA (Sutherland & MacDonald, 2006) and those patients who are prescribed steroids, amongst them clinical remission will not be attained in approximately 30%. These patients then become candidate for step 3 drugs such as immunosuppressive &/or biological agents and only 40% of these patients attain remission (Creed & Probert, 2007). Around 20% of CD and UC patients to whom biological were given required intestinal resection after 2-5 years.

This highlights the fact that there are a high proportion of patients with IBD who are not entering remission (Peyrin-Biroulet & Lémann, 2011).

1.4.3. Natural products as a therapeutic option

These limitations mentioned above warrant for therapies that could help induce and attain remission with safe and cost-effective treatment. Knowing that the trend of IBD in Asia is rising and the resources of developing countries are scarce to bear both the burden of cost as well as serious side-effects; natural products are efficacious, safe and cost effective option, in concordance with rational drug use criteria by World Health Organization (WHO, 1988).

Natural products hold a lot of promise as therapeutic aid in IBD because they can maneuver multiple targets simultaneously and are easily accessible and perceived as relatively safe when taken along regular medications. However, limited natural products enter into clinical trials owing to their lack of pharmacological rationale. Hence, in order to achieve maximum benefits to the patients and the community, ancient and modern therapeutic skills will have to work in a complementary manner (Patwardhan & Hooper, 1992). This could be advocated by combining a rich traditional knowledge about natural products with sound pharmacological studies that would enrich the library of natural products which could predict potential new candidates for IBD therapeutics. Thus, natural products hold lots of promise in IBD therapeutics.

Natural products are the globally rising alternative for people as their use as medicine and/or dietary supplements has also proven effective in keeping body healthy and free from diseases (Sen et al., 2011). In developing countries, about 75-

80% of the global population relies on traditional medicines, mainly the herbs for the healthcare purpose (Bodeker, 2001, 2006; Kamboj, 2000). The rising trend toward traditional healthcare systems is a global phenomenon indicated by the worth of global market for medicinal and aromatic plants being US$ 62 billion in the year

2002, whereas, by 2050, it is estimated to reach US$ 5 trillion (Purohit & Vyas,

2004).

In last decade or so, the advancement for identifying drugs from natural products had gone to hull because of success of genetic mapping techniques,

19 however, the high cost and limited benefit along with redundancy of the pathways, focus has again been diverted to identify natural sources as an important therapeutic means (Macha et al., 2015).

Since plant materials are known to contain multiple components they seem to be a promising option for ameliorating multiple targets. Multifactorial diseases like IBD have shown limited success with single chemical moiety despite of their specific effect. Herbal therapy on the other hand offers specificity for multiple aspects simultaneously with lower affinities due to the presence of multiple compounds. Thus of late, interest in herbal medicines for IBD has risen as it could provide with safe, effective and multi-targeted therapy (Spelman et al., 2006).

1.5. Potential of Flaxseed in IBD

After an extensive literature review, it was decided to evaluate Linum usitatisimum (Flaxseed) in animal model of IBD (Duke et a., 2002; Gruenwald et al.,1998; Evans, 2009; Rahimi et al., 2010; Usmanghani et al., 1997).

Flaxseed (Linum usitatissimum) belongs to family Linaceae (Local name:

Alsi); genus Linum. It appears as a flat and oval seed with a pointed tip with 3-6 mm length and 2-3.5 mm diameter. It is considered as a functional food (Tarpila et al.,

2004; 2005) with 40% -50% oil contents and meal, protein (23% - 34%), ash (4%) and ted viscous fiber (mucilage 5%) (Muir & Westcott, 2003). It is used for pain sores, local inflammations, pneumonia, bronchiolar inflammation, peritonitis and rheumatism (Usmanghani et al., 1997).

Flaxseed has been effective in various diseased models with an inflammatory manifestation or pathway involved such as in models of rheumatoid arthritis

(Kaithwas and Majumdar, 2010b), cancer, diabetes, cardiovascular disease, osteoporosis (Toure' & Xueming, 2010; Westcott & Muir, 2003), infections

(Christofidou-Solomidou et al., 2011a), as well as ulcer (Kaithwas & Majumdar,

2010a). Flaxseed is a potential herb to be explored for IBD therapeutics as it has a remarkable spectrum of activity against varied pathological aspects of IBD such as 20 microbes (Kaithwaset al., 2011), cytokines and oxidative stress in different diseased models (Kaithwas & Majumdar, 2010b; Toure' & Xueming, 2010; Westcott & Muir,

2003). Based on the reported effects of Flaxseed in various diseased states, it seems to hold immense potential to ameliorate multiple aspects of IBD. Amongst the many reported effects, some of the prominent reported use of Flaxseed that is in relevance to signify its potential for IBD research is mentioned below.

1.5.1. Literature search

With respect to research on IBD, following are the gaps which have been identified which need to be addressed:

The traditional use of Flaxseed has yet not been validated on any model of

IBD except two studies that studied the effect of Flaxseed diet and Flaxseed oil on colitis model of mice and failed to produce any beneficial effect under the study circumstances (Dugani., 2012; Zarepoor et al., 2014).

Since IBD is a disease with an imbalance between proinflammatory and ant- inflammatory cytokines, possible effect of Flaxseed on cytokines was reviewed.

Flaxseed showed modulated cytokines in various diseased models as summarised in

Table 1.2. However, effect of methanolic-aqueous (70:30) extract which contains both the polar and non-polar constituents has not yet been explored for effect on release of cytokines in animal model of IBD. Besides, effect of Flaxseed on IL-17 levels in acute injury like in acetic acid-induced colitis has not been studied.The antioxidant potential of Flaxseed oil and dietary Flaxseed have been proven in the in vitro (Kitts et al., 1999) and in-vivo systems (Pattanaik & Prasad, 1998), as summarized in Table 1.3, however, Antioxidant effect of Fs.Cr has not yet been evaluated in animal models of IBD.

Table 1.2: Studies of Flaxseed outcome on cytokines Form of Flaxseed Model Outcome Reference used 10% Dietary Radiation Reduced IL-6, IL-12, IL- (Christofidou- Flaxseed pneumonitis 17 and VEGF Solomidou et post-XRT in al., 2011a) mice

10% Dietary Thoracic radiation Reduced TGF-β1 (Lee et al., Flaxseed injury in mice 2009)

Flaxseed oil Streptococcus. Reduced IL1-level and (Saini et al., supplement (9 pneumoniae D39 increase IL-10 at 24 hrs 2010) weeks) serotype 2 followed by a decline after infected mice. 48 hrs

10% dietary Cholesterol- Reduced IL-6, and (Dupasquier et Flaxseed enriched diet in VCAM-1 al., 2007) mice.

Flaxseed lignan Asbestos- Reduced IL-1ß, IL-6, (Pietrofesa et complex Induced TNFα, and active TGF- al., 2015) Peritoneal 1. inflammation in Mice

Flaxseed oil Thiacloprid- Reduced TNF-α and IL-2 (Hendawi, et induced toxicity al., 2016) in rats

Flaxseed oil for 10 Liver injury mice Blocked (Wang et al., days model induced TLR4/MyD88/NF- 2016). by ethanol κcascades

Flaxseed in Endotoxin- Inhibited TNF- and IL-l (Caughey et al., domestic food stimulated 1996) preparation (4 mononuclear week) cells from healthy volunteers

Flaxseed oil High fat diet in IL-4 increased, TNF-α (Samina etal., supplemented diet mice and co- decreased 2015) culture of macrophages Diet supplemented High fat diet in Shift towards anti- (Bashir et al., with Flaxseed oil C57BL/6 mice. inflammatory (IL-4) state, 2015) co-culture of with decrease in pro- adipocytes – inflammatory TNF-α macrophages [Abbreviations: MDA = malondialdehyde; MYD88 = Myeloid differentiation primary response gene 88; NF nuclear factor kappa-light-chain-enhancer of activated B cells; TLR = toll like receptor; TGF = tumor growth factor; TNF = tumor necrosis factor; VCAM-1 = vascular cell adhesion molecule 1]. 22

Table 1.3: Studies of Flaxseed outcome on oxidative parameters

Form of Flaxseed Model Parameters Reference studied

Flaxseed or its In vivo hepatic Reduced SSG-Red (Yuan et al., 1999) lignan on antioxidant status in young rats

Long term Flaxseed S. pneumoniae D39 Reduced MDA, (Saini et al., 2010) oil supplementation serotype 2 infected NO. (9 weeks) mice.

Effect of Flaxseed Neurotoxicity Reduced MDA, (Moneim et al., oil model in rats NO. 2011a) induced by lead acetate

Effect of Flaxseed Renal toxicity Reduced MDA, (Moneim et al., oil model in rats NO, 2011b) induced by lead Prevented decrease acetate. in, CAT, SOD GST, Gpx and GSH.

Dietary Flaxseed Murine model of Reduced MDA (Lee et al., 2008) supplementation pulmonary levels and less before ischemia- IRI. ROS reperfusion injury Release (IRI) for 3 week

Flaxseed lignan Asbestos-induced Reduced MDA and (Pietrofesa et al., complex peritoneal NO activity 2015) inflammation in mice

Flaxseed oil Thiacloprid- Reduced MDA. (Hendawi et al., induced toxicity in 2016) rats

[Abbreviations: CAT = catalase; GPx= glutathione peroxidase; GSH = Glutathione; GST = Glutahtione S transferases; MDA = malodialdehyde; NO = Nitric oxide; ROS = reactive oxygen species; SOD = superoxide dismutase; SSG-Red= glutathione reductase].

Earlier, antimicrobial activity of Fs.Cr has been reported for S. typhi, B. cereus, P. aeruginosa and E. coli K-12 (Bakht et al., 2014; Gur, Turgut et al., 2006).

However, the method used was disc diffusion method which gives only qualitative analysis. Flaxseed in any form has not been evaluated for its effectiveness against

DECs, which are implicated in IBD. Besides, Flaxseed in any form has yet not been studied for its effect against methicillin-resistant Staphylococcus aureus (MRSA) and aggressive enteric pathogen, such as vancomycin-resistant Enterococcus faecalis

(VRE).

There is no study on the antidiarrhoeal activity of Flaxseed in any form except a preliminary report on Flaxseed oil that elaborated the mechanism of anti- inflammatory activity by employing castor oil-induced diarrhoea as an indicator of prostaglandin mechanism (Kaithwas & Majumdar, 2010b). This study has neither been conducted with the perspective of diarrhoea nor was any mechanism with respect to diarrhoea investigated. Thus, there is a clear gap in the literature on the effectiveness of Flaxseed in diarrhoea and possible mechanisms. In this aspect it can said that assessing effect of Flaxseed on diarrhoea is a virgin field in terms of research with no available literature. Since the traditional use of Flaxseed is predmoninatly for constipation (Duke, 2002; Gruenwald et al., 1998), there is no demarkation in literature as to which part of Flaxseed exhibits either laxative or antidiarrhoeal activity.

1.5.2. Selection of animal model of colitis

Intra-rectal (I.R.) administration of 6% AA to induce colitis causes colonic epithelial destruction within 4 hours (without inflammation), whereas, the maximum intensity is reached at 12 hrs which is characterized by influx of acute inflammatory cells (MacPherson and Pfeiffer, 1978; Bertevello et al. 2005) with alteration of cytokines causing inflammation and oxidative stress (MacPherson and Pfeiffer

1978). Hence this model will help us address our objectives of the study which

includes assessing cytokine modulation, neutrophil infiltration and antioxidant

status.

Since innate immune defect is an important aspect of IBD pathogenesis we

selected acetic acid (AA) -induced colitis model to induce colitis in BALB/c mice

which is an easy and reproducible method for induction of colitis in mice and

resembles human features of UC (MacPherson and Pfeiffer 1978).

1.5.3. Research Objectives

Multi-targeted therapy is the new approach in IBD therapeutics due to its

multifactorial etiology and failure to attain remission. Natural products are likely to

be effective in IBD therapeutics as they have multiple constituents and can target

multiple aspects of the disease simultaneously. Flaxseed has traditionally been used

in IBD with literature support for its numerous anti-inflammatory, antioxidant and

antimicrobial activities in various in vitro and in vivo settings. Hence, it was

hypothesized that

“Flaxseed is effective in IBD by targeting multiple aspects of the disease. The

mechanism involved may be through a combination of different activities

including ant-inflammatory, antioxidant, antidiarrhoeal, antispasmodic, and

antimicrobial activities”.

Based on the hypothesis multiple targets of disease were aimed to be

investigated as follows:

. Flaxseed ameliorates IBD due to its mucosal protective, ant-inflammatory and

antioxidant effects (chapter 2).

To address this hypothesis, the objective would be to test the effect of Flaxseed

extract (Fs.Cr) for its anti-inflammatory, antioxidant, mucosal protective activities

and on innate immune cytokines in an acute colitis model of mice.

. Flaxseed possesses antidiarrhoeal and antispasmodic activities (Chapter 3).

To address this hypothesis, Flaxseed extract will be tested on castor oil-induced

diarrhoea, castor oil-induced secretions and gut motility models in BALB/c mice.

Fs.Cr will further be studied for its possible antispasmodic mechanisms on isolated

rabbit jejunum.

. Flaxseed possesses antimicrobial activity against enteric pathogens implicated

in IBD and gastroenteritis (Chapter 4).

To achieve this, the in-vitro effect of Flaxseed extract will be tested against different

pathogenic strains of E. coli implicated in IBD including enteropathogenic E.coli

(EPEC), enterotoxigenic E. coli (ETEC) and enteroaggregative E.coli (EAEC) as

well as on enteric pathogens (Salmonella typhi, Bacillus cereus, Pseudomonas

aeruginosa) and antibiotic-resistant pathogens including methicillin-resistant

Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE).

. Other form of Flaxseed (oil/mucilage) has potential for IBD therapeutics

(Chapter 5).

To achieve this aim Flaxseed oil and mucilage will be tested for some of the

parameters of IBD pharmacotherapy (diarrhoea, spasm, microbes) and preliminary

screening against acetic acid-induced colitis.

1.5.4. Outline of Thesis

Subsequent chapters of thesis are based on individual objectives. Each

chapter has its own short introduction, methodology, but the references of all the

chapters are collectively placed at the end. The theme of the story of thesis is to

assess whether Flaxseed has potential to ameliorate IBD from multiple targets. These

targets are inflammation, inflammatory cytokines, gut motility and microbes

For this purpose initially Flaxseed extract (Fs.Cr) has been assessed in a colitis model of mice with regards to anti-inflammatory, antioxidant and cytokine modulatory activities against AA-induced colitis model of mice and the phytochemical standardization is also performed by external source (chapter 2).

Thereafter, effect of Fs.Cr on gut motility has been extensively elaborated with possible antispasmodic mechanisms and their relevance to IBD (chapter 3).

Fs.Cr is further assessed for its activity against pathogens implicated in IBD, and pathogens causing gastroenteritis as well as effect against antibiotic-resistant organisms (Chapter 4).

Finally, to assess a holistic picture for effectiveness of Flaxseed as well as for the reason of its edible status, Flaxseed’s oil has been studied for its potential in

IBD and has been screened at a preliminary level against AA model of colitis, antidiarrhoeal and antispasmodic activity and mechanisms as well as antimicrobial in vitro activity against EPEC, ETEC and EAEC.

CHAPTER 2

2. FLAXSEED IN COLITIS MODEL OF MICE

2.1. Introduction

Inflammatory Bowel Disease (IBD) which is the inflammation of bowel is of two types, namely ulcerative colitis (UC) and Crohn’s disease (CD). It manifests in a genetically susceptible host when exposed to either microbes or other environmental factors. UC is prototypically considered as T helper type (Th)2 and

CD is considered prototypically as Th1-associated disease. Recently Th17 cells have been identified as the common player in both disease phenotypes (Monteleone et al.,

2011a). Traditionally it has been believed that the adaptive immune system is the key contributor to the pathogenesis of IBD (Xavier & Podolsky, 2007). However, the recent discovery that cells mediating innate immunity are altered in IBD patients

(Cader & Kaser, 2013) along with proven efficacy of anti-TNF-α drugs in UC

(Rahimi et al., 2007); a crucial role for defective innate immunity has also been established for therapeutic interventions.

Innate immune pathogenesis causes activation of the inflammatory cytokine cascade that leads to chemotaxis and reactive oxygen metabolite (ROM) cascade.

This further causes oxidative stress (Keshavarzian et al., 2003) , resulting in further aggravation of the inflammation initiated tissue damage (Baumgart & Carding,

2007). The cytokine most commonly involved in innate immune pathogenesis of

IBD are IL-8, IL1- TNF-, IFN-and IL-12. IL-17A released from innate lymphoid cells (ILCs) (Cader & Kaser, 2013) has a controversial role as an effector or protector. Some studies have shown its pathogenic role (Fujino et al., 2003), however treatment with their antibodies in CD patients was not very effective

(Hueber et al., 2012). On the contrary, IL-17 A has been reported to have a protective role in different models of IBD (Eken & Singh, 2013; O'Connor Jr et al., 2009; Yang et al., 2008). IFN- another important cytokine has an established proinflammatory role in the pathogenesis of IBD(Cruickshank, McVay, Baumgart, Felsburg, &

Carding, 2004). Surprisingly, the clinical trials with IFN- antibody were also not promising, indicating that combating a single cytokine may not be able to control

29 diseases effectively as compared to animal studies where single anti cytokine therapies have proven effective. This led to testing the combination therapy of TNF-

 and IFN- antibodies or other combination of cytokines as an alternative approach to target the disease (Morrison, 2007).

However, a limitation of cytokine therapy is that it possesses serious side effects that require extensive monitoring, especially in developing countries that have burden of tuberculosis and hepatitis, which can be reactivated as a result of immune suppression (Pai et al., 2005). That’s the reason why cytokine therapy are usually kept as a third line of action when the escalating response is required, despite being the most effective in the available treatment options (Rogler, 2015).

Recently, there has been a lot of interest generated in alternative and complementary medicine for IBD (Langmead & Rampton, 2006). Natural products hold a lot of promise as they are mostly in common use and are perceived as relatively safe when taken along regular medications. Natural products have been tried in clinical trials in IBD patients based on their successful effect in different experimental settings. The use of natural products have proven effective in IBD therapeutics, for example, curcumin capsules improved patient symptoms as well as decreased the duration of corticosteroid therapy (Taylor & Leonard, 2011), whereas, in active mild to moderate UC patients along with amino salicylic acid, it induced remission in 4 weeks as compared to none of the patients receiving placebo only

(Lang et al., 2015).

Flaxseed is traditionally known as beneficial in IBD (Rahimi et al., 2010); however, its pharmacological basis for medicinal use in IBD has not yet been validated. Based on its multiple activity profile for different etiological factors of

IBD and due to its traditional use it was hypothesized that “Flaxseed extract is effective in mice model of colitis”.

The primary objective therefore was to assess the effectiveness in acetic acid

(AA)-induced colitis model in mice by studying their ant-inflammatory, antioxidant and cytokine modulatory effects. AA colitis model was used because it is an easy

30 and reproducible method in laboratory for induction of colitis and resembles human features of UC (MacPherson & Pfeiffer, 1978) with alteration of cytokines causing inflammation and oxidative stress.

2.2. Materials and Methods

2.2.1. Reagents

Acetic acid, DPX, prednisolone, periodic acid, and Schiff reagent were purchased from Sigma. DMSO and Tween-80 were purchased from Scharlau chemicals. Catalase activity, lipid peroxidation (MDA), myeloperoxidase

(Myeloperoxidase colorimetric activity assay kit-Biovision) and glutathione peroxidase assay kits were purchased from Biovision, whereas, glutathioine and superoxide dismutase assay kits were purchased from Sigma. Elisa kits and tissue extraction reagent for cytokines estimation were purchased from Invitrogen, Life sciences. Cenogenics single strip kit for occult blood test. Sigma Fast Protease inhibitor tablets were purchased from Sigma. 

2.2.2. Preparation of crude extract of Flaxseed

Linum usitatisimum. L (Flaxseed) was bought from the authentic herbal supplier located in the local market of Karachi, Pakistan. The plant name and physical characteristics matched the description when checked with http://www.theplantlist.org. It appeared as deep brown flat and oval seed with a pointed tip, and measured about 4 mm. A sample of Flaxseed was deposited in the herbarium of 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 grounded finely into powder by an electrical chopper. 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 & Gilani, 2005). It was filtered through a double layered muslin cloth and

31 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 methanolic-aqueous crude extract from the plant material. The combined filtrate was concentrated in a rotary evaporator at 40°C under reduced pressure (-760 mm Hg) to a thick, semisolid, dark brown coloured mass, labeled as the methanolic-aqueous crude extract of Flaxseed or Flaxseed extract (Fs.Cr), with approximated yield of 8% and was stored at -20 °C until used. The extract was tested for microbial contamination in vitro and for pyrogen activity in vivo. No microbial growth was observed when extract was plated on nutrient agar plate and grown aerobically at 37°C in an incubator for 16 hrs Animals were then administered the extract i.p., for 10 days to observe for pyrogen activity. No toxicity signs were observed in animals indicating that the extract was pyrogen free.

2.2.3. Working stocks

For the in vivo and ex vivo experiments, the extract was solubilised in 10%

DMSO-5% Tween-80. On the day of administration, subsequent dilutions of the solubilised extract were made in distilled water. For the sham control animals, 10%

DMSO-5% Tween-80 in distilled water was prepared. Prednisolone was dissolved in peanut oil. Acetic acid was diluted to 6% in normal saline. Peanut oil was verified in separate sets of experiments for its inert nature.

For HPLC analysis, sample of Fs.Cr (2 mg) was dissolved in mobile phase

(2 ml) and the sample was sonicated for 30 min. before injecting this solution, it was filtered through Millipore filter (0.45 m). The mobile phase consisted of methanol: acetonitrile: water: acetic acid (40:20:39:1 v/v) and the flow rate of 0.8 ml/min was used to deliver, while the temperature of the column was sustained at 25 C with an ultraviolet detection performed at 254 nm.

2.2.4. Ethical statement

The study protocol (005-Ani-BBS-13) was approved by ECACU (Ethics

Committee for Animal Care and Use) of The Aga Khan University, and experiments performed complied with the guidelines of the Institute of Laboratory Animal

Resources, Commission on Life Sciences, National Research Council (Clark et al.,

1996). The study design is in concordance with ARRIVE guidelines and a checklist has been followed while designing the study (Kilkenny et al., 2010). 2.2.5. Experimental animals and diet

Female BALB/c mice between the age of 6-8 weeks old (22-30 g) were obtained and maintained in animal house of Aga Khan University (Karachi), in the rectangular cages (47×34×18 cm3) with sawdust (changed at every 48 hrs). Animals were given standard diet consisting of bran (5 kg), fish meal (2.25 kg), flour (5 kg), molasses (150 g), nutrivet L (33 g), oil (500 g), potassium meta bisulphate (15 g), powdered milk (2 kg), salt (75 g), and for a total of 13 kg of the food material and maintained under standard conditions (temperature 22–25°C, with 70–75% humidity and 12 hrs light and dark cycle lighting regimen. Animals also had free access to water and food ad libitum, throughout the study, except that 18 hrs prior to induction of colitis, animals’ food was withdrawn. Mice were killed after 24 hrs by cervical dislocation and colonic biopsies were taken for different purposes after assessing for disease activity index (diarrhoea, weight loss and rectal bleed), macroscopic and microscopic damage.

2.2.6. Study Design

There were total of 6 groups named as sham control, diseased control (AA group), treatment groups (Fs.Cr 150 mg/kg, 300 mg/kg and 500 mg/kg) and the positive control group (Prednisolone). Because of high mortality rate (20%-30%),

12 animals in each group (except the negative control) were selected. Studies with power calculation of 80% show that it will require at least 10 mice per group in order to guarantee that this effect is detected in 80% of cases with a p value of 0.05. The 33 animals were first divided in the cages that were comparable after which they were randomized in each group moving horizontally through cages. Single blinding was done in histopathological scoring. The schedule for dosing is summarised in Fig.

2.1.

The dose of Fs.Cr was chosen based on the pilot experiments. Intraperitoneal

(i.p.) route was adopted to administer the test material in order to avoid the first pass effect and any metabolic changes due to gastric environment since the initial idea was to screen the potential in plant. At 6th day, animals were induced for the disease except the sham control group. All the groups continued to receive the respective treatments till the last day when mice were to be killed.

Figure 2.1; Study design for dosing schedules in BALB/c mice. Fs.Cr (solubilised in DMSO+Tween 80 in distilled water) was given i.p., in 3 different doses (150, 300 and 500 mg/kg) for 6 days before the induction of colitis and furthermore for the subsequent day after colitis. Similarly sham and diseased controls were given DMSO+Tween 80, i.p, in distilled water for all the days of experiment. Prednisolone (1.15 mg/kg) was used as a positive control, started from 1 day prior to induction for total of 3 days, whereas, vehicle solvent was used as sham control. After anaesthetising the mice, colitis was induced by IR administration of 0.1 ml of 6% acetic acid (AA) in all the animals except the sham control that received IR saline. DAI was measured for all the days of experiment.

[Abbreviations: DAI = disease activity index; DMSO = di methyl sulfoxide; i.p. = intraperitoneally ; IR = intrarectally; Fs.Cr = Flaxseed extract].

2.2.7. Phytochemical analysis

The crude methanolic-aqueous (30:70) extract of Flaxseed (Fs.Cr) was qualitatively analysed according to standard methods for the presence or absence of alkaloids, anthraquinones, coumarins, flavonoids, saponins, sterols, tannins, and terpenes (Evans, 2009).

High performance liquid chromatography (HPLC) analysis of Fs.Cr, and standard drugs (quercetin, luteolin, ofloxacin, metronidazole, nicotinic acid and nicotinamide) was performed externally by H.E.J. institute. Briefly, it was analysed using a Schimadzu (Kyoto, Japan) Prominence 20-AT equipped with a pump

(LC20AT), PDA detector (SPD-M20A) and Kromasil-100C18 column

(Teknokroma, 8 m, 25X0.46 mm) with membrane filter 0.45 m and syringe filter

0.45 m. Alpha-linoleic acid (ALA), linolenic acid (LA) and oleic acid (OA) are the known standards used for Flaxseed oil analysis, but since the methanolic- aqueous extract showed very few peaks at later retention times, which is the characteristic of polar compounds (Ignat et al., 2011), therefore speculated standards of non-polar compounds were used.

2.2.8. Acute toxicity study

A total of 20 mice (20–25 g) of either sex were equally divided into four groups (n = 5). The test was performed by administering Fs.Cr in different doses

(300, 500 and 1000 mg/kg; i.p.) Negative control group of mice was administered control vehicle (10 ml/kg; i.p.). The animals were allowed food and water ad libitum and were regularly observed every 30 minutes up to 6 hrs for the signs of toxicity, while the lethality was monitored up to 24 hrs. Toxicity signs assessed were piloerection, exploratory behavioral changes and blindness.

2.2.9. Induction of acetic acid colitis

Intrarectal (IR) administration of 6% acetic acid (AA) is an easy and reproducible method in laboratory for induction of colitis which resembles human features of UC (MacPherson & Pfeiffer, 1978) including diarrhoea, weight loss, rectal bleed apparently and inflammation followed by ulcer in colons internally. This model was adopted to decipher the effect of the tested material in innate immune pathogenesis that has now been considered as one of the important therapeutic modality for IBD therapeutics.

BALB/c mice were fasted for 18 hrs with free access to water ad libitum, and then anaesthetised with a mixture of ketamine (100 mg/kg), and diazepam (2.5 mg/kg) in normal saline, given i.p. To induce colitis, 0.1 ml of 6% acetic acid (6% v/v, in 0.9% saline) was administered through feeding needle intra rectally which was inserted to a distance of 5 cm proximal to the anus (MacPherson & Pfeiffer,

1978). The mice were held vertically for 45 sec to avoid any spillage till that time and animals that excreted AA from colon before 45 sec were excluded from the study. Finally, the rectum was flushed with 0.2 ml saline to neutralise the solution.

Control animals only received the normal saline enema. Disease activity index was assessed after 24 hrs and then mice were killed by cervical dislocation and subsequent colonic biopsies were taken for different purposes.

2.2.10. Disease activity Index

For the assessment of the clinical severity of colitis, the body weight, stool consistency and rectal bleeding were examined from the time before induction till the day of sacrifice in a blinded fashion. Disease activity index (DAI) was the average of these three parameters (Wirtz et al., 2005) as presented in Table 2.1. To know presence of occult blood for assessing criteria of rectal bleed, hemo-occult testing was done by using stool occult blood kit (Cynogenics). A blue colouration was an indicator of occult blood; hence, based on the intensity of colour, the hemo-occult positive samples were further categorized as mild

(light blue), moderate (light to dark blue) or severe (deep dark blue).

Table 2.1. Disease Activity Index Scoring Criteria

Parameter Scoring Criteria*

Weight loss 1-5%, 6-10%,11-20% >21% =0, 1, 2, 3, 4

Stool Normal, soft but still formed, very soft, diarrhoea = 0, 1,2,3

Bleed/occult Normal, hemooccult positive, gross bleeding = 0,2,4

DAI Weight loss + stool consistency + rectal bleeding/3

*Wirtz et al.,2005; [Abbreviation: DAI: Disease activity index]

2.2.11. Macroscopic damage

Mice were killed by cervical dislocation and dissected open to remove colon, cleaned from fat and mesentry. The wet weight of the spleen and colon of each mice of the respective group were weighed and length measured. Next, the colon was opened longitudnally along the mesentric line and the percentage of area affected was measured, for which 5 cm colon area proximal to the anus was assessed for hyperemia, with or without lesions. Scoring was done as 0, 1, 2, 3, 4, 5, 6 for the respective area affected as 0, 1-5, 5-10, 10-25, 25-50, 50-75, and 75-100% respectively (Ghafari et al., 2006). Next, semi quantification of ulcer and inflammation was done by using the previously used scoring criteria (Wallace et al.,

1992): score 0 = no ulcer/inflammation; score 1 = localised hyperaemia and no ulcer; score 2 = no hyperaemia but presence of ulceration; score 3 = both ulceration and inflammation present but at one site only; score 4 = both ulceration and inflammation but at two or more than two sites; score 5 = ulceration extending more than 2 cm”.

To estimate ulcer index (Minaiyan et al., 2006) sum of score of ulcer lesion

(Morris et al., 1989) and ulcer area was determined. Lesion score considered qualitative nature of ulcer and were scored 0 when no change in macroscopic parameters was evident, it was scored 1 when there was only mucosal erythema, whereas, it was scored 2 points when there was slight bleeding/erosion and mild mucosal edema. When there was moderate edema accompanied with bleeding ulcers/erosions it was scored 3, whereas, severe edema, erosions, ulceration and tissue necrosis was scored as 4. Area of ulcer lesion was calculated by keeping the tissue on the graph paper. Each box of graph paper was considered as 1 mm2 in area, and the area of ulcerated part occupying the number of cells was counted that gave the total ulcer area. Calculation of ulcer index was done as follows:

푳풆풔풊풐풏 풔풄풐풓풆 + 풂풓풆풂 풐풇 풖풍풄풆풓 풍풆풔풊풐풏

Further to this, colonic biopsies underwent histopathological processing and analysis.

2.2.12. Microscopic damage

Histological processing of colon tissues was done to assess colonic damage by using hematoxylin and eosin (H and E) staining. Mucus content was assessed by periodic acid-Schiff-alcian blue (PAS-Ab) staining.

Briefly, 5 cm proximal part of the excised colon from experimental mice were fixed in 10% neutral buffered formaldehyde solution for 24 hrs, followed by processing through different concentrations of graded alcohol (30 minutes) and xylene (15 min for each dip with two dips in total) and then subsequently embedded in paraffin. During embedding, the colon section of each mouse was further divided into three segments (proximal third, middle third, and distal third) and each of these was further cut into two segments so that they could be placed in a single block,

Hence, producing six different sections per animal. Subsequently tissues were sectioned at 7 μm thickness by a rotary microtome (Shandon, Thermo Scientific), mounted on clean glass slides followed by overnight drying at 37◦C and then stained with H and E & PAS-Ab staining. All tissue sections were examined in an Olympus

BX 52 microscope for characterization of histopathological changes which were scored by a histopathologist who was blinded to the groups. Photographs were taken using Olympus DP21 camera mounted over the microscope and the photomicrographs were labeled using Microsoft power point 2010 software.

Three types of microscopic scoring criteria were adopted to score the damage as all the three criteria were giving some added information. The criteria includes

Neurath (score 0-4) (Neurath et al., 1995), Vilaseca (score 0-8) (Vilaseca et al., 1990) and Kiela (score 0-3) (Kiela et al., 2005). The detailed values have been enumerated in Table 2.2. Furthermore, Goblet cells were assessed on the basis of level of mucin staining (Dorofeyev et al., 2013) to quantify the loss of goblet cells.

2.2.13. Myeloperoxidase assay

Myeloperoxidase (MPO) is a marker for neutrophil infiltration (Krawisz et al., 1984) which indicates the progression of acute inflammatory process. Hence, to 40 determine the activity of MPO in supernatant of tissue homogenate obtained from colon of treated and non-treated mice, colon tissues were cleaned from faecal material and weighed and homogenized in the dilution buffer (provided with the kit) in the ratio of 1:4 with a Polytron Teflon homogenizer (Scientific, France) at high voltage input for 30 sec at 4-8C. Homogenates were subsequently centrifuged at

14000 g for 30 min at 4C and supernatant was used to run the assay according to manufacturer’s protocol 

2.2.14. Malondialdehyde assay

Malondialdehyde (MDA) is released as an oxidation product when the lipids on the mucosal barrier encounter oxidative stress. Hence, to determine the effect of

Fs.Cr on lipid peroxidation, MDA activity was assessed in supernatant of tissue homogenate of all the experimental mice. Sample from the colon tissues were homogenized in homogenization buffer included in the kit with the help of tissue homogenizer, at 4-8°C. Subsequently, they were centrifuged at 14000 g for 30 min at 4°C and the supernatant collected was used for the assay according to manufacturer’s protocol (Lipid peroxidation; MDA colorimetric assay kit-

Biovision).

Table 2.2. Multiple scoring criteria to assess microscopic damage in the colon of mice.

Scoring type Scoring variables Microscopic Score Neurath Score 1 No leukocyte infiltration 0 Low level of leukocyte infiltration 1 Moderate level of leukocyte infiltration 2 Moderate level of leukocyte 3 infiltration, high vascular density and thickening of colon wall Transmural leukocyte infiltration, loss 4 of goblet cells, high vascular density and thickening of the colon wall. Ulceration No ulcer, epithelization 0 Small ulcers, 3 mm 1 Large ulcers. 3 mm 2 Inflammation None 0

2 Mild 1 Moderate 2

Score Severe 3 Vilaseca Modified Modified Vilaseca Depth of the lesion None 0 Submucosa 1 Muscularis propria 2 Serosa 3 Keila Score3 No evidence of inflammation 0 Low level of inflammation 1 with scattered infiltrating mononuclear cells (1–2 foci) Moderate inflammation with 2 multiple foci High level of inflammation 3 with increased vascular density and marked wall thickening Mucin scoring4 <1% of stained cells 0 1-30% of stained cells; low 1 level of staining; 30-80% of stained cells; 2 medium level of staining Up to 80% of stained cells; 3 high level of staining The semiquantitative scoring system assessed the microscopic damage in AA - induced colitic mice, treated with and without Fs.Cr, and then stained with H and E. This scoring was performed by a histopathologist who was blinded to the treatment.

1(Neurath et al., 1995), 2(Vilaseca et al., 1990); 3(Kiela et al., 2005); 4(Dorofeyev et al., 2013). [Abbreviations: AA = acetic acid; Fs.Cr = Flaxseed extract; H and E = hemotoxylin and Eosin

2.2.15. Antioxidant assay

Similar to MPO and MDA, antioxidant activity was assessed in supernatant of colon homogenate of all the experimental mice. For this purpose, glutathione peroxidase (GPx) activity colorimetric assay kit (Biovision), catalase (CAT) activity colorimetric assay kit (Biovision), superoxide dismutase (SOD) assay kit (Sigma) and total glutathione activity assay kit (Sigma) were used for the respective analysis of GPx, CAT, SOD and total glutathione. The colon tissues of treated and non- treated mice were homogenized in assay buffer provided with the respective kits, via

Teflon homogenizer, except for SOD assay. For SOD assay, sucrose lysis buffer solution was prepared [(10 mM Tris (pH 8), 0.25 M sucrose, 1 mM EDTA (pH 8)] and sonicated on an ice bath (60 W with 0.5 sec intervals for 15 min). For total glutathione assay, homogenized tissues were further deproteinised in 5- sulfosalicylic acid. All the homogenates were centrifuged at 14000 g for 15 min at

4C, and the supernatant collected was used for assay according to the manufacturer’s protocol.

2.2.16. Tissue Cytokine ELISA

Next the secretory level of cytokines were assessed in supernatant of tissue homogenate of mice pre-treated with different doses of Fs.Cr (150, 300 & 500 mg/kg) and prednisolone (1.15 mg/kg) whereas, AA group received only pre- treatment with vehicle solvent. Cytokines levels were measured at 6, 12 and 24 hrs to study their kinetics. For analysis, snap frozen colon samples were thawed, weighed and homogenized via Teflon homogenizer at 4–8C in the tissue extraction reagent (Invitrogen, Life sciences), that was simultaneously supplemented with protease inhibitor cocktail (Sigma FAST). The homogenized tissues were centrifuged at 14000 g for 20 min and the supernatant was used for the measurement of mucosal TNF-, IFN-, and IL-17 with a quantitative enzyme immunoassay kit

(Invitrogen, by Life Sciences).

2.2.17. Statistical analysis

All the data expressed are mean±standard error of mean (SEM). The statistical test applied in all the biochemical assays was One-way analysis of

s post-test, whereas, cytokine analysis was׳variance (ANOVA) followed by Tukey done by Mann Whitney test. All other graphing, calculations and statistical analysis were also carried out by using Graph-PAD software (Graph-Pad, San Diego,

California, USA) and Instat Graph pad.

2.3. Results:

2.3.1. Phytochemical and HPLC analysis

Preliminary phytochemical analysis of the methanolic-aqueous crude extract

of Flaxseed (Fs.Cr) revealed the presence of tannins, flavonoids, triterpenes,

alkaloids and coumarins, whereas, saponins were weak and anthraquinones were not

detected.

HPLC finger print analysis of Fs.Cr (Fig. 2.2) and standard drugs (Fig. 2.3

A-E) were obtained. The retention times (Rt) of Fs.Cr, 3.075 min, 3.387 min, , and

5.608 min were comparable to the tested standards, nicotinic acid (2.955 min),

nicotinamide (3.327 min), quercetin (5.774 min) respectively. There were two

unidentified peaks with retention times of 2.789 min and 3.633 min that matched

with synthetic drugs ofloxacin (2.835 min) and metronidazole (3.699 min)

respectively (Fig. 2.3 A-E).

Quantitative analysis of Fs.Cr revealed the presence of quercetin (19.05 g),

nicotinamide (12.83 g) and nicotinic acid (7.07 g). The unidentified compound

whose retention time resembled with ofloxacin was detected in highest

concentration (38.99 g), whereas, the compound with similar retention time as

metronidazole was 12.39 g.

2.3.2. Acute toxicity test

Fs.Cr was well tolerated in BALB/c mice up to the tested oral dose of 1000

mg/kg, i.p. There were no apparent signs of acute toxicity over the period of

observation including piloerection, restlessness and seizures and no deaths were

recorded up to the period observed for 10 days. d .

Figure 2.2; High performance liquid chromatography (HPLC) fingerprints of Fs.Cr. Peaks of Fs.Cr resemble with nicotinic acid (Rt=2.955), nicotinamide (Rt=3.327) and quercetin (Rt=5.774). Other unidentified compounds matching synthetic compounds ofloxacin (Rt=2.835) and metronidazole (Rt=3.699). Peaks identified in Figure indicate retention time (in min) of respective compounds.

Figure 2.3 (A-E); High performance liquid chromatography fingerprints of standards used to compare peaks of Fs.Cr. HPLC fingerprints of nicotinic acid (Rt=2.955) (A), nicotinamide (Rt=3.327) (B), quercetin (Rt=5.774) (C), synthetic compounds ofloxacin (Rt=2.835) (D) and metronidazole (Rt=3.699) (E). These peaks almost resemble to corresponding peaks of Fs.Cr in Fig. 2.2. Peaks identified in Fig. indicate retention time (in min) of respective compounds.

2.3.3. Effect on Disease activity index

The disease severity was assessed 24 hrs after the IR administration of AA in the respective groups. Since AA is able to induce acute colonic inflammation, the untreated animals underwent a significant body weight loss due to severe anorexia as evident in Fig. 2.4A. Compared to untreated group, Fs.Cr and prednisolone pre- treated groups’ showed significantly lower loss in body weight. The other aspect of assessing DAI was to evaluate stool consistency and rectal bleed; untreated group showed a decrease in stool consistency from normal to being very lose, whereas, the treated groups showed improvement in this consistency. Similarly, rectal bleed was evident in some untreated mice with most of them showing an occult blood positive test, indicating internal bleeding in colon. Compared to this, Fs.Cr treated group mostly showed no rectal bleed, however, in different doses occult blood positivity reduced. Amongst the mice who tested positive for occult blood test, untreated mice showed darkest blue colouration (+++) indicating the presence of highest occults of blood as compared to treatment groups where, Fs.Cr 150 mg/kg pre-treated group showed moderate to dark blue colour (++) and mice with doses of (300 and 500 mg/kg) had light blue colouration (+++).

Hence, the overall DAI was reduced due to Fs.Cr pre-treatment dose- dependently for 150 mg/kg (p<0.01) and 300 mg/kg (p<0.001) as can be seen in Fig.

2.4B. Hence, the untreated group showed a consistent increase in DAI, whereas,

Fs.Cr reduced the DAI with increasing doses.

Figure 2.4; The percent change in body weight (A) and disease activity index (B) of BALB/c mice in control, untreated and treated groups, 24 hrs after the induction of colitis. Fs.Cr was given in doses of 150 mg/kg, 300 mg/kg and 500 mg/kg, for 7 days, i.p., prednisolone, given for 3 days (1.15 mg/kg), and used as positive control whereas vehicle solvent (given for 7 days) was used as sham control. All mice were induced with colitis by IR administration of 0.1 ml of 6% AA, except sham control that received normal saline IR. Values given are means±SEM (n = 12). One way ANOVA was used for statistical analysis followed by Tukey’s post test. **p<0.01; ***p<0.001 compared with AA (untreated) group. [Abbreviations: AA = acetic acid; Fs.Cr = Flaxseed extract; IR = intrarectally; i.p. = intraperitoneally].

2.3.4. Effect on macroscopic damage

The first thing in macroscopic inspection evaluated was wet weight/length ratio of the mice colon and spleen. It was evident that AA group had a greater average weight/length ratio of the colon and spleen as compared to the sham control, whereas, Fs.Cr treated groups at 300 mg/kg and 500 mg/kg doses had relatively smaller ratio (p<0.001) as compared to AA group indicating a possibility for a decrease in inflammatory process as evident in Table 2.3

Apparently, the AA mice colon appeared to be flabby with evidences for bowel wall thickening and greater percentage of colon area affected with ulcers

(91±4.58%), whereas, with the increasing doses of Fs.Cr, significantly less area was affected as can be seen in Fig. 2.5A, with the percentage of area affected as

59.95±5%, 31.78±7.54% and 43.15±6.4% for 150, 300 and 500 mg/kg doses respectively. Wallace score in Fig. 2.5B shows that AA group mostly showed atleast two or more than two sites of ulceration and inflammation occasionally extending to more than 2 cm (score 4.54±0.42). This trend was reversed in Fs.Cr pre-treated groups with a score of 2.83±0.1 6, 2.22±0.12 and 2.06±0.06 for 150 mg/kg, 300 mg/kg and 500 mg/kg doses respectively.

Further to this, ulcers were quantified on the basis of type of lesion and area of ulcer covered. The same protective trend from ulcers was evident in the Fs.Cr treatment group. It was found that highest lesion score was for AA group (3.66±0.57) and with increasing doses of Fs.Cr, this score reduced to 2.25±0.05, 1.95±0.6 and

1.83±0.05 for 150, 300 and 500 mg/kg doses respectively (Fig. 2.6A).

Similarly the ulcer area covered was greatest by AA group with an average of

18.22±5mm2 for AA group and decreased with increasing doses of Fs.Cr being 9.43±1 mm2,

5.88±0.5 mm2 and 4.18±1 mm2 at 150, 300 and 500 mg/kg doses respectively (Fig. 2.6B).

The previously mentioned lesion score and ulcer area was further represented as ulcer index in Fig. 2.6C. As evident, AA group had highest ulcer index (21.88) and pre- treatment with Fs.Cr significantly reduced this ulcer index to 11.68, 7.83 and 6.01 at 150, 300 and 500 mg/kg doses respectively. Hence, Fs.Cr significantly attenuated the extent and severity of the colonic injury as compared to the AA group.

Table 2.3. Colon and spleen wet weight to length ratio

Group Colon weight and length Spleen weight and ratio length ratio (gm/cm) (gm/cm) Sham control 0.03±0.002 0.05±0.003*** Untreated (AA)# 0.09±0.004# 0.07±0.003# Fs.Cr 150a 0.07±0.005 0.07±0.007 Fs.Cr 300b 0.05±0.002** 0.06±0.007* Fs.Cr 500c 0.06±0.003*** 0.06±0.007** Prednisoloned 0.06±0.001*** 0.05±0.003***

Fs.Cr (150 mg/kg, 300 mg/kg and 500 mg/kg) was administered for total of 7 days and prednisolone (1.15 mg/kg) for 3 days, i.p.. After anaesthetizing mice, colitis was induced by IR administration of 0.1 ml of 6% AA in all the animals, except in sham control that were administered 0.1 ml saline IR. One way ANOVA was used for statistical analysis followed by Tukey’s post test. All the values are expressed as mean±SEMs (n= 12).*p<0.05; **p<0.01; ***p<0.001 compared with AA (#) control group.

[Abbreviations: AA = acetic acid; Fs.Cr = Flaxseed extract; i.p. = intraperitoneally; IR = intrarectally].

Figure 2.5; The percentage area affected (A) and length of ulceration and number of inflammation1 (B), scored according to Wallace score1 showing macroscopic damage in colon sections of control, untreated groups and Fs.Cr treated groups in BALB/c mice. Colon tissues were assessed 24 hrs after IR administration of 0.1 ml of 6% AA, except in sham control which received IR saline. Tissues were obtained from mice that were killed 24 hrs after the induction of colitis. Fs.Cr in doses of 150 mg/kg, 300 mg/kg and 500 mg/kg/day was used for treatment, given for total of 7 days; prednisolone was given in dose of 1.15 mg/kg, was used as positive control. Values given are means±SEM (n = 12). One way ANOVA was used for statistical analysis followed by Tukey’s post test. **p<0.01; ***p<0.001 compared with AA. All the drugs were administered i.p. 1Wallace et al., 1992. [Abbreviations: AA = acetic acid; Fs.Cr = Flaxseed extract; i.p. intraperitoneally; IR = intrarectal]

Figure 2.6; Type of ulcer lesion (A), area covered by ulcer (B), and the sum of A and B represented as ulcer index (C), scored for assessing macroscopic damage parameters in colon sections of control, untreated groups and Fs.Cr treated groups in BALB/c mice, 24 hrs after induction of colitis. Colitis was induced by IR administration of 0.1 ml of 6% AA, except in sham control which received IR saline. Fs.Cr in doses of 150 mg/kg, 300 mg/kg and 500 mg/kg/day was used for treatment, given for total of 7 days; prednisolone in dose of 1.15 mg/kg was used as positive control, given for 3 days whereas sham control were administered vehicle solvent. Values given are means±SEM (n = 12). One way ANOVA was used for statistical analysis followed by Tukey’s post test. **p<0.01; ***p<0.001. [Abbreviations: AA = acetic acid; Fs.Cr = Flaxseed extract; i.p. intraperitoneally; IR = intrarectally]

2.3.5. Effect on microscopic damage

When the colonic tissues of animals of all the experimental group were examined microscopically Fig 2.7 (A-F) shows the photographs of the selected tissue sections, processed for histological assessment and stained with H and E. Sham- treated mice were typical of a normal morphological appearance (Fig. 2.7A).

Compared to this, AA showed acute multifocal colitis with complete loss of goblet cells from the surface epithelium of the ulcerated site and diffused neutrophil and lymphocytic infiltration transmurally, whereas, the mildly affected areas showed partially depleted cells with mixed infiltration (Fig. 2.7B). Fs.Cr treatment reduced these morphological signs with smaller ulcers, less goblet cell depletion, reduced infiltrates and limiting the infiltrates to submucosal layer mostly (Fig. 2.7 D-F).

Hence, over all the microscopic damage was improved by Fs.Cr pre-treatment. A focused view at 400X for AA and treatment groups is presented in Fig. 2.8 A-D.

When the tissue sections were stained with PAS- Ab stain in processed colon tissues of respective groups, goblet cells depletion on the basis of mucins absence or presence as well as the type of mucin (neutral, acidic, basic) could be scored. Fig 2.8

A-D shows sham-treated mice colon section with a normal goblet cell appearance that was stained pink for neutral mucin at the mucosal layer (Fig. 2.9A). Compared to this, in the AA group goblet cells were stained as purple depicting acidic mucin in the regions of the re-epithelized mucosal layer, whereas, in ulcerated areas marked mucin depletion was evident (Fig. 2.9B). When the Fs.Cr treated groups were compared with the diseased group (AA), it not only showed relatively reduced depletion but also retention of neutral mucin at the mucosal layer (Fig. 2.9 D-F).

This over all histological damage evident by H and E was further quantified using scores by Neurath et al., (1995) and Kiela et al., (2005) which are presented in

Table 2.4 (A-B) and by Vilaseca et al., (1990) which are elaborated in Fig. 2.10A.

Level of mucin by PAS-Ab stains was also quantified through a scoring system

(Dorofeyev et al., 2013) and is presented in Table 2.4 (C) and illustrated in Fig

2.10B. It appeared that AA group only showed 30% of staining as compared to sham

54 control, whereas, Fs.Cr at the dose of 300 and 500 mg/kg retained the goblet cells up to 80% as compared to the AA group (Fig. 2.10B). Hence, the AA group showed a consistent increase in microscopic damage, whereas, Fs.Cr significantly reduced this damage leading to attenuation in the extent and severity of the colonic injury as compared to the AA group.

Table 2.4. Microscopic damage score of H and E and Periodic acid Schiff-Alcian blue-stained colon tissues

Groups (A) (B) (C) Neurath1 score Kiela2 score Mucin score3 (Max score = 4 ) (Max score = (Max score = 3) 3) Untreated 3.48±0.65# 3# 1.2±0.37# Fs.Cr 150 b 2.33±0.57* 2.44±0.09*** 1.50±0.25 Fs.Cr 300 c 1.56±0.5** 1.83±0.33*** 2.100±0.25*** Fs.Cr 500 d 1.70±0.03** 1.8±0.3*** 2.3±0.5*** Prednisolone 1.27±0.02*** 1.26±0.24*** 2.5±0.6***

Column A and B represents microscopic scoring of H and E stained colon sections and column C represents stained sections of colon scored for mucin quantification by periodic acid Schiff-Alcian blue (PAS-Ab) staining. One way ANOVA followed by Tukey’s post test was used for analysis. Values expressed are mean±SEM (n=12). **p<0.01; ***p<0.001, vs AA. # Diseased control group from which the significance was measured. Abbreviations = [Fs.Cr = methanolic-aqueous crude extract of Flaxseed in mg/kg given intraperitoneally for total of 7 days; prednisolone (1.15 mg/kg) was used as positive control given for 3 days. 1(Neurath et al., 1995); 2(Kiela et al., 2005); 3(Dorofeyev et al., 2013).

Figure 2.7; Representative microscopic images at 200x magnification of colon sections, stained with H and E. Tissues stained were obtained from sham control (A), untreated (B), positive control (C), Fs.Cr pre-treated groups, 150 mg/kg (D), 300 mg/kg (E) and 500 mg/kg (F). Fs.Cr was administered to mice for 7 days whereas, positive control groups received prednisolone (1.15 mg/kg) for 3 days, whereas, sham control group were administered vehicle solvent. All drugs were administered i.p. Colitis was induced in mice by IR administration of 0.1 ml of 6% AA under anaesthesia, whereas, sham control received IR saline. Mice were killed 24 hrs after induction of colitis, colon tissues were stored in neutral buffered formalin overnight and then processed for microscopic staining by H and E staining. [Abbreviationss: Fs.Cr = Flaxseed extract; H and E = hemotoxylin and Eosin; i.p. = intraperitoneally; IR = intrarectally].

Figure 2.8; Representative microscopic images at 400x magnification of colon sections, stained with H and E obtained from untreated (A), methanolic-aqueous crude extract of Flaxseed (Fs.Cr) pre-treated groups, 150 mg/kg (C), 300 mg/kg (D) and 500 mg/kg (E) given for 6 days for the preventive effect against acetic acid (AA)-induced colitis in BALB/c mice. Prednisolone (1.15 mg/kg) was given to positive control groups 2 days prior to induction and sham control were administered vehicle solvent. All drugs were administered i.p. Colitis was induced in mice by IR administration of 0.1 ml of 6% AA under anaesthesia, whereas, sham control received IR saline. Mice were killed 24 hrs after induction of colitis; colon tissues were stored in neutral buffered formalin overnight and then processed for microscopic staining by H and E staining. [Abbreviations: AA = acetic acid; Fs.Cr = Flaxseed extract; H and E = Hemotoxylin and Eosin; IR = intrarectal; i.p = intraperitoneal].

Figure 2.9; Representative microscopic images at 400x magnification of colon sections, stained with Periodic acid Schiff-Alcian blue (PAS-Ab) stains. Tissues were obtained from untreated (A), methanolic-aqueous crude extract of Flaxseed (Fs.Cr) pre-treated groups, 150 mg/kg (C), 300 mg/kg (D) and 500 mg/kg (E) given for 6 days before induction of colitis. Prednisolone (1.15 mg/kg) was given to positive control groups 2 days prior to induction and sham control were administered vehicle solvent; All drugs were administered i.p. Colitis was induced in mice by IR administration of 0.1 ml of 6% AA during anaesthesia, whereas, sham control received IR saline. Mice were killed 24 hrs after induction of colitis, colon tissues were stored in neutral buffered formalin overnight and then processed for microscopic staining by H and E staining. [Abbreviations: AA = acetic acid; Fs.Cr = Flaxseed extract; PAS-Ab = periodic acid Schiff-Alcian blue; IR = intrarectally; i.p. = intraperitoneally].

Figure 2.10; Microscopic damage in colon sections of mice stained with H and E, (A) and Periodic acid Schiff-Alcian blue staining (B) quantified by Vilaseca1 (ulceration, inflammation and depth of lesion) and Dorofeyev2 scoring for H and E and mucin levels respectively. Colon tissues were obtained from sham control, AA (untreated), positive control and Fs.Cr pre-treated groups. Fs.Cr was given in doses of 150 mg/kg, 300 mg/kg and 500 mg/kg, for 7 days, i.p., prednisolone, given for 3 days (1.15 mg/kg) was used as positive control whereas vehicle solvent (given for 7 days) was used as sham control. All mice were induced with colitis by IR administration of 0.1 ml of 6% AA, except sham control that received normal saline IR. Values given are means±SEM (n = 6) for each group. One way ANOVA was used for statistical analysis followed by Tukey’s post test. *p<0.05; **p<0.01; ***p<0.001 vs untreated (AA) group. 1Vilaseca et al., 1990 2Dorofeyev et al., (2013) [Abbreviations: AA = acetic acid; MDA = malondialdehyde; MPO = myeloperoxidase; Fs.Cr = Flaxseed extract; IR = intrarectally; i.p. = intraperitoneally].

2.3.6. Effect on MPO activity

MPO activity was measured in the supernatant of tissue homogenate obtained from colon of the experimental mice. As evident in Fig. 2.11A, there was a marked increase in MPO activity (400±59.5 mU/ml) in untreated group as compared to sham control (18±1.54 mU/ml) group. In contrast, mice that were treated with different doses of Fs.Cr (150 mg/kg, 300 mg/kg & 500 mg/kg) showed reduced MPO activity with a dose-dependent effect at 150 mg/kg (150.85±65 mU/ml) and 300 mg/kg (107±13.89mU/ml). Although 500 mg/kg also decreased the

MPO activity significantly (80±21.79 mU/ml), it was not significantly different from the effect produced by 300 mg/kg dose. Prednisolone, the positive control also significantly reduced the activity (69±12.16 mU/ml). Hence, the untreated group showed a consistent increase in MPO activity, whereas, Fs.Cr significantly reduced this activity, and thus giving a reason for reduced pathology of the disease as compared to the untreated group.

2.3.7. Effect on Malondialdehyde activity

Malondialdehyde (MDA) activity was measured in tissue homogenates of all the experimental mice to depict the lipoperoxidation status with and without treatment with Fs.Cr. As evident in Fig. 2.11B untreated group showed significant increase in MDA activity (77.25±4 nmol/mg) as compared to sham control

(25.97±3.16 nmol/mg). Pre-treatment with Fs.Cr, displayed a significant reduction in MDA activity in AA induced colitis model of mice. This effect was dose- dependent at 150 mg/kg (49.66±4.88 nmol/mg) and 300 mg/kg (26.36±2.14 nmol/mg). Beyond this dose no difference in the activity was observed at the tested dose of 500 mg/kg (23.09±1.85 nmol/mg). Prednisolone, the reference drug, also significantly reduced MDA levels.

Hence, Fs.Cr group showed a consistent decrease in LPO activity when compared to untreated group and therefore MDA activity was inversely proportional

61 to Fs.Cr treatment. Overall, there was an inverse relationship of LPO activity in treatment group vs. untreated group.

2.3.8. Effect on antioxidant activity

The antioxidant activity was assessed in colonic tissue supernatant of mice in all experimental groups. Amongst all the groups, untreated group exhibited a significant decrease in endogenous enzymatic and non-enzymatic (total glutathione) antioxidants as evident in Fig. 2.12 (A-D). In contrast, antioxidant activity was consistently elevated in the Fs.Cr treated group.

Similar to glutathione, CAT activity was reduced in AA group (6.53±2.37 mU/ml) as compared to sham control (10.19±2.16 mU/ml). A significant dose- dependent increase in CAT activity was evident at 300 mg/kg and 500 mg/kg doses

(24.77±2.69 mU/ml and 37.50±2.5 mU/ml, respectively) as evident in Fig. 2.12 (A-

D); however, at 150 mg/kg dose, this effect was not significant. Prednisolone also significantly increased CAT activity (41.67±4.41 mU/ml)

When assessed for effect on GPx activity in colon tissue homogenate, pre- treatment of Fs.Cr clearly increased the activity of this antioxidant enzyme as evident in Fig. 2.12B. Gpx activity was decreased in the untreated group (0.177±0.12 mU/ml) in comparison with sham control (0.24±0.11 mU/ml), whereas, Fs.Cr pre- treatment at a dose of 150 mg/kg displayed an increase in Gpx activity (1.26±0.36 mU/ml). Significant increase was evident at 300 mg/kg (2.47±0.66 mU/ml) and 500 mg/kg (4±0.54 mU/ml) doses, as compared to untreated group. Prednisolone, positive control, also showed a significant increase in activity (4.5±0.5 mU/ml).

When assessed for SOD activity, it was evident that untreated mice induced with colitis showed decrease in SOD activity (30.97±2.3%) as compared to sham control (52.5±2.5%). This trend was dose-dependent with 54±5.56% increase in

SOD activity at 150 mg/kg dose with significant effect at 300 mg/kg (87.59±6.5%) and 500 mg/kg (107.91±5.6%) as evident in Fig. 2.12C, Prednisolone also significantly increased SOD activity (107.2±4.34%; p<0.001).

Similarly, total glutathione (GSH) activity was decreased in untreated group

(76.20±6.6 nmol/ml) as compared to sham control (394.93±91.53 nmol/ml). Fs.Cr showed an increase in GSH activity as compared to untreated group as evident in

Fig. 2.12D. Although 150 mg/kg dose increased the activity as compared to untreated group, it was not significant (243.9±15.25 nmol/ml). However, Fs.Cr increased the total glutathione activity, at the doses of 300 mg/kg (300±5.77 nmol/ml) and 500 mg/kg (348.5±22.42 nmol/ml). Positive control, prednisolone also significantly increased GSH activity (308±27.64 nmol/ml). Hence, Fs.Cr group showed a consistent increase in antioxidant activity when compared to untreated group. Overall, there was an inverse relationship of antioxidant activity in treatment group vs. untreated group which may be one of the reasons for attenuating disease severity in treated mice.

Figure 2.11; Myeloperoxidase (A), and Malondialdehyde (B) activities in homogenized colon tissues of BALB/c mice obtained from sham control, AA (untreated), positive control and Fs.Cr pre-treated groups. Fs.Cr was given in doses of 150 mg/kg, 300 mg/kg and 500 mg/kg, for 7 days, i.p., prednisolone, given for 3 days (1.15 mg/kg) was used as positive control whereas vehicle solvent (given for 7 days) was administered to sham control. All mice were induced with colitis by IR administration of 0.1 ml of 6% AA, except sham control that received normal saline IR. Values given are means±SEM (n = 6) for each group. One way ANOVA was used for statistical analysis followed by Tukey’s post test. *p<0.05; **p<0.01; ***p<0.001 vs untreated (AA) group. [Abbreviations: AA = acetic acid; Fs.Cr = Flaxseed extract; IR = intrarectal; i.p. = intraperitoneal; MPO = myeloperoxidase; MDA = malondialdehyde].

Figure 2.12; Changes in activity of catalase (A), glutathione peroxidase (B), superoxide dismutase (C) and total glutathione (D) in homogenized colon tissues, obtained from controls, untreated and Fs.Cr pretreated groups. Fs.Cr was used in doses of 150 mg/, 300 mg/kg and 500 mg/kg, for 7 days, whereas, prednisolone in dose of 1.15 mg/kg was given for 3 days. Sham control was administered vehicle solvent for 7 days. All drugs were administered i.p. Colitis was induced by IR administration of 0.1 ml of 6% AA, except in the sham control that received IR saline. Values presented are means±SEM (n = 6) mice for each group. One way ANOVA was used for statistical analysis followed by Tukey’s post test. *p<0.05; **p<0.01; ***p<0.001 vs untreated (AA) group. [Abbreviations: AA = acetic acid; CAT = catalase; Fs.Cr = Flaxseed extract; GPx = Glutathione peroxidase; GSH = total glutathione; SOD = Superoxide dismutase; IR = intrarectally; i.p. intraperitoneally].

2.3.9. Effect on Interferon- and tumour necrosis factor-

levels

Fig. 2.13 (A-C) and Fig. 2.14 (A-C) shows means±SEM of cytokine IFN- and TNF- levels at 6, 12 and 24 hrs respectively, assessed from the colonic tissue homogenates of all the respective groups. The kinetics of IFN-and TNF-in AA group was highest at 6 hrs, followed by a decrease at 12 hrs and slight increase at 24 hrs; the levels of IFN- were however, 6-7 folds greater than TNF-

Compared to AA group, IFN- was consistently lower in the treatment group at all doses. At 6 hrs, IFN- levels were significantly reduced at all doses (p<0.05 and p<0.01 for 150 mg/kg and 300 mg/kg doses respectively) as compared to AA group. There was no increase in effect for IFN- levels at 500 mg/kg dose. In comparison to IFN-, TNF- levels did not show significant reduction at 150 mg/kg dose but did show significant effect at 300 and 500 mg/kg doses which apparently was not dose-dependent (p<0.001).

At 12 hrs, the level of IFN- were significantly reduced with p<0.05 for 150 mg/kg and p<0.01 for 300 and 500 mg/kg doses. TNF- levels were also reduced significantly (p<0.01), but the effect was not dose-dependent.

At 24 hrs, all the doses significantly reduced the IFN- levels (p<0.05) as compared to the AA group, whereas, reduction in TNF- levels was dose-dependent between 150 mg/kg (p<0.05) and 300 mg/kg (p<0.01). Beyond this dose, the tested

500 mg/kg (p<0.01) dose did not show any significant reduction. The control drug prednisolone produced significant reduction in TNF- levels at all the time points.

Hence, Fs.Cr group showed a consistent decrease in IFN- and TNF- responses when compared to AA group (except 150 mg/kg dose at 6 hrs for TNF-. Overall, there was an inverse relationship of IFN- and TNF- release in treatment group vs.

AA group. The cytokine levels of all the tested cytokines are presented in Table 2.5

2.3.10. Effect on IL-17 levels

Next, levels of IL-17 were assessed in tissue homogenates obtained from all the experimental groups. Fig. 2.15 (A-C) shows that in the untreated group, similar to IFN- and TNF-IL-17 level was also elevated at 6 hrs. However, in contrast to

IFN- and TNF-, it declined sharply at 12 hrs, whereas, at 24 hrs, it declined furthermore. This is in sharp contrast to the pattern observed for both IFN- and

TNF- where after 12 hrs there was an increasing trend, whereas, in case of IL-17 the levels consistently declined after 6 hrs.

Compared to AA group, IL-17 level was unchanged at 6 hrs in the treatment group, whereas, it started to show an increasing trend after 12 hrs. All the doses showed a significant increase in IL-17 at 24 hrs, when compared to the AA group. Amongst the treatment group, 150 mg/kg and 300 mg/kg doses increased IL-17 levels dose- dependently (p<0.05 vs p<0.01). There was no significant increase in IL-17 levels at the increasing dose of 500 mg/kg (p<0.01). Hence, Fs.Cr group exhibited a consistent increase in IL-17 levels at all-time points when compared to AA group.

Overall, there was an inverse relationship of IL-17 release in treatment group vs. AA group.

Table 2.5. Levels of cytokine at 6, 12 and 24 hrs with and without Flaxseed treatment in acetic acid-induced colitis in mice.

6 hrs. 12 hrs 24 hrs. IFN- AA 1238±195.2# 859±100.9# 943.2±177.8# Fs.Cr 150 605±62.17* 600.9±88.19 463.9±28.45* Fs.Cr 300 492.7±37.96** 429.9±67.09** 424.5±63.85* Fs.Cr 500 425.5±58.97** 387.6±94.56** 444.5±40.85* Sham 241.7±20.79** 213.9.9±25* 252.9±9.585* Prednisolone 337.6±18.81** 277.6±38.25*** 357.9±157.9* TNF- AA 194.1±27.36# 110.3±3.39# 142.4±4.93# Fs.Cr 150 195.6±10.91 119.5±2.516 95.14±16.21 Fs.Cr 300 79.29±4.1*** 76.12±3.208** 92.59±9.527** Fs.Cr 500 91.97±8.49*** 72.3±8.42** 99.97±12.07** Sham 67.06±9.67** 60.4±12.98** 75.4±6.12*** Prednisolone 85.1±10.27*** 75.1±10.3** 63.47±2.452*** IL-17 AA 233.2±18.41 166.1±32.26 114.2±16.29# Fs.Cr 150 192.7±19.92 144.2±33.07 175.9±31.1* Fs.Cr 300 186.1±16.13 226.1±20.72 209±29.85** Fs.Cr 500 207.1±28.02 189.5±24.75 223.8±13.69** Sham 81.3±23.38 99.68±28.4 77.52±14.02 Prednisolone 117±30.53 97.02±8.538 143.4±39.41

The values of released cytokines are expressed as mU/ml. Tissues were analysed for levels of cytokine by using Tissue cytokine ELISA kits. Fs.Cr = Flaxseed extract (mg/kg); sham control (ml/kg); AA = acetic acid group, untreated

Figure 2.13; Interferon- levels at 6 hrs (A), 12 hrs (B) and 24 hrs (C) in homogenized colon tissues of BALB/c mice obtained from controls, untreated and mice pre-treated with Fs.Cr. Doses of Fs.Cr used were 150, 300 and 500 (mg/kg/day) for 7 days, given i.p. Colitis was induced by IR administration of 0.1 ml of AA 6% except in the sham control which were instilled IR saline. Values presented are means±SEM (n = 6) mice for each group. One way ANOVA was used for statistical analysis followed by Tukey’s post test. *p<0.05; **p<0.01; ***p<0.001 vs untreated (AA) group. [Abbreviations: AA=acetic acid; Fs.Cr = Flaxseed extract; IFN-: Interferon- gamma;i.p.=intraperitoneally; IR=intrarectally].

Figure 2.14; Changes in levels of tumour necrosis factor - at 6 hrs (A), 12 hrs (B) and 24 hrs (C) in homogenized colon tissues, obtained from controls, untreated and mice pre- treated with Fs.Cr. Doses of Fs.Cr used were 150, 300 and 500 (mg/kg/day) for 6 days, given i.p., followed by induction of colitis by intrarectal administration of 0.1 ml of 6% AA, except in the sham control. Values presented are means±SEM (n = 6) mice for each group. One way ANOVA was used for statistical analysis followed by Tukey’s post test. *p<0.05; **p<0.01; ***p<0.001 vs untreated (AA) group. [Abbreviations: AA=acetic acid; Fs.Cr=Flaxseed extract; i.p=intraperitoneally; IR=intrarectally; TNF=tumour necrosis factor].

Figure 2.15; Changes in IL-17 levels at 6 hrs (A), 12 hrs (B) and 24 hrs (C) in homogenized colon tissues, obtained from controls, untreated and pre-treated with Fs.Cr. Doses of Fs.Cr used were 150, 300 and 500 (mg/kg/day) for 6 days, given i.p., followed by induction of colitis by IR administration of 0.1 ml of 6% AA, except in the sham control. Prednisolone (1.15 mg/kg) was used as positive control, whereas, vehicle solvent was used as sham control. Values presented are means±SEM (n = 6) mice for each group. One way ANOVA was used for statistical analysis followed by Tukey’s post test. *p<0.05; **p<0.01; ***p<0.001 vs untreated (AA) group. Abbreviations: Acetic acid; Fs.Cr=Flaxseed extract; i.p.=intraperitoneally; IR=intrarectally; IL=interleukin]

2.4. Discussion

These findings demonstrate, for the first time, that the crude methanolic- aqueous extract of Flaxseed (Fs.Cr) reduces the severity and extent of acute colonic damage in an AA-induced colitis model of mice. The AA model of colitis used in study resembles with multiple features of UC that includes both clinical, functional and morphological characteristics (MacPherson & Pfeiffer, 1978). The current study shows that Fs.Cr pre-treatment attenuated those features including DAI, spleen and colon wet weight and length ratio and macroscopic damage.

As known that breached epithelial barrier is an initial event in IBD that results in manifestation of disease (Xavier & Podolsky, 2007). AA model of colitis also depicts the similar pattern by causing epithelial injury with reduction in mucin secreting goblet cells, hence, compromising colonic permeability and promoting microbial translocation to the colon wall (Nakhai et al., 2007). Similar pattern of injury was evident in this diseased model as the AA group in alcian blue stained sections showed apparently few goblet cells with reduced alcian blue +ve mucin.

These results are in concordance with earlier reports where AA group showed reduced goblet cells (Fawzy et al., 2013). On the contrary, this depletion was significantly less in the Fs.Cr treated groups, showing that intestinal permeability was reduced in the treatment groups. Further evaluation through H and E staining showed the loss of surface columnar epithelial lining and a transmural infiltration of lymphatic cells and neutrophils in AA colitic groups which is in accordance with previous reports (Bertevello et al., 2005). One of the therapeutic targets for IBD is enhancing the epithelial integrity by increasing the barrier protection (Hering and

Schulzke, 2009) and reducing goblet cells depletion and increasing mucin levels is one of the ways to achieve that objective, since mucin levels are reduced in human

IBD (Van der Sluis et al., 2006; Zaph et al., 2007). Hence, one of the reasons for mucosal protective effect of Fs.Cr is by reducing goblet cell depletion and neutrophil infiltration. This could be further correlated with previous reports where intestinal mucin suppressed colitis (An et al., 2007). The fact that Fs.Cr promotes epithelial 72 protection is in consistence with a very recent report that showed Flaxseed lignan secoisolariciresinol diglucoside (SDG) exhibited mucosal protective effect against dextran sodium sulphate (DSS)-induced colitis, by increasing the expression of trefoil factor 3 (TFF3) (Xu et al., 2016), which is released by goblet cells and has a role in restitution of gut mucosa (Kjellev, 2009). It is a possiblility that besides restoring goblet cells and mucin levels, Fs.Cr may also have mediated its mucosal protective effect by increasing expression of TFF3.

To further probe into the mechanisms responsible for this protective effect

Fs.Cr was studied for the effect on parameters of mucosal injury induced by AA.

The parameters were selected on the basis of relevance to IBD and AA model. AA causes physical injury to the colonic epithelium causing an entry of acid in soluble protonated form leading to extensive intracellular acidification (Nakhai et al., 2007) which possibly activates monocytes and macrophages which are known to release

IL-1, IL-6, and TNF-These cytokines further induce chemokines’ release that recruit neutrophils which further cause immune stimulation (IL-17, IL-10) and release potent cytotoxic oxidants that further amplify the chemotaxis (Fournier &

Parkos, 2012). This subsequently cause a predominance of reactive oxygen species

(ROS) over antioxidants (CAT, SOD, GSH and GPx) initiating a ROM cascade, which results in lipid peroxidation (LPO) (Baumgart & Carding, 2007), the end product of which is MDA (Yamada & Grisham, 1991). Similar abnormalities are evident in IBD patients showing evidences of enhanced oxidative stress and that’s why antioxidants have a role as an adjuvant therapy in IBD (Rogler, 2015). In this study, inflammation and oxidative stress was enhanced in AA groups as verified by biochemical activities of colonic tissues for MPO and MDA respectively as well as decrease in antioxidant (SOD, GPx, CAT and total glutathione) activity. Fs.Cr was able to prevent mucosal injury by decreasing colonic MPO, MDA and enhanced antioxidant activities against the highly oxidative stress environment induced by

AA. These results are in concordance with earlier reported antioxidant effects of

Flaxseed oil in other diseased models (Lee et al., 2009; Pietrofesa et al., 2015).

Hence, this aspect of the data indicates that besides barrier protection, Fs.Cr reduces infiltration of inflammatory cells as well as LPO and enhances antioxidant activity which may limit mucosal damage.

Levels of TNF-were measured by immunoassay since it is the upstream cytokine leading to neutrophil recruitment in AA colitis models of mice (Bertevello et al., 2005) and also has a prominent role in IBD pathogenesis (Xavier & Podolsky,

2007). In the current study too, untreated group showed elevated TNF- levels at 6,

12 and 24 hrs, whereas, pre-treatment with Fs.Cr reduced their levels significantly at all the time points. This supports that neutrophils may have been activated by chemotactic signals from the proinflammatory cytokines upstream as supported by literature too (Fournier & Parkos, 2012) and Fs.Cr was able to reduce those signals.

One of the possibilities of reduced TNF-levels in Fs.Cr treated groups could be via PDE-4 inhibition as PDE-4 receptors are also present on monocytes and have shown inhibition of TNF-(Gantner et al., 1997). Consistent with this the ex-vivo studies conducted for this thesis (chapter 3) indicate Fs.Cr to exhibit PDE-4 inhibitory like activity. Flaxseed have also shown to inhibit NO via iNOS by downregulation of IFN- and TNF in DSS model of mice(Kawaguchi et al.,

1999). Hence, Fs.Cr inhibited TNF-and possibly in this way limited the neutrophil infiltration.

The possibility of Fs.Cr to affect other innate immune cytokines involved in

IBD pathogenesis was further probed. As known that IFN-is expressed in AA colitis models (Bertevello et al., 2005) and has a critical role in IBD immunopathogenesis (Xavier & Podolsky, 2007), hence, its levels were measured.

In the current studied model, the level of IFN-was roughly 5 folds more than TNF-

 through all time periods, which is in concordance with other studies (Bertevello et al., 2005). However, the pattern of IFN-kinetics has yet not been reported in AA colitis model. In the untreated (AA control) group, highest levels of IFN- were evident at 6 hrs, followed by reduction in 12 and 24 hrs. It is quiet probable that IFN-

 released from neutrophils has been due to signals from dendritic cells (DCs) as earlier reported in mice model of injury (Li et al., 2010) and in DSS-induced colitis

(Tokieda et al., 2015). On the other hand, Fs.Cr pre-treated mice showed significantly low levels of IFN- in colonic tissue homogenates at all the time points.

Hence, Fs.Cr may have inhibited the release of IFN- by suppressing the pathway that is mediated by DCs. The inhibitory effect on both TNF- and IFN- has a very important implication as neutralisation of both of these produced an additive effect in DSS-induced colitis mice (Obermeier et al., 1999) as well as prevented the development of colitis in severe combined immunedeficient (SCID) mice after transfer of specific T cells (Powrie et al., 1994).

IL-17 is another important cytokine involved in innate immune pathogenesis in mice (Fournier & Parkos, 2012). IL-23/IL-17 axis acts upstream of IL12/IFN- axis in mice model of injury and imperative for IL-12/IFN-γ-mediated tissue injury

(Tokieda et al., 2015). In the current study, IL-17 levels in untreated mice were significantly higher than the sham control at 6 hrs, whereas, reduced at 12 and 24 hrs. This correlates with the fact that IFN- levels were highest at 6 hrs and then decreased, although not as significantly as IL-17. This supports that possibly at 6 hrs, DC activation followed IL23/IL-17 and IL12/IFN- axis pathway. A surprising effect however, was that Fs.Cr treated mice showed a consistent increasing trend for

IL-17 which was significantly greater than untreated mice at 24 hrs. It is quite plausible that in diseased arm, pathways originating from DC were activated and

Fs.Cr suppressed them; however, it may have stimulated IL-17 secreting innate lymphoid cells (ILC-17) that may have increased the levels of IL-17 at 12 and 24 hrs. This idea is based on literature evidence about ILC-17 cells (Hwang &

McKenzie, 2013) which are predominantly occupied in gut mucosa and are known to maintain intestinal epithelial integrity (Hartigan-O-Connor et al., 2011) and their loss may result in reduced IL-17 and subsequently disease progression (Estes et al.,

2010). This alternative increase and decrease is speculated and illustrated diagrammatically in Fig. 2.16. 75

Figure 2.16: Possible sources of innate IL-17 in AA-induced colitis and effect of Fs.Cr at 6, 12 and 24 hrs. It is speculated based on the kinetics of IL-17 levels that in the initial 6 hrs, AA increases IL-17 possibly due to lamina propria DCs activation which after sensing the microbes directly triggers IL-23/IL-17 axis upstream of IL-12/IFN- axis. The IL-23 released from DCs in the presence of IL-6 and TGF- stimulate neutrophils to release TNF-  along with IL-17A. This IL-17 A stimulates NKT cells to release IFN- which are already activated by DC via IL-12/IFN- axis. The released IFN- from NKT cells stimulate neutrophils and CD4+NKT cells. At 12 hrs onwards, IL-17A producing innate lymphoid cells (ILC-3) may attribute for IL-17 release. AA-induced damage inhibit the ILC-3 induced IL-17, whereas, Fs.Cr promote the secretion of IL-17 from ILC3 (possibly with IL-22 which is also released from ILC-3 cells) and hence, may account for its protective role in colitis. [Abbreviations: Fs.Cr = Flaxseed extract; AA= acetic acid 6%; DC = dendritic cells; ILC-17 = IL- 17A producing innate lymphoid cells; IFN = interferon; IL= interleukin; PMN = polymorpho nulear; TNF = tumour necrosis factor; NKT = Natural Killer T cells; TGF = tumour growth factor. Blue line indicates “stimulation” and red line indicates “inhibition”].

This is an interesting observation also because IL-17 has a controversial role, either as a protector or effector (Fuss, 2011). Hence, this aspect of data indicates that Fs.Cr mediates its ant-inflammatory effects in AA -induced colitis model of mice by reducing the levels of IFN- and TNF- throughout 24 hrs, and increasing IL-17 levels at 24 hrs. This finding is very important because currently multiple cytokine therapy are the recommended therapeutic future of IBD

(Morrison, 2007).

In short, this study highlights that crude methanolic-aqueous extract of

Flaxseed (Fs.Cr) ameliorates the severity of acetic acid-induced colitis model in mice by reducing IFN-TNF- and increasing IL-17 levels with simultaneous antioxidant and anti-inflammatory activities that result in mucosal protective effects.

Hence, Flaxseed targets multiple aspects of the disease and therefore has a potential for translational research. Since diarrhoea and spasms are one of the major debilitating symptoms of IBD, it was therefore aimed to explore role in both these states. Hence, in the next chapter Fs.Cr’s antidiarrhoeal and antispasmodic effects and possible mechanisms are elaborated in detail.

CHAPTER 3

3. FLAXSEED IN DIARRHOEA AND SPASMS

3.1. Introduction

Inflammatory Bowel Disease (IBD) has a heterogeneous pathogenesis in which multiple triggers act simultaneously. Amongst them, disruption of epithelial barrier preceding abnormal pattern of gut contractility (Bassotti et al., 2014) are considered as some of the initial perpetuators of IBD. If these are not limited, they could be persistent source for mediating inflammatory cascade in IBD patients.

Possibly that’s why restoring vasculature and disturbance of blood flow by controlling gut contractility (Banner & Trevethick, 2004) are proposed as one of the novel approaches for targeting IBD.

Diarrhoea and abdominal pain are the most common symptoms of

Inflammatory Bowel Disease (IBD) which affect the patients’ quality of life immensely (Shah & Hanauer, 2006). Diarrhoea is evident in approximately 50% of

Crohn’s disease (CD) flare-up patients and in almost 100% of ulcerative colitis (UC) patients. IBD-induced diarrhoea occurs as a consequence of barrier defect (Binder,

2009) which causes passive diffusion of ions and water from the circulation to the intestinal lumen as well as alteration in the motility (Andus & Gross, 1999), thereby causing leak flux diarrhoea and subsequently to exudative diarrhoea (Hering &

Schulzke, 2009). That is the reason why one of the major symptoms of IBD, particularly UC, is bloody diarrhoea (Cotran et al., 2009).

It is generally believed that the abdominal pain in IBD is associated with inflammatory cascade. Although it is partly true, that resolution of inflammation reduces the pain intensity, but in 20–50% of patients, disabling abdominal pain during their clinically and endoscopic proven remission phase, is still reported

(Edwards et al., 2001; Farrokhyar et al., 2006) with a poor correlation of abdominal pain with disease activity index (nja Schirbel et al., 2010). Hence, it seems that there may be an additional functional motility disorder persistent in IBD patients, independent of inflammatory cascade, which may attribute to both the spasms and diarrhoea in IBD patients and might be closely related to Irritable Bowel Symdrome

(IBS) (Kern Jr et al., 1951). Consistent with this, roughly 60% of patients with CD 79 and 30% of patients with UC report IBS-like symptoms which is why it’s important that pain management be customized (Srinath et al., 2012).

Hence, because of these aspects, antidiarrhoeals are the required therapy for longer periods of time which, in case of UC patients, could be for months or in some cases even lifetime, leading to side effects such as distention, bloating, nausea and vomiting (Grahame-Smith & Aronson, 2002). Besides these effects, chronic use of antidiarrhoeal could also lead to constipation which sometimes cause toxic megacolon which can be life threatening (Brown, 1979).

As IBD is accompanied with both the inflammatory components (Xavier &

Podolsky, 2007) and abnormal patterns of contractility (Bassotti et al., 2014), some of the novel therapeutic approaches for treatment of IBD include restoring vasculature and disturbances of blood flow by controlling contractility (Banner &

Trevethick, 2004) and inflammation. Increased cAMP levels are known to restore mucosal blood flow and reduce gut motility (Willingham & Pastan, 1975) and their levels could be increased by phosphodiesterase enzyme (PDE) inhibition since PDE enzyme is responsible for the lysis of cAMP into inactive form. Anti-inflammatory potential of PDE enzyme inhibitors have also been identified (Oldenburger et al.,

2012) and since they are found in mammalian intestine as well as in inflammatory and immune cells. This approach has advantage of targeting the inflammation as well as diarrhoea and spasms since PDE inhibitors (PDEIs) increase cAMP levels

(Jones et al., 2002) causing the reduced compression of the vasculature and restoring mucosal blood flow (Zimmerman et al., 2012) and potentially inhibit the release of proinflammatory mediators from immune cells (Banner & Trevethick,

2004). Targeting PDE-4 therefore, has immense potential in IBD therapeutics because it approaches the disease by bypassing complex immunoregulatory mechanisms involving antigen receptor-specific mechanisms (Kumar et al., 2013) .

Chapter 2 highlights the role of Flaxseed extract (Fs.Cr) in ameliorating the severity in IBD model of mice, by reducing oxidative stress, increasing antioxidant activity, and reducing pro-inflammatory cytokines. The current study was planned

80 to identify Flaxseed’s potential in ameliorating the debilitating symptoms of IBD i.e. diarrhoea and abdominal pain, as well as, the potential mechanisms involved.

In this chapter, t is evident that Fs.Cr is effective as an antidiarrhoeal and antispasmodic agent with dual inhibition of PDE-4 and Ca++ channels.

3.2. Materials and Methods

3.2.1. Standard drugs

The reference drugs, acetylcholine chloride, carbamyl choline chloride, loperamide, potassium chloride and verapamil hydrochloride were purchased from the Sigma Chemicals Co, St. Louis, MO, USA. Luria-Bertani broth and nutrient agar powders used for antimicrobial assays were also purchased from the Sigma chemicals. Physiological salt solutions that were made by chemicals including calcium chloride, EGTA, magnesium sulfate, glucose, magnesium chloride, potassium chloride, sodium bicarbonate, sodium chloride, and sodium dihydrogen phosphate were obtained from Merck, Darmstadt, Germany. DMSO and Tween-80 used for solubilisation were purchased from Merck, Darmstadt, Germany and

Scharlau chemicals, Barcelona, Spain, respectively. Charcoal meal transit that required acacia and starch powder as well as vegetable charcoal were purchased from BDH Laboratory Supplies, Poole, England. All the chemicals that were used were of the analytical grade.

3.2.2. Animals

BALB/c mice (20–25 g) and locally bred rabbits (1–1.5 kg) that were of either sex were used for this study. Animals were housed at the animal house of The

Aga Khan University, maintained at 23– 25°C. They were kept in plastic cages (47 x 34 x 18 cm) with sawdust (changed at every 48 hrs) and fasted for 18 hrs before the experiment, whereas, they were given tap water and standard diet routinely.

Animals had free access to water but food was withdrawn 18 hrs prior to

81 experiments. Rabbits were then anaesthetised by Florane and then sacrificed by cervical dislocation gently, whereas, mice were sacrificed by cervical dislocation.

The study protocol (005-Ani-BBS-13) was also approved by ECACU (Ethics

Committee for Animal Care and Use) of The Aga Khan University.

3.2.3. Working stocks

For the in vivo and ex vivo experiments, on the day of experiment, the extract was solubilised in 10% Dimethyl sulfoxide (DMSO) and 5% Tween-80 and subsequent dilutions were made in distilled water.

Tyrode's solution (mM) was prepared by using KCl (2.68), NaCl (136.9),

MgCl2 (1.05), NaHCO3 (11.90), NaH2PO4 (0.42), CaCl2 (1.8), and glucose (5.55), prior to experiments. Ca++ free Tyrode’s was composed of (mM): KCl (50), NaCl (91.04), MgCl2 (1.05), NaHCO3 (11.90), NaH2PO4 (0.42), glucose (5.55), and EGTA (0.1).

3.2.4. Castor oil-induced diarrhoea

Antidiarrhoeal effect of Fs.Cr was tested on castor oil-induced diarrhoea in mice by slight modification of the method previously used (Bashir et al., 2011). Mice fasted for 18 hrs before the experiment, were divided into 6 groups (sham control, diseased control/untreated, positive control and treatment groups in 3 different doses). Each group consisted of 5 mice that were housed in individual cages. The 1st group labelled as sham control received saline in vehicle solvent (10 ml/kg), whereas, the 2nd group (untreated) also received control solvent to be later followed by castor oil administration. The 3rd group (positive control) received loperamide

(10 mg/kg), whereas, the 4th, 5th and 6th (treatment) groups received increasing concentrations of 150, 300 and 500 mg/kg of Fs.Cr respectively. All the drugs were administered orally using feeding needles. 1 hr after the treatment, all the groups except the sham controls, received 10 ml/kg of castor oil, orally. After 4 hrs, the cages were inspected for the frequency as well as for consistency of the stool was scored as described previously (Wirtz et al., 2005) as detailed below: score 0: no 82 diarrhoea; score 1: soft but formed stool; score 2: very soft stool; score 3: diarrhoea.

For each group, total score was calculated as sum of individual scores where individual score was calculated by multiplying the number of diarrhoeal droppings with the respective score. Percent reduction i.e. how much the tested treatments reduced the control (castor oil-induced) diarrhoeal score was calculated by using equation as follows:

퐌퐞퐚퐧 퐜퐚퐬퐭퐨퐫 퐨퐢퐥 퐝퐢퐚퐫퐫퐡퐨퐞퐚퐥 퐬퐜퐨퐫퐞 (퐜퐨퐧퐭퐫퐨퐥)−퐦퐞퐚퐧 퐬퐜퐨퐫퐞 퐨퐟 퐭퐫퐞퐚퐭퐦퐞퐧퐭 퐠퐫퐨퐮퐩 × ퟏퟎퟎ 퐌퐞퐚퐧 퐜퐚퐬퐭퐨퐫 퐨퐢퐥 퐝퐢퐚퐫퐫퐡퐨퐞퐚퐥 퐬퐜퐨퐫퐞 (퐜퐨퐧퐭퐫퐨퐥)

3.2.5. Enteropooling Assay

To study the mechanism of antidiarrhoeal activity, intestinal fluid accumulation test known as castor oil-induced enteropooling assay was used, that would tell whether Fs.Cr produced its antidiarrhoeal effect by reducing secretions or not (Capasso et al., 1994). BALB/c mice fasted for 18 hrs were divided similarly as mentioned previously, into 6 groups. The negative control group and the diseased control group (untreated) received vehicle solvent orally by feeding needle, whereas, the positive control group received atropine 10 mg/kg i.p. (Mehmood et al., 2011).

Treatment groups received orally 150, 300 and 500 mg/kg of the Fs.Cr respectively.

After 1 hr, animals in all the groups except the negative control group were administered 10 ml/kg of castor oil, orally, to induce secretions, whereas, the negative control group were given saline instead of castor oil (10 ml/kg, orally). 30 min later, the mice were killed by cervical dislocation; subsequently, the entire intestine was removed and weighed with care making sure there was no leakage of fluid. The results were calculated as follows:

Pi/Pm x 1000 where, Pi is the weight of the intestine and Pm is the weight of the animal in grams.

3.2.6. Charcoal meal gut transit test

To further elaborate whether Fs.Cr has antidiarrhoeal activity by its effect on gut motility, the method modified by Bakare et al., (2011) was followed, in which motility was induced by using castor oil and then effect was checked on marker meal transit. BALB/c mice fasted for 18 hrs were classified into groups in the similar way as previously described in enteropooling assay and dosed orally through a feeding needle. 1 hr after the administration of vehicle, different doses of Fs.Cr (150, 300 and 500 mg/kg, P.O.), and control drug loperamide (10 mg/kg, P.O.), 10 ml/kg of castor oil was administered orally to all groups except the negative control group that received saline. 1 hr later, 0.3 ml of freshly prepared 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 travelled by the charcoal meal from pylorus to the caecum 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 = LM/LSI x 100 where: PI = Peristaltic Index; LM = Length of charcoal meal; LSI = Length of small intestine.

The percent inhibition relative to the control was also calculated as follows:

Percent inhibition = [(control – test)/control] x 100

3.2.7. Isolated rabbit-jejunum preparations

Experiments on isolated rabbit jejunal preparations were carried out as described previously (Gilani & Cobbin, 1987). Briefly, 2 cm of intestinal segments were suspended in tissue bath (10 ml volume) containing Tyrode's solution, aerated with carbogen (a mixture of 95% oxygen and 5% carbon dioxide) and the temperature of the bath was maintained at 37°C. To equilibrate each tissue segment, a preload of 1 g was applied on it.

Isotonic transducers (50–6360), Harvard Apparatus, Holliston, MA, USA) were used to record the intestinal responses isotonically. These transducers were coupled with Power Lab data acquisition system (model ML-845, AD Instruments,

Sydney, Australia) which was connected with computer that had chart software

(version 5.3) installed. Each tissue was equilibrated for minimum of 30 min prior to the addition of any drug. Subsequently, acetylcholine (Ach) at a submaximal concentration (0.3 μM) was administered in the bath and as soon as the contraction was evident in the chart reader, the bath fluid was replaced with normal Tyrode’s solution. ACh was added to stabilize the contractions as well as assess the appropriate functioning of the tissues. It is known that the rabbit jejunum exhibits spontaneous rhythmic contractions under these experimental conditions that allows to test the spasmolytic activity without the use of an agonist (Gilani et al., 2000).

Hence, the same method was adopted to test spasmolytic activity in isolated rabbit jejunal tissues which were contracting spontaneously.

3.2.8. PDE inhibitory (PDEI) activity

Fs.Cr was tested against CCh (1 M)-induced sustained contractions in rabbit jejunal preparations. As CCh is known to induced contraction in smooth muscles by PDE enzyme inhibition (Kaneda et al., 2005) therefore the substance which inhibits this contraction is considered as PDE blocker. Hence, for testing this effect, the jejunal tissues were prepared in the manner as mentioned in section 3.2.7., and once the tissues were stabilised with ACh submaximal dose, CCh (1 µM) was added to the tissue bath. Once the sustained contraction was achieved, concentration- dependent inhibitory responses were constructed by cumulative dosing of Fs.Cr

(Van Rossum, 1963). The inhibitory effect/relaxation response of jejunal preparations that were pre-contracted with CCh, was expressed as a percent of the control responses. PDEIs are potent in inhibiting CCh-induced contractions as compared to against high K+-induced contractions (Kaneda et al., 2005), therefore

85 when such a pattern was evident for Fs.Cr against CCh-induced contraction it was further tested for presence of PDE-inhibitor like effect.

The PDE inhibitory (PDEI) effect was confirmed by constructing isoprenaline-induced inhibitory CRCs, in the absence and presence of the Fs.Cr as described previously (Gilani et al., 2008) and elaborated as follows: Rabbit jejunal tissues were stabilised as mentioned in previous section and then incubated with

CCh (1µM), thereafter, subsequently isoprenaline CRCs were obtained against the contraction. Next, the tissues were pre-treated with different concentrations of Fs.Cr for 15 min, and then CRCs of isoprenaline were reconstructed. Leftward shift/potentiated effect of Fs.Cr compared to control isoprenaline CRCs would indicate towards the possibility of PDEI like effect as PDEIs potentiate the effect of isoprenaline (Lorenz & Wells, 1983). The same procedure was repeated for papaverine, a standard PDE inhibitor as described earlier (Choo & Mitchelson, 1978;

Sato et al., 2006; 2007). The inhibitory effect of Fs.Cr was measured against CCh- induced contractions as the percent change in jejunum spontaneous contractions.

To determine the sub-type of PDE inhibitor involved, a relationship between inhibitory effect of different PDE enzyme inhibitors was examined in presence and absence of Fs.Cr, as described previously (Sato et al., 2006; 2007). Briefly, isolated rabbit jejunal tissues were stabilised in the similar way as mentioned earlier, followed by CCh (1 µM)-induced contractions. Once these contractions were sustained, concentration-dependent inhibitory responses were constructed by cumulative dosing of respective PDE enzyme inhibitors (Van Rossum, 1963). PDE enzyme inhibitors for subtypes 3, 4 and 5 were used which were cilostazol (100 M), rolipram (3 M), and zaprinast (100 M), respectively. This was followed by restabilisation of the jejunal tissues and then the same types of concentration- dependent inhibitory responses were constructed in the presence of 0.03 mg/ml of

Fs.Cr. The results were interpreted based on whether the pre-treatment of Fs.Cr potentiated the PDE-inhibitor’s concentration-dependent inhibitory responses or not.

Since PDE is responsible for hydrolysing cyclic AMP (cAMP), hence, to further confirm that Fs.Cr mediates its effect via PDE enzyme inhibition, cAMP content of the jejunum was measured by enzyme immunoassay (Kaneda et al., 2005) using cAMP enzyme immunoassay kit for multiple species (ARBOR assays Detect

X, direct cAMP enzyme immunoassay kit, Ann Arbor, MI, USA). For this purpose

CCh-induced contraction of the jejunum was inhibited with Fs.Cr in respective different doses. The treated and untreated jejunal tissues were snap frozen in liquid nitrogen and subsequently homogenized with 1 ml of sample diluents for every 100 mg of tissue, on ice which was kept like this for 10 min and then centrifuged at

≥6000 g at 4°C for 15 min. The remaining supernatant was collected and run in the assay immediately or stored frozen at -80°C. Similar steps were repeated for tissues treated with different doss of papaverine as control. The cAMP content was expressed as picomole per ml (pmol/ml).

3.2.11. Ca++ antagonist activity

To assess the mechanism behind the spasmolytic activity, rabbit jejunal preparations were depolarized by high potassium (K+ 80 mM) according to the method described by Farre et al., (1991). The advantage of this method is that it allows determining whether the spasmolytic activity of the test substance is through

Ca++ channel blockade or some other mechanism(s). Smooth muscle contraction induced by high K+ is a consequence of Ca++ influx from extracellular fluid and therefore the substance which inhibits this high K+-induced contraction is considered to act through Ca++ channels’ blockade (Bolton, 1979).

Hence, the intestinal tissues in the isolated bath were contracted by using high K+ as spasmogen and roughly after 45 min, when the sustained contraction was achieved, Fs.Cr was dosed in cumulative fashion to obtain inhibitory responses (Van

Rossum, 1963). The relaxation of these high K+ pre-contracted tissues was expressed as a percent of the control response mediated by K+.

Ca++ antagonist activity of Fs.Cr was further confirmed in stabilized rabbit jejunal tissues by replacing their Tyrode’s solution with Ca++ free Tyrode’s solution that has a chelating agent which removes Ca++. That’s why the tissues were kept in this solution for 30 min so that Ca++ could be removed from the tissues. Thereafter this solution was replaced with K+-rich and Ca++ free Tyrode's solution, whose composition is mentioned in working stocks (Section 3.2.3.) Following an incubation period of 30 min with this K+-rich and Ca++ free Tyrode’s, Ca++ concentration-response curves (CRCs) were obtained against Ca++. This acted as control; when these control CRCs of Ca++ became super-imposable (usually takes two cycles), the tissue was then re-stabilised. To test for the possible CCB effect of

Fs.Cr, same tissues were pre-treated with different concentrations of Fs.Cr for 60 min (0.1 mg/ml and 0.3 mg/ml) and thereafter CRCs of Ca++ were reconstructed.

The same procedure was repeated for verapamil, a standard Ca++ channel blocker

(Fleckenstein, 1977).

3.2.13. Statistical analysis

All the data expressed are means± standard error of the mean (S.E.M). The statistical parameter applied in the castor oil-induced diarrhoea, 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 antimicrobial 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).

3.3. Results

3.3.1. Effect on castor oil-induced diarrhoea

Fs.Cr was evaluated for its antidiarrhoeal activity in a castor oil-induced diarrhoea model of mice. The diarrhoea was evident in the untreated group with mean diarrhoeal score being 35.7±0.81 as compared to vehicle control group that had no diarrhoea and hence no score. Pre-treatment with Fs.Cr reduced the diarrhoeal score by 39% (score: 21.6±2.85), 63.9% (score: 13±2.13) and 68.34%

(score: 11.3±1.35) at 150, 300 and 500 mg/kg doses, respectively and presented in

Fig 3.1. This effect was dose-dependent at 150 mg/kg and 300 mg/kg (p<0.01 and p<0.001 respectively). The next higher dose of 500 mg/kg did not show further change in the antidiarrheoeal effect. Hence, Fs.Cr possessed antidiarrhoeal activity against castor oil induced diarrhoea in mice. Since diarrhoea is caused due to increase in secretions and gut motility, it was therefore aimed to further assess the possibility for these mechanisms in vivo.

3.3.2. Effect on intestinal secretions

To determine whether Flaxseed mediates its antidiarrhoeal effect by reducing secretions, castor oil-induced enteropooling assay was performed in mice. Intestinal secretions in negative control mice were 77.35±1.52g, which after castor oil administration increased to 149±5.90 g in the untreated mice. These castor oil- induced intestinal secretions were reduced in the mice pre-treated with Fs.Cr by

24.12% (113±5.12 g), 28.09% (107±2.49 g) and 38.8% (91±3.06 g) at respective doses of 150, 300 and 500 mg/kg. The effect was dose-dependent for 150 and 300 mg/kg (p<0.01 and p<0.001, respectively), whereas, the next higher dose (500 mg/kg) did not cause further increase in the effect (Fig. 3.2; p>0.05)

3.3.3. Effect on gut motility

To determine whether Fs.Cr mediates its antidiarrhoeal effect by reducing gut motility, castor oil was used to induce the motility. Castor oil increased the intestinal motility as represented by the charcoal meal transit from 45.68±1.96 cm to 68.43±3.95 cm (p<0.01). Fs.Cr reduced the intestinal motility in mice by 31.66%

(46.76±3.46cm; p<0.01), 46.98% (36.28±3.73 cm; p<0.001) and 56.2% (29.97±4.30 cm; p<0.001) at the respective doses of 150, 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 as evident in Fig. 3.2B.

Figure 3.1; Diarrhea score in controls, untreated and Fs.Cr pre-treated BALB/c mice with castor oil-induced diarrhea. Diarrhoea is scored on the basis of sum of frequency and consistency of diarrhoeal droppings. Each experiment was repeated thrice. Values shown are means±SEM of 5 animals for each experimental group. (*), (**), (***) indicate p<0.05, p<0.01 and p<0.001 vs. castor oil respectively. 10% DMSO and 5% Tween-80 was the vehicle solvent used. Sham control and untreated mice were administered vehicle solvent, positive control mice received loperamide (10 mg/kg), and treatment groups received varying doses of Fs.Cr (150, 300 and 500 mg/kg doses). Castor oil (10 ml/kg) was used to induce diarrhoea, except in sham control mice that were given normal saline. All drugs were administered orally, 30 min before castor oil administration [Abbreviation: Fs.Cr = Flaxseed extract]

Figure 3.2; Effect on castor oil-stimulated fluid accumulation in small intestines (A) and on castor oil-induced charcoal meal transit in intestine from pylorus to cecum (B) in controls, untreated and Fs.Cr treated BALB/c mice. Castor oil was used to induce the secretions and motility was given orally in dose of 10 ml/kg. Intestinal fluid accumulation is expressed as (Pi/Pm) x 1000 where Pi is the weight of the small intestine and Pm is the weight of the mouse. Charcoal meal transit is expressed as LM/LSI x 100 where LM is the length of charcoal meal travelled and LSI is the length of small intestine. Values given are means±SEM of three experiments (n = 5). One way ANOVA was used for analysis followed by Tukey’s post-test. *p<0.05; **p<0.01 and ***p<0.001 vs untreated group. [Abbreviation: Fs.Cr = Flaxseed extract].

3.3.4. Effect on isolated rabbit jejunum

When Fs.Cr was studied in an isolated tissue bath assembly setup for assessing spasmodic mechanism, it exhibited dose-dependent relaxation of spontaneous, CCh or high K+-induced contractions in rabbit jejunal tissues (Fig.

3.3A). Similarly, papaverine (Fig. 3.3B) and verapamil (Fig.3.3C) used as positive control caused inhibitory effect against spontaneous, K+ or CCh-induced contraction but there was difference in pattern of inhibition in terms of potency of inhibition against various spasmogens. Like papaverine, Fs.Cr was most potent against CCh- induced contraction but was different in that, it showed some selectivity against high

K+ as compared to spontaneous contractions, whereas, papaverine showed similar inhibition against both spontaneous and high K+-induced contractions. Verapamil was distinctly potent against high K+ and showed less potency against spontaneous and CCh-induced contractions.

3.3.5. Effect on PDE enzyme

As papaverine is a known PDE enzyme inhibitor (Kukovetz and Poch,

1970), the relationship between the spasmolytic action of Fs.Cr and its PDE-enzyme inhibitory effect was studied. When tested for the presence or absence of PDEI like activity in rabbit jejunal tissues, isoprenaline inhibited CCh-induced contraction with an EC50 value of 1.77 mg/ml (95% CI: 1.20 to 2.61), and in the presence of

Fs.Cr (0.01 and 0.03 mg/ml), the curve was shifted towards left, i.e. relaxed earlier in a dose-dependent manner (Fig. 3.4A). Papaverine also shifted control isoprenaline

CRCs towards left at 1 M and 3 M doses (Fig. 3.4B), whereas, there was no shift in case of verapamil (Fig. 3.4C).

To determine the sub-type of PDE inhibitor, effect of pre-treatment with

Fs.Cr was studied on various PDE-inhibitors (Fig. 3.6A-C). Cilostazol, a selective inhibitor of PDE-3, showed a concentration-dependent inhibitory response against

CCh-induced contraction. As evident in Fig. 3.6A, the inhibitory response was enhanced by pre-treatment with Fs.Cr (0.03 mg/ml) shifting the curve to left, similar 93 to rolipram, as well as zaprinast, indicating that Fs.Cr and cilostazol possess different mechanisms, which is why the additive effect was evident.

Similarly, rolipram a selective inhibitor of PDE-4, inhibited the contraction, induced by CCh in a concentration-dependent manner. The inhibitory response was not enhanced by pre-treatment with Fs.Cr (0.03 mg/ml), whereas, it was enhanced by pre-treatment with cilostazol and also by zaprinast as evident in Fig. 3.6B, indicating that rolipram and Fs.Cr may be following the same mechanism. Zaprinast, a selective inhibitor of PDE-5, inhibited the contraction induced by CCh in a concentration-dependent manner. The inhibition response was enhanced by pre- treatment with Fs.Cr (0.03 mg/ml), rolipram and cilostazol as evident in Fig. 3.6C, indicating that Fs.Cr and zaprinast might be employing different mechanisms than

PDE-5 inhibitor.

The cAMP level measured from control CCh treated tissues was 16.89±1.42 pmol/ml, which increased to 29.11±1.62 (p<0.05), 65.99±1 (p<0.001) and

78.73±1.17 (p<0.001) pmol/l after pre-treatment with 1, 3 and 5 mg/ml of Fs.Cr respectively, as can be seen in Fig. 3.5A. The papaverine control exhibited the similar pattern of rise in the levels of cAMP with control CCh treated tissues showing cAMP levels to 21.01±1.75 pmol/ml and after treatment with 1 M and 3

M of papaverine the cAMP levels increased to 58.7±4.2 pmol/ml (p<0.05) and

91.89±3.4 (p<0.001) pmol/ml respectively, as evident in Fig. 3.5B.

3.3.8. Effect on Ca++channels

Since the high K+-induced contraction was inhibited by Fs.Cr, possibility of

Ca++ channel blocking (CCB) like activity was speculated. For this purpose Ca++

CRCs were constructed with and without Fs.Cr for confirmation whether or not the inhibitory response was mediated through CCB. The results showed that pre-treatment of the tissue with 0.1 mg/ml and 0.3 mg/ml of Fs.Cr shifted the Ca++ CRCs to the right with suppression of maximal response (Fig. 3.7A) in a dose-dependent fashion similar to that exhibited by verapamil (Fig. 3.7B) at the concentrations of 0.03 and 0.1 M. Papaverine also showed similar rightward shift at 1 and 3 M (Fig. 3.7C). 94

Figure 3.3; Inhibitory effect of Fs.Cr (A), papaverine (B), and verapamil (C) against spontaneous, CCh and high K+-induced contractions in isolated rabbit-jejunal preparations. Fs.Cr inhibited spontaneously contracting rabbit jejunal tissues dose-dependently, and inhibited CCh-induced contractions at 0.3 mg/ml and high K+-induced contraction at 1 mg/ml. The inhibitory effect against CCh seems somewhat similar to papaverine, a standard phosphodiesterase inhibitor. The Values represent means±SEM (n= 4-5).

[Abbreviations: Fs.Cr = Flaxseed extract; CCh = carbachol (1 µM); K+(80 mM) = high K+].

A Isoprenaline alone B Isoprenaline alone 100 +Fs.Cr (0.01 mg/ml) 100 +Papaverine (1 M) +Fs.Cr (0.03 mg/ml) +Papaverine ( M)

75 75

50 50

(%)

25 25

CCh-induced contraction CCh-induced 0 0

1 3 1 0.1 0.3 0.1 0.3 0.01 0.01 0.03 Isoprenaline (M) Isoprenaline (M)

C Isoprenaline alone +Verapamil (0.03 M) 100 +Verapamil (0.1 M)

(%)

CCh-induced Contraction CCh-induced

1 0.1 0.3 0.01 0.03 Isoprenaline (M)

Figure 3.4; Concentration-response curves (CRCs) of isoprenaline in the absence and presence of the increasing concentrations of Fs.Cr (A), papaverine (B) and verapamil (C) in isolated rabbit jejunal preparation. Isoprenaline inhibited CCh-induced contraction dose- dependently. Fs.Cr pre-treatment at 0.01 and 0.03 mg/ml clearly potentiates the inhibitory response of isoprenaline by shifting the curve to the left (A), similar to papaverine at tested dose of 1 µM and 3 µM. Verapamil, a standard Ca++ channel blocker, does not shift isoprenaline curve. Values represent means±SEM of all the experiments (n = 4-5). [Abbreviations: CCh = carbachol (1 M); Fs.Cr = Flaxseed extract]

Figure 3.5; Effect on cAMP levels in isolated rabbit jejunum tissues that were contracted by CCh (1 µM) and then treated with Fs.Cr (A) and papaverine (B), in different concentrations indicated. Fs.Cr at 1 and 3 mg/ml increased the cAMP levels dose- dependently (A), similar to papaverine, a standard phosphodiesterase inhibitor (B). Values are the means±SEM (n = 4–7). * and *** indicate p<0.05 and P<0.001 respectively as compared to control. [Abbreviations: Fs.Cr = Flaxseed exract; CCh = carbachol; cAMP = cyclic adenosine monophosphate]

Figure 3.6; Effect of Fs.Cr on the relaxant responses of cilostazol (100 µM) (A), rolipram (3 µM) (B) and zaprinast (100 µM) (C) in rabbit jejunum precontracted with CCh (1 M). Cumulative concentration–response curves (CRCs) for cilostazol without any drug and in the presence of Fs.Cr (0.03 mg/ml), rolipram and zaprinast (A), Cumulative CRCs for rolipram under control conditions and in the presence of Fs.Cr (0.03 mg/ml), cilostazol and zaprinast (B), and cumulative CRC for zaprinast control conditions and in the presence of Fs.Cr (0.03 mg/mL), cilostazol and rolipram (C). The maximum contraction induced by CCh in the absence of cilostazol, rolipram and zaprinast respectively, was taken as 100%. Fs.Cr potentiated inhibitory effect of Cilostazol (A) and zaprinast (C) but did not potentiate the effect of rolipram (B). Values are means±SEM (n = 4-5). [Abbreviations: Fs.Cr = Flaxseed extract; CCh = carbachol]

A B 100 Control 100 Control +Fs.Cr (0.1 mg/ml) + Verapamil 0.03 M) +Fs.Cr (0.3 mg/ml) + Verapamil (0.1M) 75 75

50 50

Control Max(%) Control 25 25

Control max. (%)

0 0

-4.5 -3.5 -2.5 -1.5 -4.5 -3.5 -2.5 -1.5 Log [Ca++] (M) Log [Ca++] (M) C 100 Control Papaverine (1 uM) 75 Papaverine (3 uM)

Control Max. (%) Control

-4.5 -3.5 -2.5 -1.5 -0.5 Log [Ca++] M

Figure 3.7; CRCs of Ca++ in the absence and presence of the increasing concentrations of Fs.Cr (A), verapamil (B) and papaverine (C) in isolated rabbit jejunal preparations. Incubation of isolated tissues with Fs.Cr shifted the curves to the right (A), similar to verapamil (B) and papaverine (C). Values represent means±SEM of all the experiments (n = 4-5).

[Abbreviations: Fs.Cr = Flaxseed extract; Ca++ = Calcium; CRCs = Concentration-response curves].

3.4. Discussion

The current study was undertaken to assess antidiarrhoeal and antispasmodic effects of Flaxseed to rationalize its use in IBD.

It was observed that pre-treatment of mice with Fs.Cr exhibited a dose- dependent reduction in the incidence and severity of diarrhoeal stool production, against the castor oil-induced diarrhaoea. The usual indicator of this type of diarrhoea is to see all or none response (Gilani et al., 2005) but in this study, diarrhoea was categorized quantitatively on the basis of both the frequency and consistency (Wirtz et al., 2005), as diarrhoea is the consequence of both increase in frequency and decrease in consistency of the stool. It was found that Fs.Cr was effective in reducing both the frequency and increasing the consistency of the diarrhoeal stool.

One aspect that needs to be discussed about antidiarrhoeal effect of Fs.Cr is that, literature suggests its use in both diarrhoea and constipation (Duke et al., 2002).

However, when Fs.Cr was tested for its laxative effect in normal mice, no significant increase in frequency and weight of faecal material was observed (data not shown).

This could be due to the fact that methanolic-aqueous extract of Flaxseed has been used in this study which excludes the insoluble fiber content, which has been proposed as producing laxative effect (Xu et al., 2012). Interestingly in this study,

(Gilani & Atta-ur-Rehman, 2005). It is speculated that possible presence of spasmogenic component may have presented this type of antidiarrhoeal activity which is particularly important in case of IBD as one of the consequence of chronic use of antidiarrhoeal agent is constipation leading to toxic megacolon (Brown, 1979) 100 which is a life threatening complication. Besides, there’s an increase in aggressive gut pathogens in IBD, and in such cases, completely abolishing the stool frequency may increase the chances of dissemination of infection, although with antibiotic this effect may be not important (Casburn-Jones et al., 2004).

Further to in vivo antidiarrhoeal effect Fs.Cr was studied in vivo for the possible mechanisms behind this antidiarrhoeal activity, which could be by reducing secretions and/or gut motility. As known that castor oil causes an increase in intestinal fluid contents and cause diarrhoea indirectly through ricinoleic acid formation, which changes the electrolyte and water transport and generates enormous contractions in the transverse and distal colon (Iwao & Terada, 1962); hence, castor oil-induced enteropooling assay and charcoal meal transit assay was used to analyse the effect of Fs.Cr on intestinal secretions and gut motility respectively. Fs.Cr reduced castor oil-induced intestinal secretions as well as inhibited castor oil-induced gut motility in mice.

To further explore possible mode of antimotility, effect of Fs.Cr was tested against spontaneously contracting rabbit jejunum where it caused dose-dependent inhibition, thus showing spasmolytic effect. It was observed in earlier studies that plants mediate their spasmolytic effect through PDE enzyme inhibition (Sato et al.,

2006) as well as by CCB (Gilani et al., 2005) and these mechanisms are also therapeutically employed in treatment of IBD.

In the current study, it was found that Fs.Cr caused complete inhibition of

CCh-induced contraction at a dose significantly lower than required to inhibit high

K+-induced contraction. Inhibition of CCh-induced contraction with such a pattern is linked with PDE enzyme inhibitory like activity E (Boswell-Smith et al., 2006;

Kaneda et al., 2005). Hence, Fs.Cr was tested in isolated jejunal tissues for the possible interaction with isoprenaline against CCh-induced contractions since

PDEIs effect is potentiated by isoprenaline (Lorenz & Wells, 1983). Expectedly,

Fs.Cr shifted isoprenaline CRC to left indicating that the relaxant effect was potentiated by the extract.

101

It was further aimed to identify the possible inhibition of PDE sub type; for this purpose, the inhibitory effect of different types of PDEIs subtypes against CCh- induced contraction in isolated rabbit jejunum was measured and then observed for potentiation of inhibitory effect in those inhibitors after Fs.Cr pre-treatment in those tissues. Since Fs.Cr potentiated the inhibitory effect of PDEIs (subtype-3 and 5;

Cilostazol and zaprinast respectively) significantly, it indicated that possibly Fs.Cr does not mediate its effect via these subtype inhibitors. Fs,Cr pre-treatment did not potentiate inhibitory effect of rolipram (subtype-4 inhibitor), indicating that perhaps, Fs.Cr and rolipram mediated their effect by inhibiting the same sub-type of

PDE enzyme and that’s why the EC50 value of rolipram did not show significant reduction with Fs.Cr pre-treatment. Furthermore, there was a potentiation of inhibitory effect of PDE-3 and PDE-5 subtype inhibitors with Fs.Cr pre-treatment as evident by a significant reduction in EC50 values (p<0.001 compared to control) indicating that Fs.Cr and PDE-3 and -5 mediated effects via different subtypes. This interpretation is further supported by previous studies where effect of one of the subtypes of PDEIs have been potentiated against CCh-induced contraction when pretreated with the other subtype of PDE inhibitor (Harris et al., 1989). Similarly in earlier studies subtype of PDEIs from an unknown compound have been determined by using the similar approach (Sato et al., 2006; 2007). This finding has a very important implication because blocking PDE-4 enzyme prevents mucosal damage and subsequently inflammatory cell infiltration (Tsujij et al., 1995), and hence has been proposed as a novel approach to target IBD (Gantner et al., 1997).

PDEIs mediate their effect by increasing the levels of cAMP (Anthony, 1996;

Kaneda et al., 2005) as PDEIs are responsible to inhibit the degradation of cAMP into inactive form. Similar was the case in the current study where CCh precontracted tissues when treated with Fs.Cr, exhibited a dose-dependent increase in the levels of cAMP. This is a very important aspect from IBD perspective because cAMP agonists are being evaluated for IBD therapeutics as they interact with the cytoskeletal system (Willingham & Pastan, 1975) and their dysregulation leads to

102 numerous barrier defects, which is the initial perpetuator of IBD (Bassotti et al.,

2014). One of these defects is alteration of tight junctions (TJs) which consequently reduces the ionic permeability of the paracellular pathway of intestinal epithelial cells (Duffey et al., 1981) and thereby causing leak flux diarrhoea. It has already been shown in previous chapter that Fs.Cr is effective in diarrhoea induced due to acetic acid colitis in mice as well as current study shows antidiraahoeal effect against castor oil-induced diarrhoea. It is speculated that the antidiarrhoeal effect of

Flaxseed may be mediated due to increased cAMP levels as a consequence of PDE enzyme inhibition.

A substance which inhibits CCh-induced contraction can be either PDE inhibitor and/or anticholinergic. However, the reason for considering a stronger

PDEI component in Fs.Cr is based on the evidence that isoprenaline curves were shifted to the left by distinctly lower dose of Fs.Cr which occurs due to increase in cAMP levels, while anticholinergics, do not show such a shift in isoprenaline CRCs.

Hence Fs.Cr mediates its antispasmodic mechanism possibly by PDE enzyme inhibitory activity at low doses, which is also supported by increased cAMP levels in the Fs.Cr treated tissues. Nevertheless, the possibility of an additional anticholinergic component cannot be ruled out at a higher dose.

In previous chapter, it is shown that the number and intensity of ulcers in colons of colitis mice which were pre-treated with Fs.Cr was markedly less as compared to the untreated colitic mice, it was therefore speculated that increased cAMP levels due to Fs.Cr treatment could be due to PDE-4 inhibition. Consistent with this, the HPLC fingerprints of Fs.Cr matched the peaks of quercetin, a potential

PDE-4 inhibitor (Chan et al., 2008) which has been reported to ameliorate severity of acetic acid colitis in rats (Dodda, Chhajed, & Mishra, 2014), whereas another selective PDE-4 inhibitor, Tetomilast, also showed success in animal models of IBD

(Loher et al., 2003) although effectiveness in clinical studies need to be established

(Schreiber et al., 2007). This aspect correlates with previous study in chapter 2 where

Fs.Cr showed ameliorating effect in AA-induced colitis in mice.

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Fs.Cr at relatively high doses has inhibitory activity against high K+-induced contractions; indicative of weak Ca++ antagonists like activity. However, the potency of Fs.Cr inhibitory activity observed in current study was 10 times more for CCh- induced contractions as compared to high K+-induced contractions, indicating that the predominant antispasmodic mechanism of Fs.Cr is via PDE inhibition. The dual action of Fs.Cr as PDE inhibitor and CCB holds therapeutic significance as pinaverium bromide, a CCB, is currently considered a choice for IBD-induced chronic pain that persists even after resolution of inflammation and resembles with the IBS profile (Awad et al., 1994).

Based on the current findings it is evident that Fs.Cr possesses antispasmodic and antidiarrhoeal effect through multiple mechanisms (a combination of PDE-4 inhibition and Ca++ antagonist), in addition to anti-inflammatory and antioxidant activities hence could be helpful in managing IBD triggers from multiple ends and warrant clinical studies.

Since one of the important triggers of IBD is pathogenic microbial evasion, and in many patients IBD is presented after an acute gastroenteritis episode (Porter et al., 2008), the next objective was to assess the effect of Fs.Cr on pathogenic microbes with focus on those pathogens which are also implicated in IBD.

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CHAPTER 4

4. FLAXSEED AND MICROBES

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4.1. Introduction

In the previous chapter it has been shown that Fs.Cr possess PDE-4enzyme inhibitory; although PDE-4 inhibitors have shown success in animal models of IBD

(Loher et al., 2003), their role in clinical studies need to be established (Schreiber et al., 2007). This may be due to the fact that IBD is a manifestation of multiple dysregulations simultaneously coming in action to aggravate the inflammation and therefore IBD needs to be controlled from different ends, so that triggers could be minimized.

One of the major limitations of current IBD therapy is failure to attain remission and relapses which might be due to the fact, that current therapeutic approach, targets the disease from one trigger, whereas, the situation perhaps demands targeting the disease from multiple aspects. Hence, of late, multiple- targeted approach has been gaining interest in IBD therapeutics since IBD is a multifactorial disease (Xavier & Podolsky, 2007). One approach that has shown improvement in clinical outcomes in patients with IBD is to reduce the aggressive microbial load since they may acts as trigger for persistent immune stimulation and could also aggravate already initiated inflammatory cascade in IBD (Campieri &

Gionchetti, 2001; Rahimi et al., 2007). Besides these aspects, defective microbes are also the underlying causes of IBD-induced diarrhoea.

Amongst the pathogens, adherent invasive E.coli (AIEC) is the most common reported pathogen from biopsies of IBD patients (Nataro & Kaper, 1998;

Smith et al., 2013) and it resembles in virulence properties with Enteropathogenic

E.coli (EPEC), enterotoxigenic E. coli (ETEC) and enteroaggregative E.coli (EAEC)

(Santos et al., 2015). Enteric pathogens known to cause gastroenteritis have also been reported to initiate and/or exacerbate IBD (Gradel et al., 2009; Rodriguez et al., 2006).

Consistent with this, reducing the pathogenic microbes have shown better clinical outcomes in IBD (Garrido-Mesa et al., 2011; Campieri & Gionchetti, 2001;

Rahimi et al., 2007) with significant success in IBD based septic complications, such 106 as abscesses and wound infections (Sartor, 2004). Despite of these benefits, antibiotics are not suggested for longer duration as a maintenance therapy or during remission phase due to an already emerging antibiotic resistance; the situation is worse for countries like Pakistan and India, where new bacterial resistance has emerged (Trivedi and Sabnis, 2009; Kumarasamy et al., 2010).

Natural products owing to their “effect-enhancing and side-effect neutralizing potential” (Gilani & Ata-ur-Rahman, 2005), have already been a choice of therapy in IBD patients in developing world (Rogler, 2015) and could prove to be useful as an antimicrobial source as well as for isolation of novel compounds.

In the previous chapter, it is shown that Fs.Cr exhibited antidiarrhoeal and antispasmodic mechanisms by predominant PDEI activity, followed by Ca++ antagonist activity. Anti-inflammatory and antioxidant effect has been evident in ameliorating acetic acid-induced colitis, presented in chapter 2. These aspects would reduce the manifestation of disease; however, a sustainable effect might also require reducing the source of triggers. Hence, if Fs.Cr has antimicrobial potential against pathogens implicated in IBD pathogenesis; reducing their load could attribute in managing one of the persistent triggers of IBD. So far, Flaxseed’s effect against pathogens involved in IBD is not addressed. Hence, the current study was aimed to determine the antimicrobial effect of Fs.Cr against various gut pathogens causing gastroenteritis as well as the ones implicated in IBD including EPEC, ETEC and

EAEC.

4.2. Materials and Methods

Microbial strains used in this study include enteropathogenic Eschericia coli

(2348/69), Enterotoxigenic Eschericia coli (H10407) and Entero aggregative

Eschericia coli. (042), Escherichia coli 018:K1:H7, strain RS218 (E. coli K1),

Salmonella typhi (ATCC 14028; S.typhi), Pseudomonas aeruginosa (P.aeruginosa),

Bacillus Cereus (B. cereus). All the microbes are clinical isolates available upon

107 request. Microbial strains were routinely grown aerobically at 37˚C in Luria-Bertani broth (LB) for 16 hrs.

The reference drugs, gentamycin and vancomycin were purchased from the

Sigma Chemicals Co, St. Louis, MO, USA. Nutrient agar and Luria-Bertani broth powders used for antimicrobial assays were purchased from the Sigma chemicals and were of the available analytical grade.

For determining the effect against enteric pathogens to cidal and static mechanisms a concentrated extract was used. 1ml (~2.08 g) of Fs.Cr was solubilised in 9 ml of 50% methanol (1:10) and then centrifuged at 14000 g for 60 min. The supernatant was collected and microfiltered, whereas, the remaining pellet was weighed and subtracted from the amount of extract taken before centrifuge (0.20 g).

The final concentration of Fs.Cr was 1.80 g/ml or 180 µg/µl. From this stock, 5.5 µl and 13.5 µl were picked for assay that corresponded to 5 mg/ml and 12.5 mg/ml.

The vehicle was prepared by adding 1 ml of distilled water with 9 ml of 50% methanol (MeOH) and then centrifuged and filtered in similar way as the extract

(~45% MeOH). Same volume of 5.5 µl and 13.5 µl was picked for vehicle control group as picked from the extract working stock.

For the in vitro antimicrobial experiments using low concentration of 50 and

100 µg/ml, the Fs.Cr stock was prepared as 1000 mg/ml in 50% methanol and then centrifuged at 14,000 g for 60 min. The supernatant was micro-filtered; the remaining pellet was weighed and subtracted from the amount of extract taken before centrifuge (1000 mg). The final concentration of Fs.Cr was 960 mg/ml; from this parent stock, 960 g/ml working stock was prepared. Fs.Cr (10 and 20 l) were picked from the working stock for the antimicrobial assay to get the total concentration to be 50 g/ml and 100 g/ml respectively. The vehicle control used was 12.5 % MeOH.

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4.2.1. Quantitative antimicrobial assay (16 hrs)

The assay was slightly modified from the earlier reported method by Khan et al., (2008) as follows: Original inoculum (OI) of bacteria (10 µl) picked after setting an optical density (o.d.) of 0.22, which corresponded to approximately 106 colony forming units (c.f.u.), was suspended with 5.5 µl and 13.8 µl Fs.Cr corresponding to 5 mg/ml and 12.5 mg/ml. For positive control groups (100% kill)

10 µl microbes were suspended with 100 µg/ml gentamycin (for Gram positive) or vancomycin (for Gram negative), whereas, for negative control 10 µl of inoculum were suspended with 13.8 µl of the vehicle solvent (MeOH). LB was used as an assay buffer to make up volume till 200 µl. A control for OI was simultaneously prepared, by adding 10 µl of inoculum in 190 µl PBS. Thereafter all the assay tubes except OI were incubated aerobically at 37°C for 16 hrs, instead of routine assay time of 2 hrs. This was done to provide bacteria with time to grow so that difference between cidal and static effect could be determined. 10 µl from OI tube was streaked on nutrient agar plates. After this incubation, microbial cultures were 10-fold serially diluted in LB and plated on nutrient agar plates and incubated further at 37°C overnight and microbial c.f.u. were enumerated

. The percentage bactericidal effect was determined as the percentage of microbes surviving relative to the OI as follows:

퐜. 퐟. 퐮. 퐢퐧 퐞퐱퐭퐫퐚퐜퐭 ퟏퟎퟎ − [( ) 퐱 ퟏퟎퟎ] 퐨퐫퐢퐠퐢퐧퐚퐥 퐢퐧퐨퐜퐮퐥퐮퐦

The conclusions were drawn based on colony count as follows:

1. If the microbial c.f.u. was less than the c.f.u. in OI, it was considered as bactericidal effect.

2. If the microbial c.f.u. was similar to OI, it meant the effect of the test material was bacteriostatic since microbes did not grow despite provision of nutritive environment.

3. If the microbial c.f.u. was significantly greater than OI it was inferred as test material having no effect.

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4.2.2. Quantitative antimicrobial assay (2 hrs)

EPEC, ETEC and EAEC were assessed by the method mentioned previously

(4.2.1), with the exception that low concentrations of Fs.Cr (50 and 100 µg/ml) were tested for the effect against these microbes by running the assay in LB as an assay buffer and incubating the assay mix for 2 hrs at 37°C instead of 16 hrs. Microbes incubated with LB and vehicle solvent served as negative control. Microbes incubated with gentamycin served as positive control (100 µg/ml). Simultaneously an original inoculum (OI) tube was also prepared that served as a control for the amount of microbes suspended in each tube. After the incubation of assayed tubes, microbial cultures were 10-fold serially diluted and plated on nutrient agar plates and incubated further at 37°C over-night. Next day, colonies were counted. Data were presented in terms of c.f.u. compared to microbial growth in original inoculum

(OI). The percentage bactericidal effects were determined as follows:

퐜. 퐟. 퐮. 퐢퐧 퐞퐱퐭퐫퐚퐜퐭 ퟏퟎퟎ − [( ) 퐱ퟏퟎퟎ] 퐨퐫퐢퐠퐢퐧퐚퐥 퐢퐧퐨퐜퐮퐥퐮퐦

The conclusions were drawn based on colony count as follows:

1. If the microbial c.f.u. was less than the c.f.u. in OI, it was considered as bactericidal effect.

2. If the microbial c.f.u. was similar to OI or greater than OI it was inferred as test material having no effect.

4.3. Results

4.3.1. Effect on enteric pathogens

To determine the antimicrobial activity of Fs.Cr against S. typhi, B. cereus,

P. aeruginosa, E. coli K1, MRSA and VRE, 106 c.f.u (od = 0.22), respective microbes were incubated with different concentrations of Fs.Cr, vehicle solvent and positive controls. Results were calculated on the basis of number of colony counts of the specific microbes.

110

When Fs.Cr was studied for its effect on S. typhi, it was evident that compared with the original inoculum (OI; 2.48E+06 c.f.u.), Fs.Cr at the concentration of 12.5 mg/ml did not kill the microbes significantly. Interestingly, it prevented the microbial growth (p>0.05) as compared to the colony counts of OI.

This indicates bacteriostatic effect evident in Fig. 4.1 with a dotted line from y axis showing almost similar count of OI as compared to 12.5 mg/ml concentration of

Fs.Cr.

Unlike effect on S. typhi, Fs.Cr at the same concentration of 12.5 mg/ml, exhibited bactericidal effect against P. aeruginosa killing them upto 99.8%

(3.17E+05 c.f.u; p<0.001) when compared with OI (1.77E+06 c.f.u). However 5 mg/ml of Fs.Cr did not produce cidal or static effect when compared to OI as microbes with 5 mg/ml Fs.Cr showed bacterial growth of 4.17E+06 c.f.u., which is greater than microbial count in OI (Fig 4.2A).

The microbial growth in OI of B. cereus was 1.58E+06 c.f.u., whereas, Fs.Cr groups exhibited a significant kill of B. cereus by killing the microbes to 2.03E+05 c.f.u. and 8.87E+04 c.f.u. with a percent reduction of 87.15±% (p<0.05) and 94.39±

% (p<0.05) at 5 mg/ml and 12.5 mg/ml respectively. Hence, Fs.Cr possessed a bactericidal mechanism against B. cereus at both the tested concentrations (Fig.

4.2B).

When tested against E. coli K1, a highly pathogenic strain of E.coli, Fs.Cr at the concentration of 12.5 mg/ml, killed 66.86±1.80% microbes with a colony count of 3.68E+05 c.f.u. (p<0.05) as compared to 1.95E+06 c.f.u. in OI. However, there was no effect at the concentration of 5 mg/ml. Hence, Flaxseed was bactericidal against E. coli K1 at concentration of 12.5 mg/ml. Fs.Cr also exhibited bactericidal effect against antibiotic resistant strains including Gram negative VRE and Gram positive MRSA. When VRE was incubated in only vehicle, the microbial count increased from 1.06E+05 c.f.u. (OI) to 1.168e+007c.f.u. (p<0.01). In the Fs.Cr treated group, the microbes reduced significantly to 7.07E+02 c.f.u at concentration of 5 mg/ml, whereas, 100% kill was evident at 12.5 mg/ml (0 c.f.u.) as evident in

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Fig 4.4A. Fs.Cr exhibited bactericidal effect against MRSA at a concentration of 12.5 mg/ml with a microbial kill into 2.61E+04 c.f.u. (86.25±0.07%; p<0.001).

Surprisingly, 5 mg/ml of Fs.Cr had no effect when compared to both OI as well as vehicle control groups (Fig. 4.4B).

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Figure 4.1; Effect on colony counts of ~106 c.f.u of Salmonella typhi when treated with and without Fs.Cr. Concentrations used for Fs.Cr were 5 and 12.5 mg/ml assay volume and the corresponding volume of vehicle solvent was used in vehicle control group. LB was used as assay buffer, incubated at 37°C for 16 hrs. 100 µg/ml gentamycin was used as positive control that exhibited 100% kill and that’s why not shown in graph. For negative control, microbes were incubated with 45%MeOH as vehicle in LB alone. Data are presented as the means±SEM of three independent experiments performed in duplicate. p-values were calculated by comparing OI and different extract concentrations using un-paired t- test. **indicate p<0.01 vs. vehicle control. P values for OI vs treatment groups have not been shown. (---)Dotted line from y axis indicates a bacteriostatic effect at 12.5 mg/ml Fs.Cr as the concentration of microbes remains the same as OI despite the availability of nutritive environment, where OI is the estimated concentration of microbe used in the experiment (od = 0.22~106 c.f.u.). [Abbreviations: Fs.Cr = Flaxseed extract; OI = original inoculum; od = optical density; LB = Luria broth ; S. typhi = Salmonella typhi; vehicle = 45% methanol]

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Figure 4.2 (A-B); Effect on colony counts of ~106 c.f.u of Pseudomonas aeruginosa (A) and Bacillus cereus (B) treated with and without Fs.Cr. Concentrations used for Fs.Cr were 5 and 12.5 mg/ml and the corresponding volume of vehicle was used in vehicle control group. Gentamycin was used as positive control (100 µg/ml) that exhibited 100% kill and that’s why not shown in graph. For negative control, microbes were incubated with vehicle = 45%MeOH methanol (vehicle solvent) in LB alone. Data are presented as the means±SEM of three independent experiments performed in duplicate. p-values were calculated by comparing OI and different extract concentrations using un-paired t- test. **indicate p<0.01 vs. OI. Dotted line (---) from y axis indicates a bacteriostatic effect at 12.5 mg/ml Fs.Cr as the concentration of microbes remains the same as OI despite the availability of nutritive environment, where OI is the estimated concentration of microbe used in the experiment (od = 0.22~106 c.f.u.). [Abbreviations: Fs.Cr = Flaxseed extract; OI = original inoculum; od = optical density; LB = Luria broth; B.cereus = Bacillus cereus; P.aeruginosa = Pseudomonas aeruginosa; vehicle = 45% methanol]

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Figure 4.3; Effect on colony counts of ~106 c.f.u of Eschericia coli K1 treated with and without Fs.Cr. Concentrations used for Fs.Cr were 5 and 12.5 mg/ml assay volume and the corresponding volume of vehicle was used in negative control with LB used as assay buffer; assay tubes incubated at 37°C for 16 hrs. 100 µg/ml gentamycin was used as positive control that exhibited 100% kill and that’s why not shown in graph. Data are presented as the means±SEM of three independent experiments performed in duplicate. p-values were calculated by comparing OI and different extract concentrations using un-paired t- test. **indicate p<0.01 vs. OI, where OI is the estimated concentration of microbe used in the experiment (od = 0.22~106 c.f.u.). [Abbreviations: c.f.u. = colony forming unit; Fs.Cr = Flaxseed extract; OI = original inoculum; od = optical density; LB = Luria broth; E.coli = Eschericia coli; vehicle = 45% methanol].

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Figure 4.4; Effect on colony counts of ~106 c.f.u of vancomycin-resistant Enterococcus faecalis (A), and Methicillin-resistant Staphylococcus aureus (B), treated with and without Fs.Cr. Concentrations used for Fs.Cr were 5 and 12.5 mg/ml assay volume and the corresponding volume of vehicle was used in negative control with LB used as assay buffer; assay tubes were incubated at 37°C for 16 hrs. 100 µg/ml gentamycin and vancomycin were used as positive control for VRE and MRSA respectively that exhibited 100% kill and that’s why not shown in graph. Data are presented as the means±SEM of three independent experiments performed in duplicate. p-values were calculated by comparing OI and different extract concentrations using un-paired t- test. *; **; *** indicate p<0.05, p<0.01 and p<0.001 vs. OI, where OI is the estimated concentration of microbe used in the experiment (od = 0.22~106 c.f.u.). Statistical significance of VRE (A) is not shown for 12.5 mg/ml of Fs.Cr because of 100% kill. [Abbreviations: Fs.Cr = Flaxseed extract; OI = original inoculum; o.d = optical density; LB = Luria broth; VRE = Vancomycin-resistant Enterococcus faecalis; MRSA = methicillin-resistant Staphylococcus aureus; vehicle= 45% methanol].

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4.3.2.1. Enteropathogenic Eschericia coli

When EPEC was incubated with vehicle solvent and LB as an assay buffer for 16 hrs at 37°C, a colony count of 1.027e+007 c.f.u. was grown in contrast to

1.20E+06 c.f.u. in OI, indicating a remarkable growth in vehicle controls due to availability of nutritive environment. Fs.Cr inhibited growth of EPEC in a concentration-dependent manner causing >98% kill as compared to OI, at both 5 and

12.5 mg/ml concentrations. In terms of colony count, EPEC suspended with 5 mg/ml

Fs.Cr inhibited the growth to 1.04E+04 c.f.u. (p<0.01 vs OI), whereas, at 12.5 mg/ml concentration only 3.84E+03 c.f.u. of EPEC survived (p<0.01), in contrast to

1.20E+06 c.f.u. in OI (Fig. 4.5 A)

In addition to this, effect of Fs.Cr was studied on EPEC at a lower concentration of 50 µg/ml and 100 µg/ml with 2 hrs of incubation time. Fs.Cr exhibited a significant kill of microbes at 100 µg/ml with 1.45E+05 c.f.u of EPEC; this colony count was significantly less (p<0.05) as compared to microbial count in

OI (Fig. 4.5B). Hence, Fs.Cr exhibited bactericidal effect at as low concentration as

100 µg/ml.

4.3.2.2. Enterotoxigenic E. coli

When ETEC was incubated with LB as an assay buffer, then microbial cultures grew from 1.05E+06 c.f.u. (OI) to 5.71E+11 c.f.u. (vehicle control). ETEC suspended with 5 and 12.5 mg/ml Fs.Cr and assayed in LB exhibited colony count of 3.61E+06 c.f.u. and 2.08E+06 c.f.u. respectively, which were not significantly different than OI colony count. Thus in presence of LB as a continuous source of nutrition for ETEC, Fs.Cr has a bacteriostatic effect (Fig. 4.6A)

The same effect was tested for a lower concentration of Fs.Cr 50 µg/ml and

100 µg/ml with 2 hrs of incubation time. Fs.Cr exhibited a very slight kill of microbe for 12.5 mg/ml concentration with 7.25E+05 c.f.u. of ETEC, which was not statistically significant as compared to OI (1.11E+06 c.f.u.) as evident in Fig. 4.6B.

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4.3.2.3. Enteroaggregative Eschericia coli

When EAEC was incubated with LB as an assay buffer, then microbial cultures grew from 1.14E+06 c.f.u. (OI) to 2.50E+11 c.f.u. (vehicle control). EAEC suspended with 5 and 12.5 mg/ml Fs.Cr and assayed in LB exhibited colony count of 4.23E+06 c.f.u. and 7.51E+05 c.f.u. respectively which were not significantly different than colony count in OI. Thus in presence of LB as a continuous source of nutrition for EAEC, Fs.Cr possessed a bacteriostatic effect evident in Fig. 4.7A.

Fs.Cr was tested for the same effect at the lower concentrations of 50 µg/ml and 100 µg/ml. Incubation time was reduced to 2 hrs to assess the bactericidal mechanism. Fs.Cr at 12.5 mg/ml concentration exhibited a negligible kill of microbe (2.77E+05 c.f.u.), Fig. 4.7B.

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Figure 4.5; Effect on colony counts of ~106 enteropathogenic Eschericia coli incubated with and without Fs.Cr for 16 hrs (A), and 2 hrs (B). Concentrations of 5 and 12.5 mg/ml of Fs.Cr were used for assay time of 16 hrs, whereas, concentrations of 50 and 100 µg/ml of Fs.Cr were used when assay time was 2 hrs (B). The same corresponding volume of vehicle solvent was used in vehicle control group in 200 l assay volume with Lurea broth (LB) as assay buffer, incubated at 37°C. Original inoculum (OI) is the approximate concentration of microbes plated before running assay (od = 0.22~106 c.f.u.). 100 µg/ml gentamycin was used as positive control not shown in graph (100% kill). The data are presented as the means±SEM of three independent experiments performed in duplicate. p-values were calculated by comparing OI and different extract concentrations using un-paired t- test. *; **; *** indicate p<0.05, p<0.01 and p<0.001 vs. OI. [Abbreviations: Fs.Cr = Flaxseed extract; OI = original inoculum; od = optical density; LB = Luria broth; EPEC = enteropathogenic Eschericia coli; vehicle = 45% methanol (A); 12.5% MeOH for B].

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Figure 4.6; Effect on colony counts of ~106 enterotoxigenic Eschericia coli incubated with and without Fs.Cr for 16 hrs (A), and 2 hrs (B). Concentrations of 5 and 12.5 mg/ml of Fs.Cr were used for assay time of 16 hrs, whereas, concentrations of 50 and 100 µg/ml of Fs.Cr were used when assay time was 2 hrs (B). The same corresponding volume of vehicle solvent was used in vehicle control group in 200 l assay volume with Lurea broth (LB) as assay buffer, incubated at 37°C. Original inoculum (OI) is the approximate concentration of microbes plated before running assay (od = 0.22~106 c.f.u.). 100 µg/ml gentamycin was used as positive control not shown in graph (100% kill). The data are presented as the means±SEM of three independent experiments performed in duplicate. p-values were calculated by comparing OI and different extract concentrations using un-paired t- test. [Abbreviations: Fs.Cr = Flaxseed extract; OI = original inoculum; od = optical density; LB = Luria broth; ETEC = enterotoxigenic Eschericia coli; vehicle = 45% methanol (A); 12.5% MeOH (B)].

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Figure 4.7; Effect on colony counts of ~106 enteroaggregative Eschericia coli incubated with and without Flaxseed extract (Fs.Cr) for 16 hrs (A) and 2 hrs (B). Concentrations of 5 and 12.5 mg/ml of Fs.Cr were used for assay time of 16 hrs, whereas, concentrations of 50 and 100 µg/ml of Fs.Cr were used when assay time was 2 hrs (B). The same corresponding volume of vehicle solvent was used in vehicle control group in 200 l assay volume with Lurea broth (LB) as assay buffer, incubated at 37°C. Original inoculum (OI) is the approximate concentration of microbes plated before running assay (od = 0.22~106 c.f.u.). 100 µg/ml gentamycin was used as positive control not shown in graph (100% kill). The data are presented as the means±SEM of three independent experiments performed in duplicate. p-values were calculated by comparing OI and different extract concentrations using un-paired t- test. [Abbreviations: Fs.Cr = Flaxseed extract; OI = original inoculum; od = optical density; LB = Luria broth; EAEC = enteroaggregative Eschericia coli; vehicle = 45% methanol (A); 12.5% MeOH (B)].

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4.4. Discussion

The current study was aimed to identify the potential of Fs.Cr against the pathogenic microbes implicated in IBD along with studying the possible cidal or static mechanism. For this purpose, LB was used as an assay buffer instead of routinely used PBS to provide a source of nutrition to microbes even after incubation with the test material for 16 hrs. Hence, if the microbes were still killed in the presence of growth medium in the 16 hrs which is the optimal time for exhibiting bacterial growth, it could be inferred as a true bactericidal effect, whereas, if the microbes did not grow beyond the originally inoculated amount, it would be inferred as static mechanism.

Amongst the pathogens studied, Fs.Cr exhibited bacteriostatic effect against

S. typhi at 12.5 mg/ml concentration, whereas, it displayed static and cidal effect against P. aeruginosa at 5 mg/ml and 12.5 mg/ml respectively. This is important finding from IBD perspective, as Salmonella positive cultures have shown higher incidence rate of IBD (Mylonaki et al., 2005) with prominent implication in UC

(Campieri & Gionchetti, 2001) and P. aeruginosa genes have also been linked to

CD (Wei et al., 2002). P aeruginosa may contribute in IBD by increasing paracellular permeability in stressful host environment that causes intestinal epithelial hypoxia (Kohler et al., 2005) and transform apical membrane into basolateral membrane, thus allowing the internalization of microbe into polarized intestinal epithelial cells which is not otherwise accessible to gut microbes (Kierbel et al, 2007). Hence, by reducing these microbes, the initiation and/or exacerbation of inflammatory cycle could be managed.

Earlier, antimicrobial activity of Fs.Cr has been reported against S. typhi and

P. aeruginosa (Bakht et al., 2014; Gur et al., 2006) but the antimicrobial assay used in current study has an edge as it gives a quantified effect with distinction between cidal and static effects, unlike previous reports using qualitative analysis.

To identify the effect of Fs.Cr against different types of E. coli, it was first tested on a highly pathogenic strain E. coli (K1); Fs.Cr was able to kill these at 12.5 122 mg/ml concentration significantly, whereas, at 5 mg/ml concentration there was neither cidal nor static effect evident. E. coli is one of the leading causes of enteric infections in developing countries (Boschi-Pinto et al., 2008) and its various virulent forms are implicated in IBD. Earlier reported antimicrobial effect of Fs.Cr against

E. coli K-12 (Bakht et al., 2014; Gur et al., 2006) cannot be compared to the current finding as E. coli K-12 is a non-pathogenic strain, whereas, in this study, E. coli K1 employed is a clinical isolate from a meningitis patient.

Thereafter, Fs.Cr was tested against EPEC whereby Fs.Cr exhibited bactericidal effect at 5 mg/ml and 12.5 mg/ml concentration. This is a very important finding because the invasive level of EPEC is more or less similar to adherent invasive E. coli (AIEC; strain; LF82) (Santos et al., 2015), which is the most common pathogen found in CD (Smith et al., 2013). EPEC also is able to sustain iodoacetamide-induced UC-like colitis in rats (Abdallah et al., 2011) and share common endpoints with IBD. Hence, it could be speculated that the invasive ability of E.coli in IBD patients may be limited due to the use of Fs.Cr which would ultimately reduce the consequence EPEC manifestation that include upregulation of

NF-(nuclear factor kappa-light-chain-enhancer of activated B cells) (Hecht,

2005) and modulation of TNF-α, IL-1β, IL-6, COX-2, and apoptosis and hence could prevent one of the triggers for immune dysregulations.

ETEC and EAEC are the types of E.coli which share virulence properties with AIEC. Interestingly ETEC and EAEC were not killed by Fs.Cr; however, the growth was inhibited, depicting a static effect. One aspect that needs to be focused w.r.t. the static effect of ETEC and EAEC is the growth pattern of their controls. The microbial growth in ETEC and EAEC control groups in presence of LB was

5.71E+11 c.f.u. and 2.50E+11 c.f.u. respectively as compared to EPEC control

(1.03E+07 c.f.u). These colony counts were extremely high as compared to all the pathogens that we studied in the current study in presence of LB as assay buffer, hence indicating an aggressive growth pattern. It is a possibility that Fs.Cr possesses cidal mechanism against EAEC because when they were incubated with Fs.Cr for 2

123 hrs, a weak cidal activity was apparent which was not evident when incubated for

16 hrs because the microbes started to grow aggressively in nutritious environment in the said time. This is the first study to report the effect of Flaxseed on EPEC,

ETEC, and EAEC and has important implications for IBD as reducing their load may eventually decrease one of the driving forces for persistent immune stimulation.

Based on the effect various pathogenic type of E.coli, it can be concluded that Fs.Cr possess different mechanisms for cidal or static effect in different types of E.coli.

Possibly that’s is why it exhibited cidal effect against E.coli K1 at 12.5 mg/ml concentration only but displayed cidal effect against EPEC at both 5 mg/ml and 12.5 mg/ml concentration. It exhibited no cidal effect against ETEC and EAEC but did show significant static effect against ETEC and EAEC.

Furthermore, Fs.Cr was evaluated on the hospital acquired drug-resistant microbes called methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE), which are also implicated in causing the infectious diarrhoea (Bartlett, 2006; Thakkar and Agrawal, 2010). Fs.Cr at 12.5 mg/ml dose, exhibited bactericidal effect against MRSA and VRE; however, a surprising effect on VRE was a 100% kill. This is the first study to report the effect of Fs.Cr against these drug-resistant strains. The 100% kill in VRE suggests the presence of constituents that could serve as a source of new and better antimicrobial compounds.

The above findings hold a lot of significance from therapeutic point of view because there’s a need for options that could be a source of new antimicrobials as well as combat resistant microbes. With recent reports of the discovery of carbapenem-resistance gene in enteric microbes, such as E. coli and K. pneumonia from South Asian countries (Oberoi et al., 2013; Kumarasamy et al., 2010), it has been predicted that we will be pushed into post-antibiotic era very soon, where once again, like pre-antibiotic era, the common microbial infections would lead into deadly outcomes (Alanis, 2005).

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The broad-spectrum efficacy of Fs.Cr is also important because at present a very few antibiotics are effective against different enteric microbial pathogens

(Smith and Coast, 2002). This effectiveness of Fs.Cr could be correlated with the resemblance of HPLC peaks of Fs.Cr (Fig. 2.2A) with two synthetic compounds, ofloxacin (Fig. 2.2E) and metronidazole (Fig. 2.2F), both of which have broad spectrum coverage against aerobic and anaerobic pathogens (Freeman et al., 1997).

However, the presence of some novel compounds is speculated based on 100% kill of VRE, which is resistant to conventional antibiotics.

Alternatively, it is possible, that multiple components present in Flaxseed, nets in a way that effect is enhanced and/or resistance to microbes is taken care of, when used in natural form. It may be important to cite the example of artemesin derived antimicrobials, which were novel discovery in terms of showing effectiveness against resistant strains of malaria but now resistance is beginning to develop to artemesin, whereas, the original plant Artemesia annua, if taken in its natural form (herbal tea), once a week has reported to prevent malria (Mueller et al.,

2004).

Previous findings in the thesis presented, indicate that Fs.Cr is effective in animal models of IBD because of its anti-inflammatory, antioxidant and antidiarrhoeal activities. In addition, antispasmodic mechanisms with PDE-4 enzyme inhibitory and Ca++ antagonist activity could also account for its effectiveness in IBD. From this study, an additional aspect of Fs.Cr directed against gut pathogenic microbes provide another evidence for its multifactorial potential to target IBD. Hence, Fs.Cr may become a choice of remedy that can offer to control the basic pathophysiological tenets of IBD by their immunomodulatory, mucosal protective and antimicrobial dysregulation.

Since Flaxseed oil is already an edible source in common use, therefore, it was further aimed to know to identify potential for Flaxseed oil for IBD therapeutics.

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Chapter 5

5. DIFFERENT FORMS OF FLAXSEED AND

POTENTIAL FOR IBD

126

5.1. Introduction

Flaxseed has been used for different purposes in different forms, such as meal (Zarepoor et al., 2014), defatted meal (Xu et al., 2012), oil (Power et al., 2016) and mucilage (Duke et al., 2002; Sastri, 1956).

A natural product derived from herbal source is known to contain both the polar and non-polar constituents (Ignat et al., 2011), and therefore different extraction solvents extract out different compounds (Handa et al., 2008). Hence, depending on nature of components responsible for activity, an extraction solvent is adopted (Vuong et al., 2013). It is known that 70:30 methanolic aqueous solvent extract out optimal constituents, both the polar and non-polar with predominance of polar constituents (Ignat et al., 2011). Non-polar constituents are extracted at greater extent in organic solvents (Lin et al.,1999; Lang and Wai, 2001). Hence, for understanding a holistic picture of a plant, it would be best to assess effectiveness of both the extract and oil, as both are speculated to possess different mechanisms, due to predominance of different nature of constituents. The different mechanisms in respective fractions (extract and oil) may net up the total potential of a plant. Thus in the current study the potential of Flaxseed oil in IBD relevant symptoms was explored with emphasis on antispasmodic mechanisms, along with in vitro antimicrobial assays against microbes implicated in IBD and preliminary screening for its effectiveness against acetic acid (AA)-induced colitis in mice.

Traditionally Flaxseed is predominantly known for its use as laxative (Duke et al, 2002). It was important to investigate which fraction of Flaxseed has predominant spasmogenic component, as predominant laxative effect could further worsen the symptoms of IBD-induced diarrhoea. Possibly that’s why, a study that used Flaxseed meal and oil to prevent dextran sodium sulfate (DSS)-induced colitis, ended up aggravating the symptoms in that model (Zarepoor et al., 2014). On the other hand, identifying the dose and form that exhibit laxative effect might be beneficial in left sided colitis which is constipation predominant. Hence, Flaxseed oil and mucilage, both were studied for spasmodic mechanisms. 127

5.2. Materials and Methods

5.2.1. Preparation of Flaxseed oil and mucilage

Approximately 1 kg ground Flaxseeds were soaked in the petroleum ether

(60-70C) (Kaithwas & Majumdar, 2010b), with slight modification that they were kept on water bath at 45C for 7 days with occasional shaking. Petroleum ether was evaporated from the oil on Rotavapor, and the oil was filtered to clarity. The oil was stored at -20ͦC in amber coloured airtight bottle. The yield of Flaxseed oil was 27% w/w with reference to dried seeds.

Flaxseed mucilage was prepared by the method described by Bhatty (1993) with slight modification. Whole Flaxseeds (100 g) were macerated in water at 80°C and then the temperature was reduced by 1C every hr, till it came to 40C, on which it was kept for next 12 hrs. It was then filtered by passing through glass wool kept in Buchman flask that was attached to vacuum pump, and then concentrated further on Rotavapor. The mucilage was precipitated from the extract by adding 80% ethanol in water (1:4 v/v) and allowed standing it for 1 hr. The precipitated mucilage was centrifuged at 6000 g for 35 min. The supernatant was discarded and the residual mucilage was weighed to be 38.17 g (gummy form). It was stored in -20C till further use.

5.2.1. Chemicals

The reference drugs, 4-Aminopyridine, acetylcholine chloride, atropine sulphate, carbamyl choline chloride, glibenclamide, histamine hydrochloride, loperamide, potassium chloride, pyrilamine maleate, and tetra-ethyl ammonium chloride (TEA) were purchased from Sigma–Aldrich Chemicals Company (St

Louis, MO, USA). Chemicals used for preparing physiological salt solutions include calcium chloride, glucose, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium dihydrogen phosphate, and were obtained from Merck, Darmstadt, Germany. DMSO and Tween-80 used for

128 solubilization were purchased from Merck, Darmstadt, Germany and Scharlau

Chemicals, Barcelona, Spain respectively. All chemicals used were of the available analytical grade.

5.2.2. Animals

BALB/c mice (20-25 g), local breed rabbits (1-1.5 kg) and guinea-pigs (300-

400g), of either gender, were used for the antidiarrhoeal and laxative study, whereas,

6-8 weeks’ old female BALB/c mice (22-30g) were used for colitis study. Animals were housed at the animal house of the Aga Khan University Medical College,

Karachi, maintained at 23-25°C. They were kept in plastic cages (47x34x18 cm3) with sawdust (changed at every 48 hrs) and fasted for 18 hrs for antidiarrhoeal experiments and for 6 hrs for determining laxative activity before the experiment, and they were given tap water and standard diet routinely. Animals had free access to water but food was withdrawn 24 hrs prior to experiments.

Rabbits (1-1.2 kg) and guinea-pigs (weighing 400-550 g; n = 6) of local breed and of either gender, were fasted for 18 hrs, were anaesthetised by Florane and then sacrificed by cervical dislocation gently. For acetic acid colitis, after 24 hrs, mice were killed by cervical dislocation and colonic biopsies were taken for macroscopic damage. The study protocol was approved by the Ethics Committee for Animal Care and Use (ECACU) of The Aga Khan University with approval # 005-Ani-BBS-13.

5.2.3. Working stocks

For the in vivo and in vitro experiments, the Flaxseed oil was solubilised in

10% DMSO and 5% Tween-80 and subsequent dilutions were made in distilled water on the day of experiment. Flaxseed mucilage was solubilised in water with frequent cycles of sonication and vortexing on the day of experiment.

For the in vitro antimicrobial experiments, both the Flaxseed oil and extract were used. Oil was already in the liquid form and could be easily drawn in minutest quantities, hence no dilution was required. 1000 µl of oil corresponding to 1000 µg 129 was centrifuged at 14000g for 60 min. There was no pellet formed, hence the oil was further microfiltered. From this 1.8 µl and 2.8 µl were picked for assay that corresponded to 9 g/ml and 14 g/ml respectively.

Fs.Cr was prepared as 1000 mg/ml with the final concentration of Fs.Cr being 960 mg/ml in similar way as mentioned in 4.2.4. From this parent stock, 960

g/ml working stock was prepared. 10 and 20 l of this Fs.Cr was picked from the working stock for the antimicrobial assay to get the total concentration to be 50

g/ml and 100 g/ml respectively.

5.2.4. Acute toxicity study

A total of 20 mice (20–25 g) of either gender were equally divided into seven groups (n = 5). Amongst them three groups were administered different doses of

Flaxseed oil (300, 500 and 1000 mg/kg; i.p.) and two groups were administered mucilage (500 and 1000 mg/kg; PO). There were two negative control groups with one group receiving vehicle for oil (10% DMSO and 5% Tween 80 in water) and the other group that served as the vehicle control for mucilage groups received water as vehicle (10 ml/kg). The animals were allowed food and water ad libitum and were regularly observed every 30 min up to 6 hrs for the signs of toxicity, while the lethality was monitored up to 24 hrs. Toxicity signs assessed were piloerection, exploratory behavioral changes and blindness.

5.2.5. HPLC fingerprints

High performance liquid chromatography (HPLC) analysis of Flaxseed oil was done at H.E.J institute of chemistry, by Professor Bina Siddiqui’s team.

Briefly, it was analysed using a Schimadzu (Kyoto, Japan) Prominence 20-

AT equipped with a pump (LC20AT), PDA detector (SPD-M20A) and Kromasil-

100C18 column (Teknokroma, 8 m, 25X0.46 mm) with membrane filter 0.45 m and syringe filter 0.45 m. 2 mg of sample was dissolved in 2.0 ml of mobile phase

130 and the sample was sonicated for 30 min. Then it was filtered through a Millipore

(0.45 m) filter prior to injection. The mobile phase consisting of CH3CN:H2O

(15:85) (0.5% CH3COOH) methanol: acetonitrile: water: acetic acid (40:20:39:1 v/v) was delivered at a flow rate of 1 ml/min, while column temperature was maintained at 25C, whereas, ultraviolet detection was performed at 254 nm.

5.2.6. Spasmodic mechanisms in isolated tissues

The effect on gut motility was tested using isolated rabbit jejunum and guinea-pig ileum preparations (Gilani et al., 1997; 2005). Rabbit jejunum exhibits spontaneous activity, thus allowing to primarily test inhibitory effect without using induced contractions, though spasmogenic effect can be tested, whereas, guinea-pig ileum, a quiescent preparation is suited best for testing spasmogenic effect (Gilani et al., 1997).

Hence for assessing the spasmogenic effect, Flaxseed oil and mucilage were tested in both isolated rabbit jejunum and guinea-pig ileum. Isolated rabbit jejunum was used for studying the modulation of spontaneous contractions, whereas, isolated guinea-pig ileum preparation was only used to assess the spasmogenic effect.

Respective tissues of 2 cm length were suspended in 10 ml tissue bath containing

Tyrode's solution and aerated with carbogen which were maintained at 37°C. Tissues were equilibrated by adding a preload of 1 g. The whole set-up of connection to the tissue assembly set up was similar as described in section 3.2.7.

Induction of contraction with Flaxseed oil or ACh above the basal tone was considered as spasmogenic effect of rabbit jejunal and guinea-pig ileum tissues, whereas, the reduction in amplitude of spontaneous contraction after addition of drug in a concentration-dependent manner was considered as spasmolytic effect and was measured with the peak response (in percentage) of the tissue before administration of an agonist (Flaxseed oil/mucilage).

Prior to assessing the spasmogenic and/or spasmolytic activity, the tissues were stabilized with repeated administration of 0.3 μM ACh, subsequently washed

131 with the Tyrode’s solution till the sub-maximal responses were evident. This was followed by constructing the ACh dose–response curves till the supramaximal responses were obtained. The dose at which there was no further increase in amplitude of ACh peak was considered as ACh maximum response (ACh max)

(Gilani and Coblin, 1986). The contractile effect exhibited by Flaxseed oil/mucilage was compared with supra-maximal dose of Ach (10 µM).

The spasmogenic effect of Flaxseed oil/mucilage were further characterized by pre-treating respective tissues for 30 min with different antagonists (atropine, pyrilamine, and combination of atropine and pyrilamine; 1 M each) simultaneously, and then the stimulatory effect was re-determined in the presence of antagonist (Ghayur & Gilani, 2005).

The spasmolytic effect was characterized in rabbit jejunal tissues by testing for inhibitory effect against the spasmogens that included CCh (1 µM), low K+ (25 mM) and high K+ (80 mM). This would indicate whether or not, there was a K+ channel opening (KCO), Ca++ antagonist or PDE enzyme inhibitory activity involved, respectively. For this purpose, the test material was added cumulatively in a concentration-dependent fashion. The inhibitory effect was plotted in similar way as for Fs.Cr mentioned in chapter 3 (percentage of control contraction).

Possible presence of K+ channel opening (KCO) mechanisms (Quast, 1993) were further assessed in rabbit jejunal tissues according to previously mentioned protocols (Gilani et al., 2000) as follows: The inhibitory CRCs of Flaxseed oil was constructed against low K+-induced contractions in the absence and presence of 4- aminopyridine (4-AP; 1 M), glibenclamide (Gb; 10 mM) and tetra- ethylammoniumchloride (TEA; 30 M), which are voltage-dependent, ATP- dependent and non-specific K+-channels blockers respectively.

5.2.7. Laxative activity in mice

For determining the laxative effect of test materials, method elaborated by

Mehmood and Gilani (2010) was used. Mice were divided into10 groups and 8

132 groups (6 animals in each) for Flaxseed oil and mucilage respectively. The 1st group was the negative control that was administered vehicle (DMSO and Tween-80 in distilled water; 10 ml/kg) instead of Flaxseed oil and water instead of mucilage. The

2nd group served as the positive control which received carbachol (CCh) in dose of

1 mg/kg; i.p. The 3rd, 4th and 5th groups were the treatment groups that received

Flaxseed orally at the respective doses of 30, 70 and 100 mg/kg, whereas, there were two treatment groups for Flaxseed mucilage that were given dose of 1 g/kg and 2.5 g/kg, orally for testing of laxative activity. Mice were fasted for 6 hrs and were placed individually in cages that were covered with filter paper.

To explore the underlying mechanism for laxative effect, separate sets of mice (group # 6–10) for Flaxseed oil and (group # 5-8) for Flaxseed mucilage, were pre-treated with atropine (10 mg/kg, i.p.) 1 hr before administration of the vehicle,

CCh or Flaxseed oil/Flaxseed mucilage. After 24 hrs, the faeces production (both dry and wet) was counted in all animals. Laxative effect was considered when the percentage of wet faeces relative to total faecal output was increased.

The wet faeces were further analyzed for the type of faeces as reported earlier

(Girard et al., 2003). The faeces were scored from the scale of 1 to 3, where score 1

= Soft but formed; score 2 = Very soft and score 3 = Diarrhoeal drops. This score was multiplied with the number of stools produced in that respective score and the total score was the sum of all the scores:

[(score 1 x n) + (score 2 x n) + (score 3 x n), where n is the number of stools in the given duration of the experiment. For a test material to be considered as laxative it ideally should increase the laxative score.

5.2.8. Castor oil-induced diarrhoea in mice

Antidiarrhoeal effect of Flaxseed oil was tested on castor oil-induced diarrhoea by the method previously used in our lab (Bashir et al., 2011). Mucilage was not tested for antidiarrhoeal activity based on ex-vivo results.

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Briefly, BALB/c mice fasted for 18 hrs before the experiment, were divided into 6 groups of 5 mice each, housed in individual cages. The first group labeled as the negative control received saline in vehicle (5% DMSO and 10% Tween-80 in distilled water; 10 ml/kg; P.O), whereas, the 2nd group (untreated) also received saline in vehicle to be later followed by castor oil administration for induction of diarrhoea. The 3rd group (positive control) received loperamide (10 mg/kg; P.O.), whereas, the 4th and 5th (treatment) groups received increasing concentrations of 100 and 300 mg/kg of Flaxseed oil respectively. 1 hr after the treatment, each animal, except those in negative control group, received 10 ml/kg of castor oil orally through a feeding needle. After 4 hrs, the cages were inspected for the frequency of the typical diarrhoeal droppings as well as the consistency of stool (Girard et al., 2003).

Briefly, the faeces were scored as score 0: no diarrhoea; score 1: soft but formed stool; score 2: very soft stool; score 3: diarrhoea. For each group, total score was calculated as sum of the individual scores. Individual score was calculated by multiplying the respective score with the number of diarrhoeal droppings.

The antidiarrhoeal effect of Flaxseed oil was also compared with Flaxseed extract (Fs.Cr) with respect to diarrhoeal score.

5.2.9. Enteropooling assay in mice

As a part of studying mechanisms of antidiarrhoeal activity of Flaxseed oil, intestinal fluid accumulation assay was used (Capasso et al., 2002). BALB/c mice fasted for 18 hrs were divided into six groups and dosed orally. The 1st group labeled as the negative control group received saline (10 ml/kg), dissolved in vehicle (5% DMSO and 10% Tween-80 in distilled water), whereas, the 2nd group

(untreated) also received vehicle followed by castor oil treatment. The third group

(positive control) received atropine (10 mg/kg; i.p.), whereas, the 4th and 5th groups

(treatment) received increasing doses of 100 and 300 mg/kg of Flaxseed oil orally, respectively. 1 hr after the control and test material administration, each animal received 10 ml/kg of castor oil orally except the negative control group, to induce

134 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 were expressed as follows:

Pi/Pm x 1000 where, Pi is the weight of the intestine and Pm is the weight of the animal in grams.

This effect was further compared with the effect exhibited by Flaxseed extract

(Fs.Cr)

5.2.10. Antimicrobial assay in vitro

For the in vitro antimicrobial experiments against microbes implicated in

IBD (EPEC, ETEC and EAEC), 1.8 l and 2.8 l (9 g/ml and 14 g/ml respectively) of Flaxseed oil was tested in a quantitative microbial assay as mentioned earlier (section 4.2.2) and adopted from method by Khan et al., (2008).

Additionally, the antimicrobial assay for extract was re-run to compare the efficacy of relatively low dose Flaxseed oil i.e. 9 g/ml and 14 g/ml with 50 g/ml and 100

g/ml dose of Fs.Cr.

The doses and controls used in antimicrobial assay against the Fs.Cr were similar as mentioned in section 4.2.2. For Flaxseed oil the similar method was used, with the exception that doses were different. Hence, 10 µl of inoculum (termed as

OI = original inoculum; od = 0.22) corresponding to approximately 106 colony forming units (c.f.u.) was suspended with 1.8 µl and 2.8 µl of Flaxseed oil, whereas, for negative control 10 µl of inoculum (OI) was suspended with 1.8 µl and 2.8 µl of

LB instead of Flaxseed oil. For positive control groups (100% kill) 10 µl of inoculum were suspended with 100 µg/ml gentamycin. These were then incubated for 2 hrs.

After this incubation, microbial cultures were 10-fold serially diluted in LB and plated on nutrient agar plates and incubated further at 37°C over-night and microbial c.f.u. were enumerated. Analysis was done in similar manner as mentioned in section

4.2.3. This effect was further compared with the effect produced by Fs.Cr at 50 and

100 µg/ml doses. 135

5.2.11. Acetic acid-induced colitis in mice

Flaxseed oil was further assessed for its efficacy against acetic acid (AA)- induced colitis. BALB/c mice were pre-treated with 300 and 500 mg/kg of Flaxseed oil, given i.p., for 5 days and for subsequent 2 days of colitis induction. After 5 days of Flaxseed oil administration, mice were fasted for 18 hrs with free access to water ad libitum, and then anaesthetized with a mixture of ketamine (100 mg/kg), and diazepam (2.5 mg/kg) in normal saline, given i.p. To induce colitis, 0.1 ml of 6% acetic acid (AA) (6% v/v, in 0.9% saline) was administered through feeding needle intrarectally which was inserted to a distance of 5 cm proximal to the anus

(MacPherson & Pfeiffer, 1978). Mortality rate was determined by using the following formula:

푴풊풄풆 풅풊풆풅 풊풏 풂 품풓풐풖풑 Mortality Rate = 퐱 ퟏퟎퟎ 푻풐풕풂풍 풏풖풎풃풆풓 풐풇 풎풊풄풆 풊풏 풕풉풂풕 품풓풐풖풑

Mice were observed 24 hrs after induction of colitis for the different indices of disease which included loss in body weight, effect on stool consistency and type of rectal bleed in similar way as elaborated in section 2.3.3; DAI was the average of these three parameters (Wirtz et al., 2005).

Mice were killed by cervical dislocation and the colon tissues were excised and stored in neutral buffered formalin for 24 hrs, and then colon sections were processed for H and E staining and the subsequent microscopic scoring was done in similar way as elaborated in section 2.3.5. The effect of Flaxseed oil on the above mentioned parameters were further compared with the effect exhibited by Flaxseed extract (Fs.Cr). 5.2.9. Statistical analysis

All the data expressed are means±standard error of mean (S.E.M) except the median effective concentrations (EC50 values), which are geometric means with 95% confidence intervals (CI). One-way analysis of variance (ANOVA) followed by

136

Tukey’s post-test was used to assess the laxative activity of plant oil and mucilage as well as for antidiarrhoeal and enteropooling assays of Flaxseed oil. The CRCs were analyzed by non-linear regression. All the graphing, calculations and statistical analysis were performed using GraphPad Prism 4 for windows (GraphPad Software,

San Diego, California, USA).

5.3. Results

5.3.1. HPLC finger print analysis

HPLC finger print analysis of Flaxseed oil was obtained (Fig. 5.1A). The retention times (Rt) of the prominent peaks and their corresponding concentrations were 21.109 min (0.27 g), 24.63 min (0.96 g), 33.197 (5.06g), 50.678 (0.29 g) and 61.715 (93.40 g) min.

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Figure 5.1; High performance liquid chromatography (HPLC) fingerprints of Flaxseed oil. Peaks identified in Figure indicate retention time (in min) of respective compounds.

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5.3.2. Spasmogenic effect of Flaxseed oil in isolated tissues

The effect of Flaxseed oil was tested in isolated rabbit jejunum to see effect in spontaneously contracting rabbit tissue preparations. At lower doses (0.03-1 mg/ml), Flaxseed oil exhibited a concentration-dependent stimulant effect on gut motility with maximum effect of 78.06% of acetylcholine (Ach) maximum, obtained at 1 mg/ml (Fig 5.2) followed by relaxation at a higher concentration as shown in

5.3A. When the stimulant effect was compared to ACh maximum response, it showed efficacy of 84.71±12.64% as evident in Fig. 5.2A.

The mechanism of spasmogenic effect in isolated rabbit jejunum was identified by testing the effect of Flaxseed oil in the absence and presence of atropine

(1 M) and/or pyrilamine (1 M). The spasmogenic effect of Flaxseed oil was partially blocked by atropine pre-treatment (p<0.01) compared to spontaneous effect of Flaxseed oil. This blockade was relatively more than observed with pyrilamine

(63%; P<0.05). When the tissue was pre-treated together with atropine and pyrilamine, the spasmogenic effect was completely blocked (Fig. 5.2). The spasmogenic effect was further tested on guinea-pig ileum. Surprisingly, Flaxseed oil up to the tested dose of 10 mg/ml did not produce any spasmogenic effect on guinea-pig ileum (data not shown).

139

100 Spontaneous + Atropine (1 M)  Pyrilamine (1 M) 75  Atropine (1 M) + Pyrilamine (1 M)

%age Ach Max * 25 **

1 0.1 0.3 0.01 0.03

Flaxseed Oil (mg/ml) Figure 5.2; Spasmogenic effect of Flaxseed oil on spontaneous contractions at low doses (0.01-1 mg/ml) in the absence and presence of atropine and/or pyrilamine in isolated rabbit jejunal preparations. The inhibitory effect due to pre-treatment with atropine or pyrilamine (1 µM) was different (p<0.05 vs p<0.01). Simultaneous pre-treatment with atropine and pyrilamine blocked the spasmogenic effect completely. Statistical analysis was carried out by using repeated measures ANOVA followed by Tukey's post-test. The values presented are means±S.E.M. of 5-6 preparations obtained from 4 animals. **p<0.05, ** p<0.01 compared to effect without blockers.

140

5.3.3. Spasmolytic effect of Flaxseed oil in isolated tissues

Fig 5.3A shows that the spasmogenic effect observed at lower doses (0.01-1 mg/ml) was followed by relaxant effect at high doses (3-5 mg/ml), it was therefore further studied against high and low dose K+ and CCh, to explore possible mechanisms attributing to the spasmolytic activity. It was found that Flaxseed oil

+ was distinctly more potent against low K -induced contractions with EC50 value of

0.60 mg/ml (CI: 0.36 - 0.99), while negligible effect against high K+ and relatively weaker inhibitory effect against CCh-induced contractions.

Since the inhibitory effect against low K+ (25 mM) is indicative of K+- channel opening (KCO) effect (Kishii et al., 1992; Quast, 1993), type of K+-channels involved was further studied. For this purpose, effect of Flaxseed oil in low K+- induced contraction was studied in the presence of different blockers of K+-channels to find out whether this effect was through voltage-dependent, ATP-dependent or non-specific K+-channels. Interestingly, the inhibitory CRCs of Flaxseed oil were shifted to the right in the presence of all three tested antagonists with most sensitive to TEA followed by Gb and 4-AP (Fig. 5.3 B).

Furthermore, due to evident inhibitory effect of Flaxseed oil against CCh- induced contractions at a higher dose, possibility of PDEI activity was also assessed, by using a higher dose of Flaxseed oil. Hence, to confirm the PDEI activity of

Flaxseed oil, isoprenaline CRCs were constructed in isolated rabbit jejunal tissues in presence and absence of Flaxseed oil (1 and 3 mg/ml). Expectedly, isoprenaline inhibited CCh-induced contraction with EC50 value was 2.33 (CI: 1.17 to 4.61);

Flaxseed oil shifted the curve to the left in a dose-dependent manner, with 1 mg/ml of Flaxseed oil shifting the curve to the left with EC50 value of 1.023 (CI: 0.75 to

1.37), whereas, at the tested dose of 3 mg/ml, the curve was further shifted towards left with an EC50 value of 0.10 (CI: 0.07 to 0.16) (Fig. 5.4A). Similar shift was evident in Fs.Cr but at a 100 times lower dose (0.01 and 0.03 mg/ml) as compared to Flaxseed oil (Fig 5.4B). Papaverine also shifted control isoprenaline CRCs

141 towards left at 1 M and 3 M doses (Fig. 5.4C). Hence, the PDEI activity of

Flaxseed oil is evident but exhibited at a very high dose, indicating the PDEI activity is the main antispasmodic mechanism of Fs.Cr whereas, the main antispasmodic mechanism of Flaxseed oil is KCO activity.

142

Figure 5.3 (A-B); Inhibitory effect of Flaxseed oil on: Low K+ -and High K+-induced sustained contractions (A); inhibitory CRCs against low K+ (25 mM)-induced contraction in the absence and presence of K+-channel antagonist (B). The K+ channel antagonists used were tetra-ethyl ammonium chloride (TEA; 10 mM), glibenclamide (Gb; 30 M) and 4- amino pyridine (4-AP; 1 M): The values expressed are mean ± S.E.M. (n = 5-6) preparations obtained from 4 animals. [Abbreviation: CCh = carbachol; CRCs = Concentration-response curves K+ = Potassium chloride- induced contractions.]

143

B Isoprenaline alone A Isoprenaline alone 100 100 +Fs.Cr (0.01 mg/ml) + Flaxseed oil (1 mg/ml) +Fs.Cr (0.03 mg/ml) + Flaxseed oil (3 mg/ml) 75 75

50 50

CCh-induced contraction (%) 25 25

CCh-induced contraction (%) 0 1 3 0.1 0.3 0.03 3 0.3 10 0.03 Isoprenaline (M) Isoprenaline (M) C 100 Isoprenaline alone Papaverine (1 M) Papaverine ( M) 75

CCh-induced contraction (%) contraction CCh-induced

0 1 0.1 0.3 0.01 0.03 Isoprenaline (M)

Figure 5.4; Concentration-response curves (CRC) of isoprenaline in the absence and presence of the increasing concentrations of Flaxseed oil (A), Fs.Cr (B) and papaverine (C) in isolated rabbit jejunal preparation, constructed against CCh (1 µM)-induced contractions. Flaxseed oil at 1 and 3 mg/ml shifted the isprenaline curve to the left, indicating a PDEI activity of Flaxseed oil at a higher dose. Symbols represent means±SEM of all the experiments (n = 4-5). [Abbreviations: Fs.Cr = Flaxseed extract; CCh = carbachol (1 µM); PDEI = phosphodieseteras inhibitor]

144

5.3.4. Laxative effect of Flaxseed oil in mice

Laxative potential of Flaxseed oil observed at lower doses is in line with its traditional use; hence, laxative effect was studied in mice. Treatment of mice with

Flaxseed oil produced a dose-dependent increase in wet faeces as evident in Fig.

5.5A. At 30 and 70 mg/kg doses, given orally, the increase in wet faeces was

38.33±5.5% and 54.66±6.43%, while further increase in dose (100 mg/kg) declined the effect with resulting value of 6.5±5.12% (n = 6), which is close to the normal value (5±4% in the saline treated group). The positive control, CCh (1 mg/kg; i.p.), produced 75.24±5.6% increase in wet faeces. Pre-treatment of animals with atropine

(10 mg/kg; i.p.), partially blocked the laxative effect of Flaxseed oil with resultant values of wet faeces reduced to 18.83±4.5%, 12.16±2.46% and 3.5±0.57% at the respective doses of 30, 70 and 100 mg/kg.

Fig. 5.5B shows analysis of wet faeces on the basis of their consistency and frequency. It was found that Flaxseed oil at doses of 30, 70 and 100 mg/kg, given orally, increased the laxative score from 1.33±0.7 (vehicle score) to 14±3.60,

23.22±3.78 and 1.66±0.57 respectively, whereas, CCh (1 mg/kg; i.p.), produced the score of 31±3.60. Animals were pretreated with atropine (10 mg/kg; i.p.), to see if the laxative effect was mediated through cholinergic pathways. The laxative score was reduced to 7.16±2.92, 7.33±3.78% and 1% for 30, 70 and 100 mg/kg respectively.

145

Figure 5.5; Effect of Flaxseed oil on laxative score in absence and presence of atropine pre- treatment (A), and on %age of wet feces (B). Flaxseed oil treated mice showed an increase in the laxative score as compared to untreated mice at 30 and 70 mg/kg dose, whereas this effect stopped at 100 mg/ml dose. All drugs were administered orally. CCh was used as positive control whereas untreated groups received vehicle solvent. All drugs were administered P.O., except CCh, that was administered i.p. Total laxative score was calculated as sum of individual scores. Individual score was calculated by multiplying the respective score based on consistency of stool with the number of wet feces where score 3 = diarrhoea; score 2 = very soft; score 1 = soft but formed; score 0 = normal stool. The values shown are mean±S.E.M.(n = 6) of 3 individual experiments. Statistical analysis was carried out by using one way ANOVA followed by Tukey's post- test; *p<0.05; ** p<0.01; ***p<0.001. [Abbreviations: CCh = carbachol; i.p. = intraperitoneally; P.O. = orally; S.E.M = Standard error of mean].

146

5.3.5. Antidiarrhoeal effect in mice

Based on the spasmolytic effect of Flaxseed oil observed at high doses in isolated preparations, Flaxseed oil was studied further in the in vivo experiment to see if it also exhibits antidiarrhoeal effect. The mean diarrhoeal score of castor oil group was observed to be 30.8+3.19 as compared to 0 score in vehicle control group after administration of castor oil. Pre-treatment of animals with Flaxseed oil showed a dose-dependent antidiarrhoeal activity as evident by decreased diarrhoeal score to

15.66±6.65 (49.35%), and 4.80±2.77 (84.41%) at respective oral doses of 150 and

300 mg/kg, orally, evident in Fig. 5.6A. The maximum effect observed at 300 mg/kg was similar to that of loperamide (10 mg/kg), a positive control used in this study but the next higher dose of 500 mg/kg did not show further increase in response.

The antidiarrhoeal effect of Flaxseed oil was further compared with that of

Fs.Cr. Both the oil and extract seemed to be equally effective antidiarrhoeal as evident in Table 5.1. However, Flaxseed oil group showed almost complete absence of diarrhoeal stool as compared to Flaxseed extract (Fs.Cr).

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Table 5.1. Comparison of antidiarrhoeal score of Flaxseed extract vs

Flaxseed oil Groups Flaxseed extract Flaxseed oil

Untreated (10 ml/kg) 35.66±0.85# 30.8±1.42#

Loperamide (10 mg/kg) 1.6±0.4*** 2.2±0.86***

150$/100$$ (mg/kg) 21.6±4.22** 15.6±2.97**

300 (mg/kg) 12.66±0.93*** 4.8±1.24***

Untreated groups are the ones that only received castor oil for induction and vehicle solvent instead of treatment.

[Abbreviations: $: Dose of 150 mg/kg was used for Fs.Cr; $$ dose of 100 mg/kg was used for Flaxseed oil]. # = castor oil control; ** = p<0.01; ***p<0.001 compared to castor oil control; Loperamide was used as positive control]

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5.3.6. Antisecretory effect in mice

Knowing that diarrhoea is the product of hyper gut motility and secretions, castor oil-induced enteropooling assay was also carried out in mice. Flaxseed oil exhibited inhibitory effect on gut secretions as compared to the untreated group that received no Flaxseed oil pre-treatment in enteropooling assay. There was a profound increase in intestinal secretions in the non-treated group with castor oil-induced secretions from 56.81±3.35 g to 107±4.50 g. Flaxseed oil pre-treatment reduced the castor oil-induced intestinal secretions by 19% (86.31±4.79 g) and 33.62%

(71.02±0.40 g) at respective oral doses of 100 and 300 mg/kg, indicating a dose- dependent inhibitory effect (Fig. 5.6 B). This effect was comparable to atropine, used as positive control which reduced the castor oil-induced secretions by 47.28% with resultant weight of 56.41±2.8 g.

When the effect on intestinal secretions was compared in mice that were treated with Flaxseed extract and Flaxseed oil, it was found that both the extract and oil equally reduced the secretions at comparable doses as elaborated in Table

5.2.

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Table 5.2. Comparison of antisecretory effect of Flaxseed extract vs Flaxseed oil

Groups Flaxseed extract Flaxseed oil

(Mean±SEM) (Mean±SEM)

Sham (10 ml/kg) 77.35±1.518 56.82±1.377

Castor oil (10 ml/kg) 152.4±6.701# 107±3.538#

Atropine (1 mg/kg) 76.91±1.893*** 55.03±2.344***

150$/100$$ 122.3±2.475** 86.32±3.8**

300 mg/kg 107±2.492*** 70.78±2.421***

$ represents dose for extract; $$ represents dose for Flaxseed oil. all values presented are means±SEM

150

Figure 5.6; Effect on castor oil-induced diarrhoea (A) and on castor oil-stimulated fluid accumulation in small intestines (B) of controls, untreated and Flaxseed oil pre- treated BALB/c mice. Castor oil used to induce the secretions and diarrhoea was given orally in dose of 10 ml/kg. Diarrhoea is scored on the basis of sum of frequency and consistency of diarrhoeal droppings. Intestinal fluid accumulation is expressed as (Pi/Pm) x 1000 where Pi is the weight of the small intestine and Pm is the weight of the mouse. All drugs were administered orally except atropine that was administered i.p. Values given are means±SEM of three experiments (n = 5).Each experiment was repeated thrice. (**), (***) indicate p<0.01 and p<0.001 vs. castor oil respectively.

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5.3.7. In vitro antimicrobial activity of Flaxseed oil

Flaxseed oil exhibited bactericidal effect against EPEC, ETEC and EAEC significantly at 9 µg/ml and 14 µg/ml; p<0.001 for all the tested doses) when compared to untreated microbes (both original inoculum and vehicle controls).

The effect of Flaxseed oil was further compared with Fs.Cr, against EPEC.

This was carried out by comparing the % kill and reduction in c.f.u. in respective treatment groups compared to OI and is presented in terms of colony counts in Table

5.3.

Flaxseed oil was effective against EPEC at both the tested doses of 9 µg/ml and 14 µg/ml (p<0.001; Fig. 5.7-A1). Compared to this, 100 µg/ml Fs.Cr was bactericidal against EPEC with colony count reduced to 1.45E+05 c.f.u. which was

89% lower than OI (p<0.05; Fig. 5.7-A2). Exhibiting a greater efficacy at 9 µg/ml,

Flaxseed oil reduced the total microbial counts by 99.8% (2.63E+04 c.f.u; p<0.001) as compared to OI. The dose of Flaxseed oil used was almost 10 times lower as compared to the Fs.Cr dose and hence, indicated greater bactericidal potency of Flaxseed oil.

Next, the effect of Flaxseed oil was compared with Fs.Cr against ETEC.

Flaxseed oil killed ETEC by 97% and 98% (p<0.001; Fig. 5.7-B1) as compared to

OI, at 9 µg/ml and 14 µg/ml respectively. Compared to this, Fs.Cr produced no effect at 50 µg/ml, whereas, at 100 µg/ml it killed the microbes by only 34% as compared to OI, which was not significant (Fig. 5.7-B2).

When the effect of Flaxseed oil against EAEC was compared with that of

Fs.Cr against EAEC, similar trend was observed as before. Flaxseed oil killed the microbes by 99% (p<0.001; Fig. 5.6-C1) at both the tested doses, whereas, Fs.Cr did not produce any bactericidal effect at tested dose of 50 µg/ml. Although, 100 µg/ml of Fs.Cr reduced the EAEC c.f.u. to 76%, this effect was not significant in terms of colony count compared to OI (Fig. 5.7-C2). Nevertheless as stated earlier that Fs.Cr exhibits a bacteriostatic effect against ETEC and EAEC as mentioned in chapter 4, which is also evident in Fig. 5.7 (A2, B2 and C2).

152

Table 5.3. Colony count of Enteropathogenic E.coli, Enterotoxigenic E.coli and Enteroaggregative E.coli treated with and without Flaxseed oil in antimicrobial assay.

EPEC (c.f.u.)

OI 1.13E+06# OI 1.36E+06#

Negative control 1.60E+06 Negative control 2.45E+06

Flaxseed oil (9 µg/ml) 1.27E+03*** Fs.Cr 50 µg/ml 8.50E+05

Flaxseed oil (14 µg/ml) 1.14E+03*** Fs.Cr 100 µg/ml 1.45E+05*

ETEC (c.f.u.)

OI 1.11E+06# OI 1.11E+06

Negative control 1.53E+06 Negative control 7.23E+06

Flaxseed oil (9 µg/ml) 2.63E+04*** Fs.Cr 50 µg/ml 2.67E+06

Flaxseed oil (14 µg/ml) 1.40E+04*** Fs.Cr 100 µg/ml 7.25E+05

EAEC (c.f.u.)

OI 1.17E+06# OI 1.17E+06

Negative control 1.67E+06 Negative control 1.25E+06

Flaxseed oil (9 µg/ml) 1.42E+03 Fs.Cr 50 µg/ml 1.30E+06

Flaxseed oil (14 µg/ml) 2.03E+03 Fs.Cr 100 µg/ml 2.77E+05

Values given are means±SEM of 3 experiments (n = 12). One way ANOVA was used for statistical analysis followed by Tukey’s post test. **p<0.01; ***p<0.001 compared with untreated group (#); [Abbreviations: Fs.Cr: methanolic-aqueous crude extract of Flaxseed; EPEC = enteropathogenic E.coli; EAEC: Enteroaggregative E.coli; ETEC: Enterotoxigenic E.coli; OI: Original Inocculum].

153

Figure 5.7; Effect on colony counts of ~106 enteropathogenic Eschericia coli (A1-A2), Enterotoxigenic Eschericia coli (B1-B2) and enteroaggregative Eschericia coli (C1-C2) incubated with and without Flaxseed oil (A1-C1) and Flaxseed extract (Fs.Cr) (A2-C2). Original inoculum (OI) is the approximate concentration of microbes plated before incubation (od = 0.22~106 c.f.u.) and act as negative control to compare the effect with treated samples. Untreated controls incubated shows almost similar colony count as OI, indicating no effect of assay buffer, hence in chart results, comparison of OI vs treated groups is shown. 100 µg/ml gentamycin was used as positive control not shown in graph (100% kill). The data are presented as the means±SEM of three independent experiments performed in duplicate. P-values were calculated by comparing OI and different extract concentrations using un-paired t- test. (*) (**), (***) indicate p<0.05, p<0.01 and p<0.001 vs. OI respectively. [Abbreviations: Fs.Cr: methanolic-aqueous crude extract of Flaxseed; EPEC = enteropathogenic E.coli; EAEC: Enteroaggregative E.coli; ETEC: Enterotoxigenic E.coli; OI: Original Inocculum].

154

5.3.8. Flaxseed oil in acetic acid-induced colitis

The effect of Flaxseed oil on AA-induced colitis was assessed by evaluating mortality rate, disease activity index (DAI) (Table 5.4) and microscopic assessment of the hemotoxylin and eosin (H and E) and periodic acid-Schiff-Alcian blue (PAS-

Ab) stained sections of colon of experimental mice of respective groups (Table 5.5;

Table 5.6).

The mortality rate of untreated mice was 58%, whereas, pre-treatment with

Flaxseed oil reduced this rate dose dependently (p<0.01 and p<0.001 for 300 and

500 mg/kg). When these parameters were compared with that of Fs.Cr pretreated groups, it was found that Flaxseed oil was not effective at tested dose of 150 mg/kg

(p>0.05), whereas, Fs.Cr showed mild effect at this dose as evident by improved mortality rate (33.33±5%) when compared to untreated controls (50±5.01%; p<0.05) as evident in Table 5.4.

Similar trend was observed for DAI, with an effect evident for Fs.Cr at 150 mg/kg but no effect for Flaxseed oil at this dose (and hence, not shown). Flaxseed oil and extract showed effectiveness at 300 mg/kg dose with apparently better effect with Fs.Cr at this dose for both mortality rate and DAI.

Interestingly, diseased parameters improved at an increased dose of Flaxseed oil to 500 mg/kg, as compared to Fs.Cr at the same dose. As evident in Table 5.4, the mortality rate reduced to 8.33±5% with Flaxseed oil at 500 mg/kg as compared to Fs.Cr groups (33±6%) at the same dose, indicating that the optimal effect with

Fs.Cr was evident at 300 mg/kg dose and it is possible that Flaxseed oil efficacy may be optimal at 500 mg/kg dose or may further increase beyond this dose.

When the microscopic damage was assessed for tissues treated with and without Flaxseed oil, in the colonic sections stained with H and E, it was found that the microscopic scores were improved dose-dependently for 300 and 500 mg/kg.

This effect was compared with that of Fs.Cr and it was found that some parameters were better for Flaxseed oil, whereas, some were better for Fs.Cr. Flaxseed oil improved the microscopic damage parameters dose-dependently at 300 mg/kg and

155

500 mg/kg doses. The dose-dependent effect was different from Fs.Cr which gave optimal effect at 300 mg/kg and increasing dose beyond that did not have an increase in effect. Comparing the microscopic damage scores, there was no over-all difference in efficacy profile of Flaxseed oil and extract. However, the type of protective effect was different for both Fs.Cr and Flaxseed oil. For instance,

Flaxseed oil exhibited a stronger effect on Neurath score (which focuses on neutrophil infiltration) as compared to Fs.Cr whereas F.Cr produced more pronounced increase on mucin levels as compared to Flaxseed oil. These differences are evident in Table 5.4 and Table 5.5. The detailed interpretation of what these scores signify is presented earlier in section 2.3.5.

156

Table 5.4. Comparison of mortality rate and Disease activity index of acetic acid induced colitis mice

Mortality rate Disease activity index1 Groups Oil Extract Oil extract Untreated 58.33±4.23# 50±5.01% 2.17±0.06 2.11±0.03# 150 mg/kg 55±12% 33.33±5%* 2.1±0.08 1.17±0.01* 300 mg/kg 33.33±6%** 25±8%** 1.80±0.02** 0.71±0.21** 500 mg/kg 8.33±5%*** 33±6%*** 0.93±0.01*** 0.97±0.23**

Values given are means±SEM of 3 experiments (n = 12). One way ANOVA was used for statistical analysis followed by Tukey’s post test. **p<0.01; ***p<0.001 compared with untreated group (#); all the drugs were administered intraperitoneally for 7 days. Colitis was induced on 6th day with intrarectal administration of 0.1 ml 6% acetic acid. Mice were assessed 24 hrs after induction of colitis. DAI = (Weight loss + stool consistency + rectal bleeding)/3; mortality rate = (mice died in a group/total number of mice in that group) x 100 . 1(Wirtz et al., 2005).

157

Table 5.5. Microscopic scoring comparison of H & E stained, and Periodic acid Schiff-Alcian blue stained, sections of mice colon induced with colitis pretreated with either Flaxseed oil or Flaxseed extract

Microscopic scoring of colon sections Ulceration1 Groups Flaxseed Oil Flaxseed Extract Untreated 1.8±0.2# 1.66±0.25# 300 mg/kg 0.8±0.16* 0.8±0.09* 500 mg/kg 0.36±0.5** 1.11±0.69 Inflammation1 Flaxseed Oil Flaxseed Extract Untreated 2.2±0.05# 2±0.01# - 2±0.2 0.8±0.09*** 500 mg/kg 1.4±0.22** 1.42±0.28 Depth of lesion1 Flaxseed Oil Flaxseed Extract Untreated 2.5±0.05# 2.60±0.07# 300 mg/kg 2±0.05** 1.17±0.05*** 500 mg/kg 1.8.0±0.1*** 1.15±0.20*** Neurath Score2 Flaxseed Oil Flaxseed Extract Untreated 3.29±0.5# 3.48±0.65# 300 mg/kg 1.8±0.16** 1.56±0.5** 500 mg/kg 1.33±0.02*** 1.70±0.03** Kiela Score3 Flaxseed Oil Flaxseed Extract Untreated 2.1±0.1# 3# 300 mg/kg 1.7±0.05 1.83±0.33*** 500 mg/kg 1.2±0.2*** 1.8±0.3*** Mucin Score4 Flaxseed Oil Flaxseed Extract Untreated 1.4±0.2 1.2±0.37# 300 mg/kg 1.98±0.05* 2.1±0.25*** 500 mg/kg 2.16±0.16** 2.3±0.5***

H and E stained sections of colon were scored according to Vilaseca et al., (1990)1, Neurath et al., (1995)2 and Kiela et al., (2005)3. PAS-Ab stained sections were used for mucin depletion/levels and scored according to Dorofeyev et al., (2013)4. One way ANOVA followed by Tukey’s post test was used for analysis. Values expressed are means±SEM (n=6). **=p<0.01; ***=p<0.001, vs AA. #Diseased control group All the drugs were administered i.p. Colitis was induced by IR administration of 0.1 ml 6% acetic acid during anaesthesia. Tissues were embedded in formalin for another 24 hrs and then processed and stained with H and E and PAS-Ab staining. [Abbreviations: IR = intrarectally; H and E = hemotoxylin and eosin; PAS-Ab = periodic acid Schiff-Alcian blue].

158

5.3.9. Spasmodic effect of Flaxseed mucilage

Flaxseed mucilage is the water soluble extract of Flaxseed and primarily involves component absorbed from seed coat. The effect of Flaxseed mucilage was tested in isolated rabbit jejunum and guinea-pig-ileum in similar way as mentioned in section 5.3.2.

Flaxseed mucilage did not exhibit any effect on spontaneously contracting rabbit jejunum, whereas, the quiescent preparation of guinea-pig ileum showed a clear spasmogenic effect, with efficacy of 86.29±10.21% (n = 6), compared to ACh maximum. Hence, the mechanism of spasmogenic effect was identified by testing the effect of Flaxseed mucilage in the absence and presence of atropine (1 M) and/or pyrilamine (1 M). It was found that the spasmogenic effect of Flaxseed mucilage was fully blocked in the presence of atropine, while partial blockade was observed in the presence of pyrilamine (63.90%) as evident in Fig. 5.7. Laxative potential of Flaxseed mucilage observed was in line with its traditional use; hence, laxative effect was studied in mice. Treatment of mice with Flaxseed mucilage produced a dose-dependent increase in wet faeces as evident in Fig 5.8A. Laxative effect was evident at relatively high doses (1 and 2.5 g/kg, P.O.), with resulting increase of 65.06±6.5% and 89.33±4.04% (n = 6) in wet faeces when compared to faeces from untreated mice (5±4% in the saline treated group). The positive control,

CCh (1mg/kg; i.p.), produced 82.57±9% wet faeces as shown in Fig. 5.8A. Pre- treatment of animals with atropine (10 mg/kg; i.p.), blocked the laxative effect of

Flaxseed oil with resultant values of wet faeces declining to 6.76±5.90% and

5±3.99% at respective doses of 1 and 2.5 g/kg.

When the data on wet faeces were further analyzed on the basis of their consistency and frequency, it was found that the Flaxseed mucilage increased the laxative score from

1.33±2.30 (vehicle score) to 16.33±5.13 and 27.66±1.52 respectively at 1 and 2.5 g/kg respectively (n = 6). CCh, used as positive control, increased the score to 27.66±3.2 as shown in Fig. 5.8 B, whereas, atropine pre-treatment declined the laxative score to 2±1.5 and 4.66±1.52 for the respective doses of 1 and 2.5 g/kg.

159

Guineapig Ileum 100 Spontaneous + Pyrilamine (1 M) + Atropine (1 M) 75

50 *

% of Ach max of Ach % 25

1 3 5 10 Flaxseed Mucilage (mg/ml)

Figure 5.8; Spasmogenic effect of Flaxseed mucilage in the absence and presence of atropine and/or pyrilamine, in isolated guinea-pig ileum preparations. The graph shows baseline spasmogenic effect of Flaxseed mucilage in quiescent ileal preparation and the partial inhibitory effect in the presence of pyrilamine (p<0.05) and complete inhibitory effect in the presence of atropine in isolated guinea-pig ileal preparations. Statistical analysis was carried out by using repeated measures ANOVA followed by Tukey's post-test. The values presented are means±S.E.M. of 5-6 preparations obtained from 4 animals. *p<0.05 vs effect without blockers.

[Abbreviations: ACh = acetyl choline maximum].

160

Figure 5.9; Effect of Flaxseed mucilage on laxative score in absence and presence of atropine pre-treatment (A), and on %age of wet feces (B). For each group total laxative score was calculated as sum of individual scores. Individual score was calculated by multiplying the respective score based on consistency of stool with the number of wet feces where score 3 = diarrhoea; score 2 = very soft; score 1 = soft but formed; score 0 = normal stool. The values shown are mean±S.E.M.(n = 6) of 3 individual experiments. Statistical analysis was carried out by using one way ANOVA followed by Tukey's post- test; *p<0.05; ** p<0.01; ***p<0.001. [Abbreviations: CCh = carbachol;].

161

5.4. Discussion

In the previous chapters, Flaxseed extract (Fs.Cr) showed efficacy against different aspects of IBD. However, the traditional use of Flaxseed is in constipation

(Duke, 2002), and therefore it was important to identify which fraction of Flaxseed possessed gut stimulant effect since laxative effect might aggravate IBD whose main symptom is diarrhoea. For this purpose, the oil and mucilage part of Flaxseed was studied, which are used more commonly as edible source. Besides, the extract has a relatively lesser percentage of non-polar constituents (Ignat et al., 2011) as compared to oil (Lin et al., 1999). Hence, Flaxseed oil and mucilage were studied for their potential in IBD therapeutics.

Flaxseed oil exhibited gut stimulatory effect at low doses followed by inhibitory effect at higher doses in isolated rabbit jejunum but a surprising aspect was that no such effect was evident in guinea-pig ileum. Contrary to this, Flaxseed mucilage exhibited the same stimulatory effect at the highest tested doses, in guinea- pig ileum, but no effect was evident in isolated rabbit jejunum. One of the reasons for this variable effect could be due to species and tissue variation. Such species and/or tissue specificity has also been reported in earlier studies (Ghayur & Gilani,

2005; Rehman et al., 2012) showing that the nature of gut stimulant effect varies from species to species as well as from tissues to tissues. Further in depth studies are required to address the tissue and species variation in their spasmodic effect. Another explanation could be that this type of varied response may be because of the difference in solvent used for extraction. Flaxseed oil was extracted by polar solvents, whereas, mucilage by non-polar solvent. It is possible that the components extracted from oil have a better affinity for receptors in rabbit jejunum as compared to guinea-pig ileum, as the population density of receptors varies within species and tissues (Brown & Taylor, 1996).

Flaxseed oil and mucilage showed varied sensitivities to two different receptor antagonists used in this study; The gut stimulant effect of Flaxseed oil in rabbit jejunum was partially blocked by atropine and pyrilamine indicating partial 162 presence of relatively greater cholinergic component followed by histaminergic component. Interestingly, when the tissue was treated with atropine and pyrilamine together, the spasmogenic effect was completely blocked. On the contrary, the spasmogenic effect of Flaxseed mucilage in guinea-pig ileum was completely blocked by atropine alone but partially blocked by pyrilamine, indicating that the cholinergic component may be stronger in case of Flaxseed mucilage. Taken together, these results demonstrate the presence of atropine-sensitive stimulatory constituent(s) in Flaxseed mucilage while partially sensitive stimulatory effect to atropine and pyrilamine in Flaxseed oil with dominant cholinergic effect. It also clarifies that the laxative effect of both the oil and mucilage are not merely because of the bulk forming capability or due to their lubricating properties as suggested by previous studies (Weiss & Fentlemann, 2003; Moghaddasi, 2011) and also involves spasmogenic mechanism exhibited through a chemical effect. The current study is the first one to provide scientific basis for these proposed mechanisms at receptor level.

One interesting observation during the course of this study with regard to spasmogenic mechanisms was that the moderate heating of Flaxseed has some relation with the spasmogenic effect, which was based on the fact that until the oil was extracted at 40 C for 7 days, no spasmogenic component was found; instead only spasmolytic effect was evident in the absence of moderate heating. Similar was the case with mucilage that was kept at high temperatures (80 °C) initially and then gradually reduced to 40°C. This is in accordance with the traditional reports that drinking a cup of Flaxseed mucilage, prepared by heating a teaspoon of Flaxseed powder into a glass of hot water, brewed and drained is an effective remedy for constipation (Moghaddasi, 2011). However, the contrasting aspect here is that once the mucilage was prepared, heating it further caused destabilization of spasmogenic component. Initially, to solubilize the mucilage, two ways were experimented. The first method involved adding mucilage in desired quantity of water and heating at water bath at the temperature range of 40-50C for 1 hr. In the second method,

163 mucilage was simply dissolved and vortexed in water and sonicated to decrease particle size of mucilage. However, spasmogenic effect was lost when the prepared mucilage was heated at water bath by first method (data not shown).

Following characterization of spasmogenic mechanisms, the spasmolytic effect of Flaxseed oil in isolated rabbit jejunum preparations was further assessed for possible mechanisms. For this purpose, tissues were depolarized with CCh, high

K+ and low K+ which resulted into sustained contractions and allowed to study the inhibitory effect. Flaxseed oil selectively inhibited low K+-induced contractions compared to high K+. The selective inhibitory effect against low K+-induced contractions indicated the possible presence of K+-channel opening (KCO) activity

(Kishii et al., 1992). KCOs are known to cause increase in K+ efflux, followed by hyperpolarization of membranes and thereby decreasing intracellular free Ca++ resultantly causing smooth muscle relaxation (Weston & Edwards, 1992); Hence, the type of K+-channels involved were probed. For this purpose, inhibitory effect of

Flaxseed oil against low K+-induced contraction was re-determined in the presence of different K+-channel blockers. It was observed that in the presence of TEA, a non- specific K+-channel blocker (Wu et al., 2004), Flaxseed oil shifted the curve to right significantly. A relatively small rightward shift was evident in the presence of Gb, an ATP-dependent K+-channel blocker (Franck et al., 1994), showing the predominant involvement of non-specific K+-channel activation and the partial involvement of the specific ATP-dependent K+-channels. Thus the spasmolytic effect of Flaxseed oil observed at high doses is primarily mediated through non- specific and ATP-sensitive K+-channel activation pathway.

KCO activity of Flaxseed oil is speculated to be beneficial in IBD as KCO are involved in free radical scavenging activity, prostaglandin E2 elevation, and prevention of the deteriorating rise in NO (Hosseini-Tabatabaei & Abdollahi,

2008b), all of which have protective role in IBD. Furthermore, the importance of

KCO in IBD could be correlated with the finding where one of the sub-types of KCO known as IK1 cells (Ca++-activated K+ currents of intermediate conductance cells)

164 were decreased in inflammed colon of Crohn's disease and ulcerative colitis patients and their low levels were attributed to the diarrhoea and colicky abdominal pain in

IBD (Arnold et al., 2003). Besides, one of the KCO “nicorandil”, have shown protective effects in an animal model of IBD (Hosseini-Tabatabaei et al., 2009) through activating K+-ATP channels, besides other mechanisms (Hosseini-

Tabatabaei & Abdollahi, 2008a). Since Flaxseed oil exhibited partial involvement of the specific ATP-dependent K+-channels, it was speculated to be effective in IBD.

Further to this, Flaxseed oil was studied for laxative and antidiarrhoeal activity in vivo. Similar to the trend evident in isolated tissue set-up, Flaxseed oil exhibited laxative activity at low doses and antidiarrhoeal activity at higher doses.

The dual activity i.e. antidiarrhoeal and laxative mechanisms of Flaxseed oil explored in the current study would help to set the dosages for alternating symptom profiles of IBD i.e. constipation and diarrhoea with dominant advantage of its antidiarrhoeal effect. However, the mild laxative effect observed at low doses blunted by high dose (contributing to antidiarrhoeal effect) needs explanation as no drug (single chemical entity) is known to exhibit both laxative and antidiarrhoeal effect. In fact, laxative drugs at high doses tend to cause diarrhoea and antidiarrhoeal drugs at high doses tend to cause constipation (McQuaid, 2007). It appears that

Flaxseed oil contains different chemicals, one with gut stimulating effect observed at lower dose, together with gut inhibitory constituents which do not allow stimulatory effect to go beyond certain limit which could been harmful.

Once it became clear that Flaxseed oil has dual activity in diarrhoea and constipation, Flaxseed oil was further screened for its effect against microbes implicated in IBD and the preliminary screening for its effect on AA -induced colitis in mice. It was found that Flaxseed oil was effective against microbes implicated in

IBD, i.e. EPEC, ETEC and EAEC. This antimicrobial effect was also compared with

Fs.Cr and it was found that Flaxseed oil had cidal effect against EPEC, ETEC and

EAEC, whereas, Fs.Cr exhibited cidal effect against EPEC but static effect against

ETEC and EAEC. Also the dose of Flaxseed oil was almost 10 times lesser than the

165 extract, which indicates that Flaxseed oil is both potent and efficacious as compared to Fs.Cr in antimicrobial effect.

Further to this, Flaxseed oil was assessed for its efficacy against acetic acid- induced colitis. Flaxseed oil improved mortality rate as compared to AA controls; it also improved DAI dose-dependently at 300 and 500 mg/kg doses. Unlike Fs.Cr that mediated its mucosal protective effect (evident by microscopic scoring) at 150 mg/kg dose, there was no effect of 150 mg/kg dose of Flaxseed oil. Unlike Fs.Cr that showed optimal effect at 300 mg/kg dose, interestingly, Flaxseed oil exhibited a dose-dependent mucosal protective effect between 300 mg/kg and 500 mg/kg dose.

This indicates that in Fs.Cr, the optimal dose is 300 mg/kg, because increasing the dose beyond this did not increase the efficacy, whereas, Flaxseed oil could be tested for a higher dose beyond 500 mg/kg as the effect started to show up from 300 mg/kg dose and was escalating at 500 mg/kg doses. The difference in efficacy

(antimicrobial) and mechanistic profile (antispasmodic) as well as the evidence form

HPLC that shows predominance of non-polar constituents in oil and polar constituents in the extract, indicates that both Flaxseed oil and Fs.Cr mediate their effect due to different mechanisms.

Flaxseed oil’s dual efficacy in diarrhoea and constipation, its antimicrobial activity against microbes implicated in IBD and antispasmodic mechanisms mediated by KCO and PDEI, along with improved mortality rate and DAI in acetic acid-induced colitis model indicates clearly that it could be a reasonable choice for

IBD therapeutics and shall be explored further for possible mechanisms in IBD model of mice. This finding has an important implication for clinical studies, since

Flaxseed oil is an already available edible source and could be tested in IBD patients as a remedy.

The current study showing effectiveness of Flaxseed oil and the studies presented in previous chapters, indicate that Flaxseed as a whole has potential for

IBD therapeutics and therefore taking Flaxseed as a whole may provide benefits obtained from both the oil and extract. Nevertheless, an optimal method further

166 needs to be identified that ensures maximum absorption from Flaxseed in the body via oral route.

So far, methanolic-aqueous extract of Flaxseed (Fs.Cr) has not been studied for its anti-inflammatory potential in any animal models including an IBD model.

Hence, this is the first study to assess the effect of Fs.Cr in an IBD model of mice.

Flaxseed diet (10%) and Flaxseed oil have been studied for their anti-inflammatory potential and have shown varied effects, with both anti-inflammatory (Razi et al.,

2010, 2011; Ghule et al., 2011; Saini et al., 2010) and pro-inflammatory effects

(Zarepoor et al., 2014; Toth et al., 2015). Earlier, 10% Flaxseed diet failed to ameliorate the severity of dextran sodium sulfate (DSS)-induced colitis model of mice (Zarepoor et al., 2014). This was rather surprising particularly, because the dietary components of Flaxseed (linolenic acid, kaempferol, ALA, sodium butyrate, secoisolariciresinol diglucoside/SDG) have demonstrated anti-inflammatory activities in different models of colitis (Hassan et al., 2010; Naito et al., 2006; Park et al., 2012; Tyagi et al., 2012; Vieira et al., 2012; Xu et al., 2016). One explanation for the ineffectiveness of Flaxseed diet in the study by Zarepoor et al., (2014) could be insufficient absorption of bio-active constituents of Flaxseed. However, the question arises that if absorption was compromised leading to reduced bioavailability of bio-active constituents; how could Flaxseed meal produce anti- inflammatory effect in diseased models of lung ischemia (Razi et al., 2010; 2011) and renal ischaemia-reperfusion injury (Ghule et al., 2011) in mice. One probable explanation could be that the type of inflammation mediated in these lung injury models may have triggered different inflammatory pathways (Kalogeris et al., 2012) that could be managed by the absorbed bio-active constituents, whereas, the inflammatory pathways triggered in IBD models may require different type of anti- inflammatory components which may not have been absorbed from the gut into the blood circulation. This is understandable because the level of inflammation mediated in chemical-induced IBD models of mice, allows bacterial translocation across the colonic mucosa (Nakhai et al., 2007); thus causing an interaction between resident

167 gut associated lymphoid tissues (GALT) and microbes, and hence generating an lipopolysaccharide (LPS)-like immune response (Broz et al., 2012), since microbes are rich in LPS. Possibly that’s why, in an oxidative model of lung injury, Flaxseed diet exhibited anti-inflammatory effect in hyperoxia and radiation-induced models but not against LPS-induced lung injury model of mice (Kinniry et al., 2006).

Another possibility for effectiveness in current study and ineffectiveness in the earlier study by Zarepoor et al., (2014), could be lack of optimal carrier. It was observed while preparing the Fs.Cr that it has to be solubilised in 10% DMSO and

5% Tween-80, vortexed for 10 minutes to ensure maximum exposure of the extract to the organic solvents, followed by addition of water. Even after this, the extract needed to be kept in sonicator, (with ice in it) for an hour to break the particles.

However, if the extract was first mixed with water, followed by DMSO and Tween-

80, it became hard to an extent, that adding organic solvent did not solubilise it.This indicates that there are absorption issues with Flaxseed; hence, sub-optimal conditions of administration may be the reason for aggravation of DSS-induced colitis by Flaxseed meal (Zarepoor et al., 2014). This is the reason that an intraperitoneal route was adopted in the current study to first, explore the potential of the whole plant and then optimize it for usage. From the literature it is indicated that whole Flaxseed meal may have some hindrance in absorption due to which the active bioactive constituents could not mediate their protective effects. From the current study it clear that Flaxseed does have a potential to mediate anti- inflammatory effect in IBD model of mice, though the best form of Flaxseed and the optimal carrier for administering still needs to be identified.

After establishing that Fs.Cr has effectiveness in IBD model of mice, other mechanisms that could possibly play a role in ameliorating IBD were assessed. Since whole array of causes of IBD cannot be ideally assessed in one model, some ex-vivo and in vitro means were adopted to further explore Fs.Cr’s effectiveness against diarrhea and spasms and their possible mechanisms.

168

Although, antidiarrhoeal effect of Fs.Cr was observed while assessing disease activity index in AA-induced colitis model of mice, a mechanistic basis for this effect was needed as diarrhoea in AA-induced colitis is merely due to physical injury, whereas, in IBD both the breached epithelial barrier and alteration in gut contractility are evident (Bassotti et al., 2014). That is why, restoring vasculature and disturbances of blood flow by controlling gut contractility (Banner &

Trevethick, 2004) are proposed as one of the novel approaches for targeting IBD.

Knowing that diarrhoea is a product of hypermotility and hypersecretion, experiments were conducted to assess the antidiarrhoeal effect of Fs.Cr against castor oil-induced diarrhoea. It was found that Fs.Cr had a predominant antidiarrhoeal activity against castor oil-induced diarrhoea in mice due to decrease in intestinal secretions and reduction in the gut motility. Interestingly in this study,

Fs.Cr did not halt the stool frequency completely when diarrhoea was induced; it could be speculated that possible presence of spasmogenic component may have presented this type of antidiarrhoeal activity. This aspect is considered a merit, in terms of avoiding the side-effect of constipation, as seen with antidiarrhoeal drugs like loperamide (Grahame-Smith & Aronson, 2002). These observations are in consistence with earlier reported alternating mechanisms of natural products such as being effective against both constipation and diarrhoea as evident in psyllium husk (Mehmood et al., 2011) and ginger (Ghayur & Gilani, 2005). It seems that nature has a built-in mechanism to off-set the exaggerated effect of one constituent by neutralizing it with an opposing constituent (Gilani & Atta-ur-Rehman, 2005), without compromising the desired effect.

This is the first study that reports the antidiarrheal effect of Flaxseed. Earlier, a preliminary report on Flaxseed oil employed castor oil-induced diarrhoea as an indicator of prostaglandin release (Kaithwas & Majumdar, 2010b) and hence, was neither conducted with the perspective of diarrhoea nor was any mechanism with respect to diarrhoea investigated.

169

Upon investigation for possible antispasmodic mechanisms involved in isolated rabbit jejunum, it was found that Fs.Cr exhibited phosphodiesterase enzyme inhibitory (PDEI) activity, which was observed at lower doses, while a Ca++ antagonist activity was observed at 10 times higher doses, thus highlighting that the predominant action of Fs.Cr for antispasmodic effect was mediated via PDE enzyme inhibition. Further to this, it was identified that this PDEI activity was possibly mediated by inhibiting PDE-4 enzyme. It is possible that one of the protective mechanisms by which Fs.Cr mediated its protective effect against AA-induced colitis was via PDE-4 inhibition as PDE-4 receptors are present on monocytes which are responsible for release of TNF- while PDE-4 inhibitors have been shown to mediate their anti-inflammatory effect by inhibiting monocytes with subsequent inhibition of TNF-(Gantner et al., 1997).

Another consequence of PDE inhibition is increase in the levels of cyclic

AMP (cAMP) (Anthony, 1996; Kaneda et al., 2005); this is a very important aspect from IBD perspective because cAMP is known to interact with cytoskeletal system which maintains intact epithelial barrier (Willingham & Pastan, 1975).

Dysregulation cAMP causes breached barrier, which is one of the initial perpetuators of IBD (Bassotti et al., 2014); it is possible, that one of the mechanisms by which

Fs.Cr promoted mucosal repair in AA-induced colitis model of mice was by increasing cAMP levels. Consistent with this, Flaxseed lignans showed improvement in DSS-induced colitis by increasing expression of markers of epithelial barrier promoters such as zonna occludens 1 and TFF3 (Xu et al., 2016) and hence there’s a possibility that cAMP agonist action of Fs.Cr may have prompted this kind of cytoskeletal repair.

170 persistent immune stimulation and could also aggravate already initiated inflammatory cascade in IBD (Campieri & Gionchetti, 2001; Rahimi et al., 2007).

Hence, Fs.Cr was tested in the in vitro study against the gut pathogens implicated in IBD. So far Flaxseed in any form has not been studied against these pathogens, hence for the first time Flaxseed in the form of extract (Fs.Cr) showed effectiveness against Enteropathogenic Eschericia coli (EPEC), enterotoxigenic E. coli (ETEC) and enteroaggregative E.coli (EAEC), which are the microbes that share virulence properties with adherent invasive E.coli (AIEC) (Santos et al., 2015), which is the strain of E.coli isolated from biopsies of ileal CD patients and possess multiple properties of different types of E.coli (Boudeau et al., 1999; Smith et al.,

2013).

It was also effective against broad range of gut pathogens that cause gastroenteritis and are known to aggravate the inflammatory cycle including

Salmonella typhi, vancomycin-resistant Enterococcus faecalis, methicillin-resistant

Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa.

Antimicrobial activity of Fs.Cr has also been reported against S. typhi and P. aeruginosa (Bakht et al., 2014; Gur et al., 2006) but the antimicrobial assay used in current study has an edge over previously reported assays, as it gives a quantified effect with distinction between cidal and static effects. This effectiveness of Fs.Cr could be correlated with the resemblance of HPLC peaks of Fs.Cr with two synthetic compounds, ofloxacin and metronidazole, both of which have broad spectrum coverage against aerobic and anaerobic pathogens (Freeman et al., 1997). However, the presence of some novel compounds is speculated based on 100% kill of VRE, which is resistant to conventional antibiotics. Antimicrobial effect of Fs.Cr would also preventing the possibility of side effect of disseminated infection that may occur with use of antimotility agents alone (Casburn-Jones & Farthing, 2004).

Overall, Fs.Cr reduced proinflammatory cytokines TNF- and IFN- and enhanced IL-17; reduced neutrophil infiltration and oxidative stress in AA-induced model of colitis. The in vitro study analysis showed its efficacy against broad

171 spectrum and antibiotic-resistant pathogens. The ex vivo study analysis showed that

Fs.Cr produced its antispasmodic effect mediated via PDEI and Ca++ antagonist pathways, along with increased cAMP levels in tissues, possibly mediated by PDE-

4 inhibition. Hence, Fs.Cr has ability for targeting multiple etiological aspects of

IBD.

After the detailed analysis of extract, Flaxseed oil was also studied for some of the parameters of IBD. This was done because Flaxseed oil is a known edible source, and have possibility to exhibit a different profile of activity as compared to methanolic-aqueous extract, since oil possesses only non-polar constituents (Lin et, al., 1999). Another aspect that prompted to include Flaxseed oil screening in the current study settings was the predominant literature evidence for its use in constipation (Duke et al., 2002). Since the extract did not show laxative effect, it was important to investigate which fraction of Flaxseed has gut stimulant component, as predominant gut stimulant component could worsen the symptoms of IBD-induced diarrhoea. Possibly that’s why, a study that used Flaxseed meal to prevent DSS- induced colitis, ended up aggravating the symptoms in that model (Zarepoor et al.,

2014), because whole seed have reported laxative effect (Xu et al., 2012).

Nevertheless, laxative effect of Flaxseed might be beneficial in left sided colitis which is constipation predominant. Hence, Flaxseed oil was tested both in the in- vitro and in-vivo models.

It was observed that at lower doses, Flaxseed oil exhibited gut stimulant activity (cholinergic and mild histaminergic) which was reversed at high doses. This was a very interesting finding because laxative drugs at high doses cause diarrhoea, whereas, increasing Flaxseed oil dose after a certain concentration did not increase laxative effect, in fact, blocked it; which means it has inhibitory components. Hence, at high doses, Flaxseed oil exhibited antispasmodic effect mediated by strong K+- channel opening (KCO) activity that involved non-specific and ATP-sensitive K+- channel activation pathway. Flaxseed oil also exhibited weaker PDEI activity; this was in contrast to Fs.Cr, which showed no gut stimulant effect, with a predominant

172

PDEI activity. The predominant KCO activity of Flaxseed oil may not only be useful as an antispasmodic but also could be useful in IBD because KCO are involved in scavenging free ROS, prevent deteriorating rise in nitric oxide and increase prostaglandin E2 (Hosseini-Tabatabaei & Abdollahi, 2008a; 2008b; 2009).

Further to this, Flaxseed oil was assessed in the in-vivo model to see if the dual laxative and antidiarrhoeal activities exist. Similar to the ex-vivo data, Flaxseed oil showed laxative activity in mice at lower doses which was reversed at higher doses when tested against castor oil-induced diarrhoea.

Interestingly enough, Flaxseed oil seemed to have a better efficacy and potency profile as an antimicrobial when compared to Fs.Cr. Flaxseed oil exhibited bactericidal effect against EPEC, ETEC and EAEC, whereas, Fs.Cr only exhibited cidal effect against EPEC and had a static effect against ETEC and EAEC. The effect mediated by Flaxseed oil were evident at a dose which was 10 times lesser than used for Fs.Cr; this indicates that oil may be using a different antimicrobial mechanism.

Earlier, antimicrobial effect of Flaxseed oil has been shown against E.coli (Kaithwas et al., 2011) which is a non-pathogenic strain and hence, cannot be compared.

173

6. General Discussion

174

Inflammatory Bowel Disease (IBD) is a multifactorial disease and it has multiple tenets which attribute to its pathogenesis. Amongst them, the ones that can be maneuvered are immune and microbial dysregulations, oxidative stress and/or barrier dysfunction (Xavier & Podolsky, 2007). All of these triggers act simultaneously and/or subsequently to cause an inflammatory cascade which manifests as mucosal damage, diarrhea, spasm and rectal bleed (Bamias et al.,

2005).

immune defect

IBD

microbial Barrier defect defect

In order to suppress IBD, multiple triggers need to be managed simultaneously (Bamias et al., 2005); the current IBD pharmacotherapeutic paradigm stands expensive and associated with debilitating side-effects with high remission failure rates (Pai et al., 2005; Sutherland & MacDonald, 2006; Creed &

Probert, 2007). Possible reason for failing remission state could be that, the current therapies are aimed to address an individual target, whereas, the nature of disease is such that perhaps, it requires to be controlled by multiple targets. With current practices, anti-inflammatory agents are the mainstay of therapy, but the issue with

IBD is that inflammation is mediated via simultaneous triggers from multiple routes.

Possibly that’s why maintenance phase can be many times unsuccessful, ending up in relapse (Peyrin-Biroulet & Lémann, 2011).

Natural products comprise of multiple bio-active constituents (Spelman et al., 2006) and hence, could control multiple targets of a disease simultaneously, with

“effect enhancing and side-effect neutralizing” combinations (Gilani & Atta-ur-

175

Rehman, 2005). Hence, in diseases that need multiple targets, natural products may prove better. In traditional Iranian medicine, Flaxseed has been used in IBD (Rahimi et al., 2010) and it has reported to possess anti-inflammatory (Caughey et al., 1996;

Jangale et al.,2013), antimicrobial (Christofidou-Solomidou et al., 2011a; Kaithwas et al., 2011) and antioxidant activities (Kitts et al., 1999; Pattanaik & Prasad, 1998) in various in vitro and in vivo models/assays. Hence, it was hypothesized that

Flaxseed is effective in IBD model of mice by targeting multiple aspects of the disease. For this purpose, effect of Flaxseed was assessed on inflammation, mucosal damage, and oxidative stress, as well as, against aggressive microbes which encompass the main triggers, whereas effect in diarrhoea and spasms were studied in detail as these are the main manifestations of IBD.

For this purpose, the crude methanolic-aqueous (70:30) extract of Flaxseed

(Fs.Cr) was studied, as it is known that methanolic-aqueous solvent extracts out relatively both the polar and non-polar constituents (Ignat et al., 2011), though still not complete representation of Fs.Cr in IBD model of mice. When studied in acetic acid (AA)-induced colitis model, Fs.Cr proved to be effective in ameliorating the severity of colitis by multiple mechanisms that include anti-inflammatory, antioxidant and mucosal restoring pathways. Since Fs.Cr has not been studied so far in animal or humans, the current findings on animal model are therefore compared with the reported literature on Flaxseed meal and oil, although oil only represents the non-polar component (Lin et al., 1999; Lang and Wai, 2001).

When studied for its anti-inflammatory potential, Fs.Cr was found to reduce myeloperoxidase (MPO) activity in colonic homogenates which supported further the microscopic findings, where Fs.Cr pre-treated groups showed reduced neutrophil infiltration as compared to untreated groups. The reduced MPO finding is in-line with previously reported effect of Flaxseed in other inflammatory models of mice when given in the form of meal (Razi et al., 2010, 2011; Ghule et al., 2011) and oil

(Saini et al., 2010). Surprisingly, in some studies, the dietary supplementation of

176

Flaxseed meal increased MPO activity (Toth et al., 2015; Zarepoor et al., 2014) and did not show protective effect.

The levels of TNF- and IFN- were monitored to assess the effect of Fs.Cr on pro-inflammatory cytokines and it was found to be inhibited at 6, 12 and 24 hrs.

This may form the basis for its effectiveness in AA-induced colitis model in the current study. The inhibitory effect of Flaxseed on TNF- is in accordance with previously reported effects mediated by Flaxseed diet (Caughey et al., 1996;

Christofidou-Solomidou et al., 2011) and Flaxseed oil (Bashir et al., 2015; Sargi et al., 2012; Hendawi et al., 2016; Baranowski et al., 2012), whereas, another study

(Machado et al.,(2015) showed that Flaxseed meal given to overweight adolescents could not prevent the rise of TNF-.The reduced level of IFN-due to Fs.Cr treatment in the current study is in line with previous reports where Flaxseed in the form of oil reduced IFN- in animals Han et al., On the contrary, Flaxseed diet has shown earlier not affecting IFN- level in animals (Lessard et al., 2003; cFarmer et al., 2010) and in humans (Machado et al., 2015; Wallace et al., 2003).

In the current study, IL-17 was also measured; so far the role of IL-17 is still unclear as being protective or detrimental (Fuss et al., 2011). This is the first study that elaborates on the kinetics of IL-17A in AA-induced colitis model of mice as well as effect of Fs.Cr on its release. It was found in the current study that IL-17 was elevated at 6 hrs in untreated colitis model and reduced significantly at 12 and 24 hrs. Opposed to this, at 6 hrs, Fs.Cr pre-treatment reduced IL-17 level and at 12 and

24 hrs, there was a significant increase in IL-17 level. The elevated IL-17 level in

Fs.Cr group at 12 and 24 hrs, corroborated with the protective effects including anti- inflammatory and antioxidant activities.

Earlier, dietary Flaxseed has shown to reduce IL-17 levels in lung homogenates of radiation-induced pneumonopathy mice, which corroborated with protective effects in that model (Christofidou-Solomidou et al., 2011), indicating a pathogenic role of IL-17 in that diseased model and protective effect of Flaxseed mediated via reduced IL-17. These effects cannot be correlated with the current

177 findings because lung injury model was the chronic model of inflammation

(Christofidou-Solomidou et al., 2011) and the release pattern of IL-17 in chronic models may be mediated via Th-17 cells, which have a different release profile altogether (Annunziato et al., 2007).

The simultaneous effect of Fs.Cr on multiple cytokines with inhibition of some (TNF-, IFN-) and elevation of some (IL-17) advocates that Fs.Cr may mediates its protective effect by inhibiting release from certain inflammatory cells rather than only acting as anti-cytokine agent. This explanation seems to be justified specifically for IL-17, since this cytokine had an opposing effect at different time points. This indicates a possibility that Fs.Cr may not be inhibiting or increasing IL-

17 directly; rather, it may be acting by inhibiting different types of immune cells that would come in action in different time points, that would be responsible to control

IL-17 release. This is speculated because if Fs.Cr was acting as an antibody to IL-

17, then IL-17 A would have been inhibited at all the times points and hence no elevated levels could have been possible. Based on literature evidence in mice models of injury and current findings, a hypothetical model has been elaborated in

Chapter 2, Figure 2.16.

Fs.Cr also mediated mucosal protective effect by reducing oxidative stress, as evident by reduced malondialdehyde (MDA) activity and increase in catalase

(CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and total glutathione (GSH) activity. This is the first study to report the antioxidant effect of

Fs.Cr, as well as, the first study to show effectiveness of Flaxseed in reducing oxidative stress in an in-vivo model of colitis. However, effect of Flaxseed diet

(10%) and oil have shown to reduce oxidative stress in-vitro, and in vivo set-ups

(Moneim et al., 2011a; 2011b; Saini et al., 2010; Hendawi et al., 2016). Earlier, dietary Flaxseed has shown to reduce glutathione (Yuan et al., 1999), MDA activity and enhanced antioxidant activity (Lee et al., 2008). Flaxseed in the form of oil has also shown to increase CAT, SOD, GST glutathione-S-tranferase (GST), GPx and

GSH activities as compared to the diseased control (Moneim et al., 2011b; Jangale

178 et al., 2013). Flaxseed diet (10%) have also shown to exhibit antioxidant effect against some specific lung injury models; however, there was no effect in a lung injury model induced by LPS instillation (Kinniry et al., 2006). This contradictory effect could be attributed to the ineffective absorption of bioactive constituents in the form of diet. However, a question arises that, in the same study, the antioxidant effect was evident when the lung injury was induced by other agents (radiation and hyperoxia). It seems that Flaxseed diet was unable to retain its antioxidant effect against LPS-induced injury, whereas, it was effective against other types of injury.

It can be speculated that if Fs.Cr was administered instead of Flaxseed diet, it might have shown effectiveness against LPS-induced stimulation, possibly because AA- model could be correlated with an LPS-induced model of lung injury; this is based on the fact that AA cause bacterial translocation due to the injury to mucosa (Nakhai et al., 2007) and the gut microbes which have specific LPS in them, would interact with the immune cells in Lamina propria (LP) of the gut (Tlaskalová-Hogenová et al., 2004). Secondly, in this thesis, it is already mentioned that Fs.Cr was an effective bactericidal against different types of Gram negative pathogens (Chapter4), and therefore, has the potential to combat injury induced by such types of stimuli.

Another protective effect of Fs.Cr in AA-induced colitis model of mice was significantly reduced depletion of goblet cells with an increase in mucin levels, as compared to untreated group. Increased mucin levels have protective role in human

IBD (Van der Sluis et al., 2006; Zaph et al., 2007) and their elevated levels have shown to suppress colitis in animal model of IBD (An et al., 2007), possibly due to enhanced epithelial integrity (Hering and Schulzke, 2009). Therefore, the retention of goblet cells and increase in mucin levels might have caused mucosal protective effects. The mucosal protective effect is further supported by clinical aspects including reduced diarrhea and rectal bleed. This is the first study to report effect of

Flaxseed on goblet cells depletion and mucin levels. After publication of the finding in current study (Palla et al., 2016), a study appeared in the literature showing mucosal protective effect of Flaxseed lignan secoisolariciresinol diglucoside (SDG)

179 against DSS-induced colitis, mediated by increasing the expression of trefoil factor

3 (TFF3) (Xu et al., 2016), which is a peptide released by goblet cells and has a role in restitution of gut mucosa (Kjellev, 2009). It is possible that besides restoring goblet cells and mucin levels, Fs.Cr might also have mediated its mucosal protective effect by increasing expression of TFF3. It is however, important to highlight the fact that Flaxseed lignans are the part of Flaxseed (Tarpila et al., 2005) and do not represent the total activity possessed by Flaxseed, which is further proven by the literature where different constituents of Flaxseed have exhibited protective effect against colitis models of mice (Hassan et al., 2010; Naito et al., 2006; Park et al.,

2012; Tyagi et al., 2012; Vieira et al., 2012).

Hence, Fs.Cr by virtue of its mucosal restoring, anti-inflammatory and antioxidant properties, as evident by reduced goblet cell depletion, increased mucin, reduced oxidative stress and modulation of multiple cytokines mediates its protective effect against AA-induced colitis model of mice.

This is the first study to assess the effect of Fs.Cr in an IBD model of mice.

Earlier, Flaxseed diet (10%) and Flaxseed oil have been studied for their anti- inflammatory potential and have shown varied effects, with both anti-inflammatory

(Razi et al., 2010, 2011; Ghule et al., 2011; Saini et al., 2010) and pro-inflammatory effects (Zarepoor et al., 2014; Toth et al., 2015). Earlier, 10% Flaxseed diet aggravated the severity of dextran sodium sulfate (DSS)-induced colitis model of mice (Zarepoor et al., 2014). This was rather surprising, particularly, because the dietary components of Flaxseed (linolenic acid, kaempferol, ALA, sodium butyrate, secoisolariciresinol diglucoside/SDG) have been shown to exhibit anti- inflammatory activity in different models of colitis (Hassan et al., 2010; Naito et al.,

2006; Park et al., 2012; Tyagi et al., 2012; Vieira et al., 2012; Xu et al., 2016). One explanation for the ineffectiveness of Flaxseed diet in the study by Zarepoor et al.,

(2014) could be insufficient absorption of bio-active constituents of Flaxseed.

However, the question arises that if absorption was compromised leading to reduced bio-availability of bio-active constituents; how could Flaxseed meal produce anti-

180 inflammatory effect in diseased models of lung ischemia (Razi et al., 2010; 2011) and renal ischaemia-reperfusion injury (Ghule et al., 2011) in mice. One probable explanation could be that the type of inflammation mediated in these lung injury models may have triggered different inflammatory pathways (Kalogeris et al., 2012) that could be managed by the absorbed bio-active constituents, whereas, the inflammatory pathways triggered in IBD models may require different type of anti- inflammatory components which may not have been absorbed from the gut into the blood circulation. This is understandable because the level of inflammation mediated in chemical-induced IBD models of mice, allows bacterial translocation across the colonic mucosa (Nakhai et al., 2007); thus causing an interaction between resident gut associated lymphoid tissues (GALT) and microbes, and hence generating a lipopolysaccharide (LPS)-like immune response (Broz et al., 2012), since microbes are rich in LPS. Possibly that’s why, in an oxidative model of lung injury, Flaxseed diet exhibited anti-inflammatory effect in hyperoxia and radiation-induced models but not against LPS-induced lung injury model of mice (Kinniry et al., 2006).

Another possibility for effectiveness in current study and ineffectiveness in the study by Zarepoor et al., (2014), could be lack of optimal carrier. It was observed while preparing the Fs.Cr that for it to be soluble, 10% DMSO and 5% Tween-80 were both required and the extract couldn’t be solubilised completely in any one of them; it also had to be further vortexed for 10 minutes to ensure maximum exposure of the extract to the organic solvents, followed by addition of water. Even after this, the extract needed to be kept in sonicator, (with ice in it) for an hour to break the particles. However, if the extract was first mixed with water, followed by DMSO and Tween-80, it became hard to an extent, that adding organic solvent did not make it soluble. This indicates that there are absorption issues with Flaxseed; hence, sub- optimal conditions of administration may be the reason for aggravation of DSS- induced colitis by Flaxseed meal (Zarepoor et al., 2014). This was the reason that the intraperitoneal route was adopted in the current study to first, explore the potential of the whole plant and then optimize it for usage. From the literature, it is

181 indicated that whole Flaxseed meal may have some hindrance in absorption due to which the active bioactive constituents could not elicit their protective effects. The best form of Flaxseed and the optimal carrier for administering still need to be identified. From the current study, it has been clarified that Flaxseed does have a potential to mediate anti-inflammatory effect in IBD model of mice.

After establishing that Fs.Cr has effectiveness in IBD model of mice, other mechanisms that could possibly play a role in ameliorating IBD were assessed. Since whole array of causes of IBD cannot be ideally assessed in one model, some ex-vivo and in vitro means were adopted to further explore the effectiveness of Fs.Cr against diarrhea and spasms and its possible mechanisms.

Although, antidiarrheal effect of Fs.Cr was observed while assessing disease activity index in AA-induced colitis model of mice, a mechanistic basis for this effect was needed as the diarrhea in AA-induced colitis is merely due to physical injury, whereas, in IBD, both the breached epithelial barrier and alteration in gut contractility are evident (Bassotti et al., 2014). That is why, restoring vasculature and disturbances of blood flow by controlling gut contractility (Banner &

Trevethick, 2004) are proposed as one of the novel approaches for targeting IBD.

Knowing that diarrhea is a product of hypermotility and hypersecretion, experiments were conducted in mice to assess the antidiarrheal effect of Fs.Cr against castor oil- induced diarrhea. It was found that Fs.Cr had a predominant antidiarrheal activity against castor oil-induced diarrhea in mice due to decrease in intestinal secretions and reduction in the gut motility. Interestingly in this study, Fs.Cr did not halt the stool frequency completely when diarrhea was induced; it could be speculated that possible presence of spasmogenic component might have prevented the accessive antidiarrheal activity. This aspect is considered a merit, in terms of avoiding the side- effect of constipation, as seen with antidiarrheal drugs like, loperamide (Grahame-

Smith & Aronson, 2002). These observations are in consistence with earlier reported alternating mechanisms of natural products, such as being effective against both constipation and diarrhea as evident in psyllium husk (Mehmood et al., 2011) and

182 ginger (Ghayur & Gilani, 2005). It seems that nature has a built-in mechanism to off-set the exaggerated effect of one constituent by neutralizing it with an opposing constituent (Gilani & Atta-ur-Rehman, 2005), without compromising the desired effect.

This is the first study that reports the antidiarrheal effect of Flaxseed. Earlier, a preliminary report on Flaxseed oil employed castor oil-induced diarrhea as an indicator of prostaglandin release (Kaithwas & Majumdar, 2010b) and hence, was neither conducted with the perspective of diarrhea nor was any mechanism with respect to diarrhea investigated.

Upon investigation for possible antispasmodic mechanisms involved in isolated rabbit jejunum, it was found that Fs.Cr exhibited phosphodiesterase inhibitory (PDEI) activity, which was observed at low doses, while a Ca++ antagonist activity was observed at 10 times higher doses, thus highlighting that the predominant action of Fs.Cr for mediating antispasmodic effect was by PDE inhibition. Further to this, it was identified that this PDEI activity was possibly mediated by inhibiting PDE-4 enzyme. It could be a possibility that one of the protective mechanisms by which Fs.Cr mediated its protective effects against AA- induced colitis was via PDE-4 inhibition as PDE-4 receptors are present on monocytes which are responsible for release of TNF- and PDE-4 inhibitors have shown to mediate their anti-inflammatory effect by inhibiting monocytes with subsequent inhibition of TNF-(Gantner et al., 1997).

Another consequence of PDE inhibition is increase in the levels of cyclic

AMP (cAMP) (Anthony, 1996; Kaneda et al., 2005); this is important aspect from

IBD perspective because cAMP is known to interact with cytoskeletal system which maintains intact epithelial barrier (Willingham & Pastan, 1975). Dysregulation of cAMP causes breached barrier, which is one of the initial perpetuator of IBD

(Bassotti et al., 2014); it is possible, that one of the mechanisms by which Fs.Cr promoted mucosal repair in AA-induced colitis model of mice was by increasing cAMP levels. Consistent with this, Flaxseed lignans showed improvement in DSS-

183 induced colitis by increasing expression of markers of epithelial barrier promoters such as Zonna occludens 1 and TFF3 (Xu et al., 2016) and hence there’s a possibility that cAMP agonist action of Fs.Cr may have prompted this kind of cytoskeletal repair.

After establishing the efficacy of Fs.Cr in colitis model of mice and addressing its potential as antispasmodic and antimotility agent, Fs.Cr was further explored for its effect against microbes since they also have an established role in immune pathogenesis of IBD (Rescigno, 2008). Microbes may act as trigger for persistent immune stimulation and could also aggravate already initiated inflammatory cascade in IBD (Campieri & Gionchetti, 2001; Rahimi et al., 2007).

Hence, Fs.Cr effect was tested in vitro against the gut pathogens implicated in IBD. So far Flaxseed in any form has not been studied against these pathogens, hence for the first time Flaxseed in the form of extract (Fs.Cr) showed effectiveness against Enteropathogenic Eschericia coli (EPEC), enterotoxigenic E. coli (ETEC) and enteroaggregative E.coli (EAEC), which are the microbes that share virulence properties with adherent invasive E.coli (AIEC) (Santos et al., 2015), which is the strain of E.coli isolated from biopsies of ileal CD patients and possess multiple properties of different types of E.coli (Boudeau et al., 1999; Smith et al., 2013).

It was also effective against broad range of gut pathogens that cause gastroenteritis and are known to aggravate the inflammatory cycle including

Salmonella typhi, vancomycin-resistant Enterococcus faecalis, methicillin-resistant

Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa.

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However, the presence of some novel compounds is speculated based on 100% kill of VRE, which is resistant to conventional antibiotics. Antimicrobial effect of Fs.Cr would also preventing the possibility of side effect of disseminated infection that may occur with use of antimotility agents alone (Casburn-Jones & Farthing, 2004).

Overall, Fs.Cr reduced pro-inflammatory cytokines TNF- and IFN- and enhanced IL-17; reduced neutrophil infiltration and oxidative stress in AA-induced model of colitis. In vitro analysis showed its efficacy against broad spectrum and antibiotic-resistant pathogens. Ex vivo analysis showed that Fs.Cr produced its antispasmodic effect by PDEI and Ca++ antagonist activity, along with increased cAMP level in tissues, possibly mediated by PDE-4 inhibition. Hence, Fs.Cr has ability for targeting multiple etiological aspects of IBD.

After the detailed analysis of extract, Flaxseed oil was also studied for some of the parameters of IBD. This was done because Flaxseed oil is a known edible source, and have possibility to exhibit a different profile of activity as compared to methanolic-aqueous extract, since oil possess only non-polar constituents (Lin et, al., 1999). Another aspect that prompted to include Flaxseed oil screening in the current study settings was the predominant literature evidence for its use in constipation (Duke et al., 2002). Since the extract did not show laxative effect, it was important to investigate which fraction of Flaxseed has gut stimulant component, as predominant gut stimulant component could worsen the symptoms of IBD-induced diarrhea. Possibly that’s why, a study that used Flaxseed meal to prevent DSS- induced colitis, ended up aggravating the symptoms in that model (Zarepoor et al.,

2014), because whole seed have reported laxative effect (Xu et al., 2012).

Nevertheless, laxative effect of Flaxseed might be beneficial in left sided colitis which is constipation predominant. Hence, Flaxseed oil was tested both in-vitro and in-vivo.

It was identified that at low doses Flaxseed oil exhibited gut stimulant activity (cholinergic and mild histaminergic) which was reversed at high doses. This was a very interesting finding because laxative drugs at high doses cause diarrhoea,

185 whereas, increasing Flaxseed oil dose after a certain concentration did not increase laxative effect, in fact, blocked it; which means it has inhibitory components. Hence, at high doses, Flaxseed oil exhibited antispasmodic effect mediated by strong K+- channel opening (KCO) activity that involved non-specific and ATP-sensitive K+- channel activation pathway. Flaxseed oil also exhibited weaker PDEI activity; this was in contrast to Fs.Cr, which showed no gut stimulant effect, with a predominant

Further to this, Flaxseed oil was assessed in the in-vivo assay to see, if it exhibits dual activity (laxative and antidiarrheal). Similar to ex-vivo data, Flaxseed oil showed laxative activity in mice at low doses which was reversed at higher doses when tested against castor oil-induced diarrhoea.

2011) which is a non-pathogenic strain and hence, cannot be compared.

Further to this, Flaxseed oil was assessed for its efficacy against AA-induced colitis. Flaxseed oil improved DAI dose-dependently at 300 and 500 mg/kg doses.

Unlike Fs.Cr that mediated its mucosal protective effect (evident by microscopic scoring) at 150 mg/kg dose, there was no effect at the dose of 150 mg/kg of Flaxseed oil. Unlike Fs.Cr that showed optimal effect at 300 mg/kg dose, Flaxseed oil exhibited a dose-dependent mucosal protective effect between 300 mg/kg and 500

186 mg/kg doses. This indicates that the optimal dose of Fs.Cr, is 300 mg/kg, because increasing the dose beyond this did not increase the efficacy, whereas, Flaxseed oil could be tested for a dose beyond 500 mg/kg as the effect started to show up from

300 mg/kg dose and was escalating at 500 mg/kg doses.

The dual efficacy of Flaxseed oil in diarrhea and constipation, its antimicrobial activity against microbes implicated in IBD and antispasmodic mechanisms mediated by KCO and PDEI, along with improved mortality rate and

DAI in acetic acid-induced colitis model indicate that it could be a reasonable choice for IBD therapeutics and needs to be explored further for possible mechanisms in

IBD model of mice. This finding has an important implication for clinical studies, since Flaxseed oil is already available edible source and could be tested in IBD patients as a remedy.

The difference in antimicrobial efficacy and mechanistic profile for antispasmodic activity as well as the evidence from HPLC peaks that shows predominance of non-polar constituents in oil while polar constituents in the extract, indicates that both Flaxseed oil and Fs.Cr mediate their effect due to different mechanisms. The efficacy of extract and oil against IBD model of mice along with different pattern of activity indicates that Flaxseed as a whole has potential for IBD therapeutics and therefore taking Flaxseed in its natural form is likely to provide benefits obtained from both the oil and extract. Nevertheless, an optimal method further needs to be identified that ensures maximum absorption of bio-active constituents from Flaxseed in the body.

6.1. Conclusion:

From the thesis, it is clear that Flaxseed extract (Fs.Cr) is effective in ameliorating the severity of AA-induced colitis in BALB/c mice by virtue of its anti- inflammatory, antioxidant and mucosal restoring mechanisms. The modulation of multiple cytokines further advocates its potential in IBD therapy. Its antibacterial effect against bacteria implicated in IBD suggests reducing one of the persistent

187 triggers of inflammation. The antidiarrhoeal activity of Fs.Cr reduced the symptoms of IBD, by decreasing motility and secretions; the presence of multiple mechanisms indicates that Fs.Cr may have a direct association in reducing many causes of IBD, attributed to increase in cAMP and PDE-4 inhibition which have role in mucosal restoration and reduction of inflammation.

The currently available edible source of Flaxseed i.e. Flaxseed oil also has potential for managing IBD, evident by effectiveness in AA-induced model of colitis, mucosal protective effects, antidiarrhoeal, antispasmodic and a strong antimicrobial effect.

This indicates the potential of Flaxseed as a whole to ameliorate IBD, and can be used as a remedy for treatment of IBD as they possess effect against basic triggers that include inflammation, immune modulation, oxidative stress, aggressive microbes and against the major manifestation of IBD that include diarrhea and spasms. Hence, Flaxseed has constituents that can ameliorate multiple targets of IBD by multiple mechanisms.

6.2. Limitations of the study

There are a few limitations associated with this study. One of the limitations is that AA colitis model was used as an innate immune model. This is though an easy and reproducible model, but amongst the chemical models of colitis, DSS is a more sophisticated model.

In the AA model of colitis, only IL-17 was measured; simultaneous measurement of IL-23, IL-6 and TGF- could have given much more information on pathogenic nature of IL-17, where as, measurement of IL-22 could have indicated towards the type of cells involved in release of IL-17.

Also, IBD is a chronic disease and a simultaneous chronic model could have given a complete picture of its efficacy in colitis. Another limitation of this study was using an i.p. route for administration of the test material since the anti- inflammatory effect was the best when given through this route.

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6.3. Future plans

The future plan includes knowing how to increase the bioavailability of active constituents from the whole seeds to produce more significant effects and to compare the efficacy of various fractions of Flaxseed.

In AA-induced colitis model, correlation of IL-17 with its protective and pathogenic effect would be established by measuring the simultaneous levels of IL-

23, IL-6 and TGF- as well as IL-22. In addition, DSS-induced colitis, which is a relatively more sophisticated model of innate immune colitis, will be used and pattern of IL-17 and other innate immune cytokines will be studied, and the effect of Fs.Cr, Flaxseed meal and Flaxseed oil on its release and also on the release of other immune cytokines.

The mechanism of Fs.Cr on T cells pathway with special emphasis on Th-17 cells will be focused, and it would be tried to identify the effect of Fs.Cr on a more sophisticated model of innate immune colitis (knockout models) as well as adaptive immune colitis, to specifically identify protective and effecting status of IL-17. For this purpose, TNBS model would be selected.

The current study lacks the reporting of Fs.Cr’s effect on commensal microbes and future studies may be directed to determine the effects on them, although one study by using Flaxseed meal and oil has shown such protective effects

(Zarepoor et al., 2014). Furthermore, studies would be directed to test effect of Fs.Cr on AIEC as well as gastroenteritis causing broad-spectrum of Gram negative pathogens including Vibrio cholera, Campylobacter jejunii, Shigella flexinerii,

Proteus mirabilis as well as viral and parasitic pathogens causing enteric infections.

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8. Vita

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Amber Hanif Palla, Ph.D

C-30, Dhoraji Colony, Karachi, Sindh. 74500 Telephone: 0334-3653650; Email:[email protected];

SUMMARY Effective teachers have always inspired me, and the love and passion for teaching pharmacology developed ever since I started to study this subject. The depth of teaching was further clarified when I started my Ph.D studies, where I understood the importance of integration of research and its importance in teaching the theoretical concepts.

SKILLS Pharmacology and Pharmacy Practice Research expertise in basic and applied sciences Animal handling and dissection Diabetes, Hospice, and Stroke Patient Care Natural products Drug screening Patient evaluation/intervention Effective public speaker Expert in developing inventory systems Diagnostic tools familiarity

CERTIFICATIONS: ACLS Certification by AHA (American Heart Association), ---- in Process BLS Certification by AHA ------in process

WORK HISTORY  Assistant Professor, Pharmacology and Pharmacy Practice, Barrett Hodgson University, Karachi Jan 2017- ongoing  Teaching Assistanceship, Pharmacology (part-time), The Aga Khan University Karachi Sep 2009- Sep 2013  Co-operative Teacher, Pharmacology (part-time), Karachi University, Jan 2009- June 2009  Assistant Professor, (Pharmacology, Faculty of Pharmacy), Jinnah University for Women, Karachi, Mar 2008-Jan 2009  Pharmacist, Drug and Poison Information Center, Department of Pharmacy, Aga Khan University Hospital Sep 2006-Apr 2007.

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 Clinical cardiology clerkship, NICVD hospital, Jun 2006-Jul 2006.  Trainee Pharmacist, Department of Pharmacy, The Aga Khan University Hospital Jan 2003-Jan 2004.

EDUCATION  Ph.D (Health Sciences); Specialization: Pharmacology, Aga Khan University and Medical College, Karachi, 2009-2016  Masters in Pharmacy (Pharmacology), University of Karachi, Karachi, Sindh (C.GPR: 3.91) 2003-2006  Bachelors in Pharmacy, University of Karachi, Karachi, Sindh (C.GPR: 3.49) 1999-2002

PERSONAL DATA DATE OF BIRTH: 10th Aug 1981; PLACE OF BIRTH: Karachi; LANGUAGES: Urdu, English, Memoni, French; MARITAL STATUS: Married; CHILDREN: 2; LICENSE #: 2325 (26.3.2005). Pharmacy Council of Sindh

PUBLICATIONS: 1. Palla, Amber Hanif., Iqbal, Najeeha Talat., Minhas, Khurram., Gilani, Anwar-ul-Hassan. (2016). Flaxseed extract exhibits mucosal protective effect in acetic acid induced colitis in mice by modulating cytokines, antioxidant and antiinflammatory mechanisms. International Immunopharmacology, 6 (38), 153-166. (IF: 2.472)

2. Palla, Amber Hanif, & Gilani, Anwar-ul-Hassan (2015). Dual effectiveness of Flaxseed in constipation and diarrhea: Possible mechanism. Journal of Ethnopharmacology, 169, 60-68. April (IF: 2.998)

3. Palla, Amber Hanif., Khan, Naveed Ahmed., Bashir, Samra., Iqbal, Junaid., Gilani, Anwar-ul-Hassan, (2015). Pharmacological basis for the medicinal use of Linum usitatissimum (Flaxseed) in infectious and non-infectious diarrhea. Journal of Ethnopharmacology, 160, 61-68. (IF: 2.998)

4. Palla, Amber Hanif., Khan, Rafeeq Alam., Gilani, Anwar-ul-Hassan., & Marra, Fawzia. (2012). Over prescription of antibiotics for adult pharyngitis is prevalent in developing countries but can be reduced using McIsaac modification of Centor scores: a cross-sectional study. BMC Pulmonary Medicine, 12(1), 70. (IF: 2.760)

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ABSTRACTS

1. Palla, Amber Hanif., Khan, Rafeeq Alam., Gilani, Anwar-ul-Hassan., & Marra, Fawzia. (2012). Antibiotics are prescribed inappropriately to adult pharyngitis patients and McIsaac modification of Centor score is the answer to reduce unnecessary antibiotic prescriptions in low socio-economic areas”. Journal of Pakistan Medical Association 62 (5), S8.

2. Palla, Amber Hanif., Ehsan, Salwa., Raza, Shamim, Shiekh, Latif. (2007). Impact of pharmacist-run therapeutic drug monitoring in a tertiary care hospital. 2nd International Critical Care Symposium Abstract Book, AKU.

3. Palla, Amber Hanif., Sheikh, Latif., Qadeer, Abdul Sajid. (2004). Utilization pattern of omeprazole in outpatient setting after the availability of cheaper generic products. Community Pharmacy Section posters (CPS)-P003. World Congress of Pharmacy and Pharmaceutical Sciences; 64th

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