effect of cinnamomum cassia bark...

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EFFECT OF CINNAMOMUM CASSIA BARK EXTRACT ON

GLUCOSE HOMEOSTASIS IN ALLOXAN-INDUCED DIABETIC

RATS IN COMPARISON WITH DIRECT RENIN INHIBITOR

(ALISKIREN) AND DIPEPTIDYL PEPTIDASE-IV INHIBITOR

(SITAGLIPTIN)

A thesis submitted in partial fulfillment for the requirements for

the degree of Doctor of Philosophy in Physiology

by

Navaid Kazi

2

Department of Physiology

Faculty of Medicine & Allied Medical Sciences

Isra University, Hyderabad, Sindh

December 2015

EFFECT OF CINNAMOMUM CASSIA BARK EXTRACT ON

GLUCOSE HOMEOSTASIS IN ALLAXON-INDUCED DIABETIC

RATS IN COMPARISON WITH DIRECT RENIN INHIBITOR

(ALISKIREN) AND DIPEPTIDYL PEPTIDASE-IV INHIBITOR

(SITAGLIPTIN)

by

Navaid Kazi

Supervisor/Co-supervisors

3

Prof. Din Muhammad Shaikh (Supervisor)

Professor of Physiology

Prof. Ahmed Azmi (Co-supervisor)

Professor of Physiology

Prof. Mumtaz Qureshi (Co-supervisor)

Professor of Biochemistry

DEDICATION

4

THIS THESIS IS DEDICATED TO MY PARENTS, WIFE AND CHILDREN

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CERTIFICATE

This is to certify that DR. NAVAID KAZI S/O ABDUL WAHEED KAZI has carried out

research work on the topic “EFFECT OF CINNAMOMUM CASSIA BARK EXTRACT ON GLUCOSE

HOMEOSTASIS IN ALLAXON-INDUCED DIABETIC RATS IN COMPARISON WITH DIRECT RENIN

INHIBITOR (ALISKIREN) AND DIPEPTIDYL PEPTIDASE-IV INHIBITOR (SITAGLIPTIN)” under my

supervision and that his work is original and his thesis is worthy of presentation to Isra University

for awarding the degree of “Doctor of Philosophy” in the subject of Physiology.

Prof. Din Muhammad Shaikh

Supervisor _____________________

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Department of Physiology Signature of Supervisor

Isra University

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ACKNOWLEDGEMENT

With the deep and profound sense of gratitude and thanks to the almighty ALLAH

for giving me the chance for completing this thesis, I am greatly indebted to my respected

Supervisor, Prof. Dr. Din Muhammad Shaikh and Co-supervisor, Prof. Dr Ahmed Azmi

and Prof. Dr Mumtaz A Qureshi for their cooperation, guidance and constructive criticism

in the successful completion of this thesis and without their help, this manuscript was not

possible to complete. I am grateful to Prof. Dr. Ghulam Qadir Kazi, the Vice Chancellor

Isra University for his whole heartedly valuable co-operation and support.

My thanks are also to all my colleagues, friends and well wishers for giving me

the support and help during the course of conduct of this study.

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ABSTRACT

BACKGROUND: Cinnamon is a small evergreen tree belonging to the family

Lauraceae. Cassia is a member of plant family known as Cinnamon. It is also known as

"Java cinnamon", Saigon cinnamon”, "Padang cassia" or as the "Chinese cinnamon".

Cinnamon plays role in the management of diabetes and its complications, and also used

as an herbal remedy. Cinnamon is one of spice having a long history of use as flavoring

agent, preservative and pharmaco-logical remedies.

OBJECTIVES OF STUDY:

To determine effects of cinnamon cassia bark extract (CCBE) on glucose

homeostasis in alloxan induced diabetic rats.

To compare the effect of CCBE vs. Sitagliptin and Aliskerin on glucose homeostasis

in alloxan induced diabetic rats.

To evaluate the anti oxidant activity of CCBE in alloxan induced rats

To compare the anti oxidant effect of CCBE vs. aliskerin and sitagliptin in alloxan

induced diabetic rats.

To observe the protective effects of C. cassia on the histology of Islets of Langerhans

of Pancreas in comparison to aliskerin and sitagliptin in alloxan induced diabetic rats.

SUBJECTS AND METHODS: An experimental – analytical study was conducted at the

Isra University, Al-Tibri Medical College and Liaquat University. Sixty Wistar Albino rats

of either sex were selected according to inclusion and exclusion criteria and were divided

into 6 groups comprising of 10 rats in each group which were further subdivided into 5 in

each sub group. Cinnamomum cassia barks extract (ethanolic) (CCBE), aliskerin and

sitagliptin were given in proper doses. Animal were handled under close supervision

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during experimentation. The blood samples and pancreatic tissue were stored (-80oC)

and processed in a systemic way. Stem bark of Cinnamomum cassia (cinnamon) was

collected from the local market and authenticated by Botany Department of Sindh

University. Blood glucose, serum insulin, superoxide dismutase and glutathione

peroxidase were determined according to standard methods. Pancreatic tissue was

stained with H & E for microscopic examination. Collected data was analyzed on SPSS

version 21.0. (IBM, corporation, USA) P-value of significance was taken at 0.05.

RESULTS: Blood glucose lowering activity of CCBE was observed at both low

and high doses of 0.3g/day and 0.6g/day respectively. Glucose homeostasis

potential of CCBE was found comparable to sitagliptin. CCBE in combination with

sitagliptin revealed more synergistic effect on blood glucose levels. Serum insulin

was elevated by CCBE. Antioxidant and tissue protective effects of CCBE were

also observed compared to other groups. Superoxide dismutase (SOD) and

glutathione peroxidase (GPX) were found elevated in CCBE. The CCBE treated

animals revealed intact tissue architecture, pancreatic acini, and visible Islets of

Langerhans at both low and high doses.

CONCLUSION: It is concluded that the Cinnamomum cassia bark extract exerts

glucose lowering effects primarily by stimulation of insulin from β-cells of the islets

of Langerhans. Microscopic picture of pancreatic histology revealed intact tissue

in Cinnamomum cassia treated animals. Regenerating pancreatic tissue and

most probably the pancreatic β-cells mass was visibly observed

KEYWORDS: Cinnamomum cassia Sitagliptin Aliskerin Glucose

homeostasis Alloxan Diabetic Rats

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LIST OF ABBREVIATION

ABBREVIATION

TERM

ACE

AD

AGT

AMPK

ARBs

AT-1

ATMC

ATP

BMI

BPA

BUN

CA

CAE

cAMP

CAT

CCBE

CD 26

DCT

DM

DNA

Angiotensin converting enzyme

Anno Domini

Angiotensinogen

Activated Monophosphate protein kinase pathways

Angiotensin receptor blockers

Angiotensin 1 Receptors

Al Tibri Medical College

Adenosine tri phosphate

Body mass index

Bisphenol A

Blood urea Nitrogen

Cinnamaldehyde

Cinnamomum Aldehyde extract

Cyclic Adenosine Mono phosphate

Cinnamaldehyde treated

Cinnamomum Cassia Bark extract

Cluster of differentiation 26 marker.

Distal convoluted tubule

Diabetes Mellitus

Deoxyribonucleic acid

11

DPP - IV

DRI

DUHS

EC50

ED50

EDTA

EGF

ER

FAO

FRAP

GFR

GIP

GLP

GLUT 2

GPX

GSH

GSSG

HbA1c

HDL

IDF

IFN -

IRS

JECFA

J G cells

LDL

L/min

LSD

MDA

MDP

Dipeptidyl Peptidase- IV

Direct rennin inhibitors

Dow University of health sciences

Effective concentration

Effective dose

Ethylene diamine tetra acetic acid

Endothelial derived growth factor

Endoplasmic reticulum

Federation of agriculture organization

Ferric reducing activity of plasma

Glomerular filtration rate

Glucose dependent insulinotropic peptide

Glicentin like peptide

Glucose transporter -2

Glutathione peroxidase

Reduced Glutathione

Oxidized Glutathione

Glycosylated Hemoglobin

High density lipoprotein

International Diabetes Federation

Interferon gamma

Insulin Receptor substrate

Joint expert committee on food additives

Juxta Glomerular Cells

Low density lipoprotein

Liter per minute

Lysergic acid diethyl amide

Melondialdehyde

Mesoscale discovery plate

12

Mg / liter

MHCP

MICs

g/ liter

mRNA

MSD

MSP

NADPH

NBT

NO

NWFP

OGTT

OP

PBST

PCOs

PDE

PDX-1

PGE2

PI

PNDS

RAAS

RBC

SGOT

SGPT

SOD

SPSS

STZ

TBARS

US- FDA

Milligram per liter

Methyl hydroxyl chalcone polymer

Minimal inhibitor y concentration

Microgram per liter

Messenger Ribonucleic acid

Metabolic assays mouse /rat insulin kit

Medoff sliding plates

Nicotinamide dinucleotide phosphate

Nitrobule tetrazolium

Nitric Oxide

North west frontier province

Oral glucose tolerance test

Octyphenol

Phosphate buffered saline tween

Poly cystic ovarian syndrome

Phosphor - di - esterase inhibitors

Pancreatic and duodenal homeobox

Prostaglandin Eicosanoid

Phosphatidyl – inositol

Pakistan National Diabetes Survey

Renin Angiotensin Aldosterone Mechanism

Red blood cells

Serum glutamic oxaloacetate transaminase

Serum glutamic pyruvate transaminase

Superoxide dismutase

Statistical Package for the social sciences

Streptozotocin (induced diabetes)

Thiobarbituric acid reactive substances

United Sate Food and drugs administration

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WHO World health organization

TABLE OF CONTENTS

Page #

ACKNOWLEDGEMENT--------------------------------------------------------------- iv

ABSTRACT------------------------------------------------------------------------------- v

LIST OF ABBREVIATION-------------------------------------------------------------

TABLE OF CONTENTS--------------------------------------------------------------

vii

ix

LIST OF TABLES----------------------------------------------------------------------- xii

LIST OF FIGURES--------------------------------------------------------------------- xiii

LIST OF GRAPHS----------------------------------------------------------------------

LIST OF PHOTOMICROGRAPHS

xiv

xvii

CHAPTER – I ---------------------------------------------------------------------------

01

1. INTRODUCTION--------------------------------------------------------------- 01

2. AIMS/OBJECTIVES ---------------------------------------------------------- 3. RATIONALE OF STUDY----------------------------------------------------- 4. Hypothesis-----------------------------------------------------------------------

04

05

06

14

CHAPTER – II ------------------------------------------------------------------------- 07

LITERATURE REVIEW -------------------------------------------------------------- 07

1. Pancreas ------------------------------------------------------------------------ 1.1. Beta cell biology (Islets of Langerhans)-------------------------

07

07

2. Physiology of glucose homeostasis -------------------------------------- 2.1. Source of blood glucose-------------------------------------------- 2.2. Fate of blood glucose level---------------------------------------- 2.3. Significance of glucose homeostasis---------------------------- 2.4. Factors regulating glucose homeostasis------------------------

2.4.1. Insulin----------------------------------------------------------- 2.4.2. Glucagon------------------------------------------------------- 2.4.3. Catecholamines---------------------------------------------- 2.4.4. Cortisol---------------------------------------------------------- 2.4.5. Growth hormone----------------------------------------------

10

11

13

14

15

15

16

17

17

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3. Incretin hormones-------------------------------------------------------------

3.1. The entero – insular axis------------------------------------------- 3.2. History of incretins---------------------------------------------------- 3.3. The incretin system--------------------------------------------------- 3.4. GLP -1 and Beta cell proliferation-------------------------------- 3.5. GLP -1 and Beta cell neogenesis-------------------------------- 3.6. Dipeptidyl Peptidase IV ( DPP – IV )----------------------------- 3.7. Dipeptidyl Peptidase Inhibitor -------------------------------------

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18

18

19

23

23

24

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4. Renin Angiotensin Aldosterone System----------------------------------

4.1. History of discovery -------------------------------------------------- 4.2. Synthesis of circulating renin---------------------------------------

4.2.1. Decreased Stretch of afferent arteriole---------------- 4.2.2. Decreased delivery of salt to the macula densa------------------------------------------------------------ 4.2.3. Adrenergic stimulation -------------------------------------

4.3. Physiology of RAAS ------------------------------------------------- 4.3.1. Alternative Enzymatic pathways-------------------------- 4.3.2. ACE can act on substrates other than Ang I 4.3.3. Angiotensin II can be converted to

Angiotensin III & IV ------------------------------------------ 4.3.4. Components of the RAAS also reside within

25

25

26

27

28

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local tissues --------------------------------------------------- 4.3.5. Angiotensin receptors---------------------------------------

4.4. Direct Renin inhibitors- Aliskerin ---------------------------------- 4.4.1. Aliskerin--------------------------------------------------------- 4.4.2. Mechanism of action of Aliskerin ------------------------ 4.4.3. Aliskerin was co – developed by Swiss

Pharmaceuticals----------------------------------------------

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29

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35

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5. Cinnamomum Cassia -------------------------------------------------------- 5.1. Over view -------------------------------------------------------------- 5.2. Historical prospective of C. Cassia as medicine-------------- 5.3. Cinnamom used as traditional medicine ------------------------ 5.4. Adult posology & mode of administration ---------------------- 5.5. Non clinical pharmacological data-------------------------------- 5.6. Toxicology---------------------------------------------------------------

5.6.1. Teratogenicity ------------------------------------------------ 5.6.2. Reproductive organs --------------------------------------- 5.6.3. Carcinogenicity----------------------------------------------- 5.6.4. Genotoxicity & mutagenicity------------------------------- 5.6.5. Acute toxicity-------------------------------------------------- 5.6.6. Sub acute & chronic toxicity-------------------------------

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36

40

41

42

43

47

47

47

47

47

48

48

CHAPTER – III -------------------------------------------------------------------------

50

MATERIALS AND METHODS ---------------------------------------------------- 50

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3.1. Study Design ------------------------------------------------------------- 50

3.2. Study setting -------------------------------------------------------------- 50

3.3. Duration of study--------------------------------------------------------- 50

3.4. Sample size --------------------------------------------------------------- 3.5. Grouping of animals -----------------------------------------------------

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50

3.6. Inclusion criteria --------------------------------------------------------- 50

3.7. Exclusion criteria---------------------------------------------------------- 53

3.8. Data collection procedure --------------------------------------------- 53

3.9. Animal housing & experimental details ----------------------------- 53

3.10. Preparation of ethanol based Cinnamomum Cassia ----------- 53

3.10.1. Plant material ------------------------------------------------- 3.10.2. Collection of plant material--------------------------------- 3.10.3. Preparation of plant extract-------------------------------- 3.10.4. Dryness of Cinnamom Stem Bark----------------------- 3.10.5. Check level of Cinnamom Stem Bark dryness---------------------------------------------------------

3.10.6. Sifting of Cinnamom bark powder------------------------ 3.10.7. Preparation of Cinnamom Stem Bark extract 3.10.8. Filtration of mixture ------------------------------------------ 3.10.9. Separation of extract from ethanol----------------------- 3.10.10. Re – Separation of ethanol from extract---------------- 3.10.11. Storage of extract-------------------------------------------- 3.10.12. Animal Sacrifice and Blood sampling------------------- 3.10.13. Determination of Glutathione peroxidase—----------- 3.10.14. Determination of Superoxide dismutase--------------- 3.10.15. Insulin Detection

3.11. Data Analysis--------------------------------------------------------------

54

54

54

54

55

55

55

56

56

56

57

57

58

58

59

60

CHAPTER – IV ------------------------------------------------------------------------- 61

17

RESULTS -------------------------------------------------------------------------------- 61

CHAPTER – V -------------------------------------------------------------------------- 109

DISCUSSION --------------------------------------------------------------------------- 109

CHAPTER – VI ------------------------------------------------------------------------- 131

CONCLUSION -------------------------------------------------------------------------- 131

CHAPTER – VII ------------------------------------------------------------------------- 132

RECOMMENDATIONS --------------------------------------------------------------- 132

REFRENCES ---------------------------------------------------------------------------- 133

APPENDIX - I---------------------------------------------------------------------------

APPENDIX- II----------------------------------------------------------------------------

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LIST OF TABLES

Chapter Description Page

IV –1 Analysis of variance ( ANOVA ) ------------------------------------ 64

IV –2 Blood glucose level ( mg/dl) in animal groups ------------------ 65

IV-3 Serum Insulin level ((U/ml) in animal groups ------------------ 66

IV-4 Superoxide Dismutase (U/ml) in animal groups --------------- 67

IV-5 Glutathione Peroxidase (U/ml) in animal groups ------------- 68

IV-6 Blood Glucose , Serum Insulin, Superoxide Dismutase &

Glutathione levels in group” A” animals -------------------------

69


Glutathione levels in group” B” animals

70


Glutathione levels in group “C” animals

71


Glutathione levels in group “D” animals

72

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Glutathione levels in group “E” animals

73


Glutathione levels in group “F” animals 74

LIST OF FIGURES

Figure Description Page

II – 1 Physiological Anatomy of Pancreas, duct system & Islets of

Pancreas 09

II – 2 Utilization of Blood Glucose by different tissues & organs shown

as micromole of glucose ----------------------------------- 12

II – 3 Fate of glucose metabolism ---------------------------------------- 13

II – 4 Incretin effect in normal healthy & diabetic subjects -------- 21

II – 5 Incretin effect in normal healthy & diabetic subjects --------- 22

20

II – 6 Release of Renin & stimulation of Angiotensinogen into

Angiotensi I & Angiotensin II--------------------------------------- 34

II – 7 Release of Renin & stimulation of Angiotensinogen into

Angiotensi I & Angiotensin II --------------------------------------- 35

II – 8 Physiological effect of Renin Angiotensin Aldosterone System --

----------------------------------------------------------------- 36

II – 9 Physiological effect of Renin Angiotensin Aldosterone System --

----------------------------------------------------------------- 37

II – 10 Cinnamomum Cassia -------------------------------------------- 40

LIST OF GRAPHS

IV-1 Bar graphs showing blood glucose distribution among all groups

as a cumulative on different days in all groups -------

75

IV-2 Bar graphs showing insulin secretion among all groups as a

cumulative on different days in all groups --------

76

IV-3 Bar graphs showing Superoxide Dismutase activity as a

cumulative on different days in all groups ------------------------

77

21

IV-4 Bar graphs showing Glutathione peroxidase activity as a

cumulative on different days in all groups -----------------------

78

IV-5 Bar graphs showing blood glucose level in sub groups A1 & A2----

----------------------------------------------------------------------

79

IV-6 Bar graphs showing blood insulin level in sub groups A1 & A2------

---------------------------------------------------------------------

80

IV-7 Bar graphs showing Superoxide dismutase level in sub groups A1

& A2-----------------------------------------------------------

81

IV-8 Bar graphs showing Glutathione peroxidase activity in sub groups

A1 & A2----------------------

82

IV-9 Bar graphs showing blood glucose level in sub groups B1 & B2----

-----------------------------------------------------------------------

83

IV-10 Bar graphs showing blood insulin level in sub groups B1 & B2------

-------------------------------------------------------------------

84

IV-11 Bar graphs showing Superoxide dismutase activity in sub groups

B1 & B2-----------------------------------------------------------

85


B1 & B2----------------------------------------------------------

86

IV-13 Bar graphs showing blood glucose level in sub groups C1 & C2----

-----------------------------------------------------------------------

87

22

IV-14 Bar graphs showing blood insulin level in sub groups C1 & C2------

--------------------------------------------------------------------

88


C1 & C2---------------------------------------------------------

89


C1 & C2---------------------------------------------------------

90

IV-17 Bar graphs showing blood glucose level in sub groups D1 & D2----

----------------------------------------------------------------------

91

IV-18 Bar graphs showing blood insulin level in sub groups D1 & D2------

------------------------------------------------------------------

92


D1 & D2--------------------------------------------------------

93

IV-20 . Bar graphs showing Glutathione peroxidase activity in sub

groups D1 & D2----------------------------------------------------------

94

IV-21 Bar graphs showing blood glucose level in sub groups E1 & E2----

------------------------------------------------------------------------

95

IV-22 Bar graphs showing blood insulin level in sub groups E1 & E2------

---------------------------------------------------------------------

96


E1 & E2-----------------------------------------------------------

97

23


E1 & E2----------------------------------------------------------

98

IV-25 Bar graphs showing blood glucose level in sub groups F1 & F2-----

-----------------------------------------------------------------------

99

IV-26 Bar graphs showing blood insulin level in sub groups F1 & F2-------

---------------------------------------------------------------------

100


F1 & F2----------------------------------------------------------

101


F1 & F2----------------------------------------------------------

102

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LIST OF PHOTOMICROGRAPH

IV – 1 Group A1. (Control). Normal Architecture or Pancreas, is visible

and islet of Langerhans are also visible. X100 (H & E)-------------

--------------------------------------------------------------

103

IV – 2 Group A2 (Alloxan induced Rats) completely disturbed

Architecture of pancreas, islet of Langerhans is not properly

visible. X100 (H&E)----------------------------------------

103

IV – 3 Group B1 (CCBE 0.3g/dl). Islets of Langerhans are partially

visible but smaller in size X100 (H&E)-----------------

104

IV – 4 Group B2 (CCBE 0.6g/dl) Anatomical architecture of pancreas

is visible, with islets of Langerhans showing moderate recovery.

X400 (H&E)-----------------------------------

104

IV – 5 Group C1 (Aliskerin) Anatomical Architecture of pancreas is

disturbed; islets of Langerhans are also invisible. X100 (H&E)---

-------------------------------------------------------------------

105

IV– 6. Group C2 (Aliskerin) Anatomical architecture of pancreas is

disturbed; islets of Langerhans are also invisible. X100 (H & E)--

-----------------------------------------------------------------

105

IV– 7 Group D1 (Sitagliptin) Anatomical architecture of pancreas is

partially disturbed; with mild recovery of islets of Langerhans.

X400 (H & E)-------------------------------------------

106

IV– 8. Group D2 (Sitagliptin) Anatomical architecture of pancreas is

partially disturbed; with mild recovery of islets of Langerhans.

X400 (H & E)-------------------------------------------

106

IV– 9 Group E1 (Low dose CCBE+Aliskerin) Anatomical architecture

of pancreas is mildly distorted with partial recovery of islets of

Langerhans. X200 (H&E)------------------

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107

IV– 10 Group E2 (Low dose CCBE+Sitagliptin) Anatomical architecture

of pancreas is nearly well defined; islets of Langerhans are

moderately visible. X100 (H & E)-------------

107

IV– 11 Group F1 (High Dose CCBE+Aliskerin) Anatomical architecture

of pancreas is mildly disturbed with partial recovery of islets of

Langerhans. X200 (H & E)----------------

108

IV– 12 Group F2 (High Dose CCBE+Sitagliptin) Anatomical

architecture of pancreas is well defined; islets of Langerhans

are almost recovered. X200 (H & E)--------------

108

CHAPTER I

1. INTRODUCTION

Diabetes mellitus (DM) is a syndrome characterized by chronic

hyperglycemia caused by relative or absolute insulin deficiency. (1, 2) The

chronic hyperglycemia, in long term causes damage in the target organs like eye,

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nerves, kidney, heart and blood vessels. (3, 4) According to international diabetes

federation (IDF), the number of diabetics at the age of twenty is going to rise from

285 million in 2013. (4) The Pakistan ranks six position regarding diabetes burden

in whole world. (5) According to an estimate of Shera, et al. there are 15%

Pakistani’s with diagnosed DM millions more who remain undiagnosed / unaware

of having DM.(6) Pakistan National diabetes survey (PNDS) revealed that for

each diagnosed case of DM there are 2 cases of undiagnosed DM and 3 cases of

impaired glucose tolerance approximately. (6, 7)

The incidence of type-2 DM is increasing rapidly around the world. DM is

the most common of the endocrine disorder in which homeostasis of

carbohydrate and lipid metabolism is improperly regulated by insulin. (2, 8, 9)

Insulin is the ideal treatment for diabetes in the conditions where blood glucose

levels cannot be controlled by oral hypoglycemic agents. The application of

insulin and administration of oral hypoglycemic agents has involved in the

management of diabetes. Despite the adverse drug effects the researchers tried

to adopt some hypoglycemic agents of plant origin particularly in the developing

countries. (10) It has been noted that spices play a major role in the

management of diabetes and its complications, and also used as an herbal

remedy. (6,11)

Cinnamon is one of such spices having a long history of use as spice,

preservative, medicinal use and flavoring additive, (12) and is associated with

increased risk of various ailments. (13) Cinnamon is a small evergreen tree

belonging to the family Lauraceae. It has two varieties Cinnamomum cassia and

Cinnamomum zylanicum having both in vitro and in vivo anti-diabetic effect. (14)

Aliskiren is the first agent in a new class of orally effective direct rennin inhibitors

28

approved for hypertension treatment. (15) In contrast to conventional RAS

blockers angiotensin- converting enzyme (ACE) inhibitors and Angiotensin II type

1 receptor blockers (ARBs), Aliskerin blocks RAAS by directly inhibiting plasma

rennin activity and preventing the formation of both Angiotensin I and Angiotensin

II as demonstrated by basic and clinical findings. (16, 17) Data from the AVOID

trial suggest that the addition of Aliskerin to an ARB provides an additive anti-

proteinuric effect compared to that of the ARB alone. (18) From the ALTITUDE

study (19), the potential cardiorenal benefits and safety of Aliskerin in a broad

range of high-risk patient with TD2M remain controversial.

Dipeptidyl Peptidase-IV (DPP-IV), also known as adenosine deaminase

complexing protein 2, degrades both glicentin-like peptide (GLP) and glucose

dependent insulinotropic peptide (GIP) to metabolites which are physiologically

inert. Competitive inhibition of DPP- IV by drugs increases the half-life of GLP

and GIP. The available DPP- IV inhibitors include Sitagliptin, linagliptin, alogliptin,

and vildgliptin. The first four are approved in the USA for the treatment of T2DM,

while vildagliptin has been approved for use in Europe and Latin-America.

Several other agents of DDP-IV inhibitors are in phase III clinical trials and

include dutogliptin and gemigliptin. With daily doses ranging from 100mg for

Sitagliptin to 5mg for saxagliptin and linagliptin, the drugs are all similar in their

efficiency in lowering Hb A1c levels, safety profile, and patient tolerance, (20)

DPP –IV inhibitors result in a mean decrease in HbA1C ranging between 0.5%

and 1%.(21). The present study is proposed to study the effects of Cinnamomum

cassia bark extract on glucose homeostasis in comparison to sitagliptin and

aliskerin in alloxan induced diabetic rat model.

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2. OBJECTIVES OF STUDY

To determine effects of cinnamon cassia bark extract (CCBE) on glucose


To compare the effect of CCBE vs. Sitagliptin and Aliskerin on glucose


30

To evaluate the anti oxidant activity of CCBE in alloxan induced rats

To compare the anti oxidant effect of CCBE vs. aliskerin and sitagliptin in

alloxan induced diabetic rats.

To observe the protective effects of C. cassia on the histology of Islets of

Langerhans of Pancreas in comparison to aliskerin and sitagliptin in alloxan


3. RATIONALE OF STUDY

Currently, the Pakistan is suffering a serious epidemic of DM, with millions

of diagnosed cases of DM and thousands of new cases. Cinnamomum cassia in

comparison with the present study is intended to investigate the effect of direct

rennin Inhibitor (Aliskiren) and Dipeptidyl-peptidase IV inhibitor (Sitagliptin), on

31

the glucose metabolism, physiology and cellular effects at the level of beta cell

pancreas in alloxan induced diabetic rat model.

The rational of study is to develop a therapeutic approach for managing

glucose homeostasis by repairing the injury of endocrine pancreas with the help

of Cinnamomum cassia (Herb) in comparison with direct rennin inhibitor

(Aliskiren) and Dipeptidyl-peptidase IV inhibitor (Sitagliptin) in alloxan induced

diabetic rat model.

4. HYPOTHESIS

The comparison between Cinnamomum cassia with direct rennin

(Aliskiren) and Dipeptidyl peptidase- IV inhibitors in glucose homeostasis will be

studied

32

Null Hypothesis: Cinnamomum cassia does not have anti diabetic effect on

alloxan induced diabetic rats.

Alternative Hypothesis: Cinnamomum cassia has anti diabetic effect in alloxan


CHAPTER II

LITERATURE REVIEW

1. PANCREAS

33

The pancreas is a retroperitoneal organ which lies horizontally extending

from C loop of duodenum to the hilum of spleen. Anatomically, pancreas is

divisible into head, neck, body and tail. The head of pancreas lies sandwiched in

the C loop of duodenum, posterior to hanging transverse colon. The anterior

surface of body of pancreas is covered by peritoneum. The tail lies in lieno -renal

ligament of spleen. (22). Physiologically, pancreas has exocrine and endocrine

parts, serves two major functions:

(i) Exocrine part: comprises of 85% of pancreatic tissue, produces digestive juice

rich in enzymes. The digestive juice is secreted by acinar cells. The pancreatic

digestive juice enters second part of duodenum through duct; and

(ii) Endocrine part: The blood regulation of glucose level, which is achieved by

endocrine cells of the islets of Langerhans.

Embryologically, pancreas is derived from a ventral and dorsal pancreatic

bud at the junction of fore and mid gut. The ventral bud gives rise to head and

uncinate process of pancreas, while dorsal bud becomes pancreatic body and

tail. Pancreatic ventral bud has a separate duct called “duct of Wirsung” and

“duct of Santorini” is that of dorsal bud in adult life. (22).

1.1. βeta cell Biology- Islets of Langerhans

The pancreas contains around 1 million of Islet of Langerhans in a

normal adult. The size of Islet of Langerhans varies from 40-900µm. The larger

Islet of Langerhans lie very close to arteriole and venules, whilst smaller one lie

embedded deep within pancreatic core tissue. Most of the Islet of Langerhans

contain 300-4000 cells of five major types;

i. α-(alpha) cells: secrete glucagon, an antagonistic hormone of insulin.

34

ii. β-(beta) cells: produce insulin which is the main blood glucose regulating

hormone of endocrine pancreas.

iii. β-(beta) cells: release amylin, which counter regulates the insulin secretion

and functions.

iv. β-(beta) cells: secrete pancreastatin. The pancreastatin decreases release

of insulin and somatostatin, increases release of glucagon, and inhibits the

exocrine secretions.

v. δ- (delta) cells: produce somatostatin. The somatostatin is general

inhibitory hormone. It inhibits secretion of gut and endocrine pancreas.

vi. ε- (Epsilon) cells: secrete Gherlin hormone that both release and augment

physiological functions of insulin. (22)

35

Figure. II-1. Physiological anatomy of Pancreas, duct system and Islets of

Pancreas. Adapted from: Samikannu, (23)

2. PHYSIOLOGY OF GLUCOSE HOMEOSTASIS

36

Blood glucose varies throughout 24 hours. It is affected by multiple factors

like tissue metabolism, glucose supply and hormonal factors. Normal blood

glucose in post-prandial period peaks to 200 mg/dl and in fasting is up to 100

mg/day. Thus blood glucose level is maintained by various physiological factors

within a narrow range defined as normoglycemia.

Normoglycemia is tightly controlled by co- and counter regulatory

mechanisms of neural and hormonal systems. Blood glucose of ≤20mg/dl

produces complete inhibition of insulin release, and stimulates counter regulatory

hyperglycemic hormone called the glucagon.

Central gluco-receptors of hypothalamus are regulated by blood glucose

level, thus hypothalamus plays a central role in glucose homeostasis. Whenever,

blood glucose declines, the counter regulatory mechanisms come into action and

include;

Sympathetic nerve stimulation

Anti-insulin hormones

o Catecholamine

o Glucagon

o Cortisol

o Growth hormone—etc.

Counter regulatory mechanism maintain a normal glucose homeostasis by

counter regulating the insulin. Thus there is balance between insulin and anti -

insulin hormones to maintain a normal balance of blood glucose. (24)

2.1. Source of Blood Glucose:

37

Diet is the most important sole source of blood glucose. But during inter-

digestive phases, the liver in particular plays major role by i ts glycogen contents.

Liver maintains normoglycemia through;

Glycogenolysis: is defined as the lysis of liver glycogen.

Gluconeogenesis: is the synthesis of de-novo glucose from non-carbohydrate

sources such as pyruvate, lactate and glycerol etc.(24, 25).

38

Figure. II-2. Utilization of blood glucose by different tissues and organs shown as

micromole of glucose. Adapted from SHRAYYEF, et al(24)

2.2. Fate of Blood Glucose level

39

Blood glucose is utilized by various body cells, tissues, organs and

systems. The inter-digestive periods are essential as the external sources of food

are not available. Some body organs like liver perform functions of maintaining

blood glucose homeostasis. Disposal of glucose occurs through a number of

limited metabolic pathways, as below;

Glucose is metabolized by glycolysis, Krebs cycle and oxidative

phosphorylation. During aerobic conditions final product of glucose is

pyruvate, while under anaerobic conditions lactate is the final product. But

lactate may be converted into pyruvate once oxygen is available.

Glucose is broken down by glycolysis and other pathways to release energy,

which is then captured in form of high energy phosphate bonds like ATP

(adenosine triphosphate).

Lactate and pyruvate may enter various cycles to yield energy.

(24-26).

The fate of blood glucose level is shown in figure II-3.

Figure. II-3. Fate of glucose metabolism. Adapted from SHRAYYEF, et al (24)

2.3. Significance of Glucose Homeostasis

40

Blood glucose is an obligatory food for the extraction of energy by all cells

of human body. Certain organs are completely glucose dependent and insulin

independent like;

Neurons

Kidneys – medullary part

Red blood cells

Retina

Schwann cells

Enterocytes

The above tissues are affected at the most during hypo- and/or

hyperglycemic conditions. With prolonged starvation, ketone bodies are produced

by liver and released into blood. Ketone bodies cross blood brain barrier and are

utilized by neurons also.

Blood glucose levels <55 mg/dl disturbs brain physiology. Glucose levels

<20 mg/dl may result in permanent damage of various parts of brain. The

resulting conditions are coma, convulsions, brain damage, amnesia, and death.

(24-29).

2.4. Factors Regulating Glucose Homeostasis

41

Neurohormonal factors are involved in the blood glucose homeostasis.

There is balance between hypoglycemic hormone, insulin vs. hyperglycemic

hormones (anti-insulin hormones) as glucagon, cortisol, growth hormone,

catecholamine and thyroxine, etc. (25, 27, 28-30)

Physiological effects of various hormones on glucose homeostasis are

described as under:

2.4.1. Insulin

Insulin is a peptide hormone comprising of 51 amino acids, arranged in

two chains. It is secreted by the β- cell of “Islet of Langerhans” of Pancreas.

Insulin is called as the “hormone of abundance” because of availability of

food is handled by it. When food reaches in stomach, the insulin secretion and

synthesis are switched on.

Insulin stores the food in reservoirs like liver, skeletal muscle and adipose

tissue.

Insulin functions through its membrane receptor. Its receptor shows tyrosine

kinase activity.

Glucose transporters are translated and inserted in cell membrane, which

internalize the blood glucose into intracellular compartment.

Insulin exerts following effects on the blood glucose level;

o Transport of blood glucose into cell

o Glycogenolysis is inhibited in liver.

o Glycogen synthesis is stimulated

o Gluconeogenesis is inhibited

o Lipogenesis stimulation

42

o Lipolysis inhibition

Physiological effects of insulin are shown in table II.

Insulin release is increased by 3-4 times after meal intake. On the contrary,

blood glucose <50mg/dl causes a 80-90% reduction in insulin release.

Carbohydrates, fat and protein meals equally stimulate insulin release. Food

intake stimulates gut related local hormones, known as incretins, which circulate

through local blood vessels and stimulate insulin release.

(24, 28-31)

2.4.2. Glucagon

Glucagon is synthesized and secreted by the α-cells of Islets of

Langerhans.

Glucagon is anti-insulin hormone

It is peptide in structure i.e. composed of amino acids.

Most potent stimulus is hypoglycemia

Many other also regulate blood glucose

Glucagon receptors are present on cell membrane, and associated with

adenylyl cyclase.

Glucagon stimulates glycogenolysis

Stimulates gluconeogenesis

Stimulates Lipolysis

(24, 25, 30, 32)

2.4.3. Catecholamine

43

Catecholamines are potent anti-insulin hormones, which include

epinephrine, nor-epinephrine and dopamine. Catecholamines are released from

adrenal medulla. They are the anti-insulin hormones released in response to

hypoglycemia. Catecholamine increase blood glucose through

Glycogenolysis

Stimulates gluconeogenesis

Stimulates Lipolysis

Add free glucose to blood vessels.

(24, 25, 28-30, 31)

2.4.4. Cortisol

Cortisol is a steroid hormone synthesized and secreted by adrenal cortex.

Hypoglycemia is a potent stimulus for Cortisol release

Cortisol stimulates glycogenolysis

Stimulates gluconeogenesis.

(24, 25, 28-30, 31)

2.4.5. Growth hormone

Anterior pituitary glands produce growth hormone in response to

hypoglycemia. Growth hormone stimulates glycogenolysis and gluconeogenesis.

(24, 25, 28, 29, 32)

44

3. INCRETIN HORMONES

3.1. The Entero-Insular Axis

Food intake causes release of certain factors from intestinal mucosa which

stimulate endocrine pancreas to release insulin which was first conceptualized to

explain that the oral glucose stimulates more insulin release compared to

parenteral glucose load. Such gut related factors were collectively called as

incretin. (24)

3.2. History of incretins

Bayliss and Starling (1902) first time presented concept that a hormone

(secretin) released from intestinal mucosa stimulates the exocrine pancreas.

Intestinal mucosa extract was administered in patients with type 1 DM, but failed

to improve blood glucose homeostasis.

La Barre (1932) introduced the term ‘incretin’ for factors extracted from the

upper gut mucosa, which caused hypoglycemia. A proposal for therapeutic role of

incretin for DM was introduced by him.

The studies of Leow et al. (1939) concluded the existence of incretins as

“suspicious”. This was followed by a silent gap for incretins for 30 years till 1970.

With the advancement in molecular biology after 70s, the incretin

hypothesis was researched aggressively. Finally, it was proved that the incretins

do exist which revolutionized the DM treatment. (33-35)

3.3. The Incretin System

45

In response to food intake, the gut related L and K cells of intestinal

mucosa release incretin hormones. Incretins stimulate secretion of insulin in

presence of food in intestinal lumen. The insulin becomes available even before

glucose enters into blood commonly called feed forward mechanism, but the

events occur in glucose dependent manner so that no risk of hypoglycemia does

occur.

Incretins regulate blood glucose level through multiple targets; they slow

gastric emptying, inhibit glucagon secretion, reduce satiety and aid in losing

weight. At present two bio molecules are termed under umbrella of incretins;

Glucagon-Like Polypeptide-1 (GLP-1) and

Gastric Inhibitory Peptide (GIP).

It is reported that type 2 DM subjects with controlled glycaemic status

exhibit an effective incretin-effect in response to glucose load compared with

normal healthy counterparts as shown in II-5. (33-36)

Exogenous administration of GLP-1 increases insulin release sufficient to

normalize plasma glucose levels, while GIP in supra physiological doses, exhibits

reduced insulinotropic effect with no effect on blood glucose levels. Therefore,

therapeutic strategies for type 2 diabetes mellitus are focused on the use of GLP-

1 analogues but not with GIP, but the drawback of GLP-1 is its short half life of

just 2 minutes, because it is rapidly cleared from blood by the enzyme Dipetidyl

Peptidase-4 (DPP-4). Recently, synthetic DPP-4 resistant incretins are available

by sophisticated biological techniques. (33-36)

For the glucose lowering potential of incretins, some intact β-cells must be

present to produce glycaemic control; therefore incretins are useful in type 2 DM,

but not in type 1 DM. The negative results of incretins on blood glucose levels

during 1920-1940 were due to the fact that they were used in type 1 DM having

46

no β-cells mass at all. Therefore, one of important therapeutic options was

missed. The GLP-1 mimetic agents resistant to DPP-4 are available with long half

life and effective glycaemic effects. The two GLP-1 mimetics introduced in clinical

practice include the; Exenatide and Liraglutide.

Exenatide, was first introduced as new class of drug in United States in

2005 but in 2007 in Europe. Liraglutide was approved for drug use in Europe in

2009 and in the Japan and United States in 2010. (33-36)

47

Figure. II-4. Incretin effect in normal healthy and diabetes mellitus. Adapted

From SEEWODHRY, et al. (33)

48

Figure. II-5. Incretin effect in normal healthy and diabetes mellitus.

Adapted from SHRAYYEF, et al. (24)

3.4. GLP-1 and β Cell Proliferation

49

GLP-1 over expresses homeobox gene-1 (PDX-1) of the β-cell-specific

transcription factor related to pancreas and duodenum. The gene is implicated in

the expression of insulin gene, glucokinase gene, GLUT2 gene, and also

differentiation of β-cell. The incretins increases β -cell proliferation in in-vitro

experiments via phosphatidyl-inositol (PI)-3-kinase and protein kinase C signaling

interactions.

GLP-1 induces genes and proto-oncogene’s which are implicated in the

cell apoptosis and cell growth like; junD, c-jun, nur77 and c-fos. GLP-1 causes

induction of DNA synthesis as detected by incorporation of tritiated thymidine

incorporation in insulinoma and also in islets of Langerhans in rat model.

Exenadin-4 is a long acting GLP-1 agonist. Exenadin-4 induces β-cells

proliferations and β-cells neogenesis from duct progenitor cells of pancreas type

2 diabetic rat model. (33, 36-41).

3.5. GLP-1 and β cell Neogenesis

The de-novo development of islets of Langerhans cells and β-cells

neogenesis in adult pancreas are highly controversial reports, but evidence

based conclusions showed it happening, especially in transgenic mice and rodent

models.

The In-vivo models have demonstrated that the ductile cells are capable of

giving rise to new β-cells and also the interferon gamma (IFN-γ) over expression.

Wrapping of pancreatic duct with plastic sheaths in order to induce inflammation

with concomitant administration of EGF and gastrin is reported to induce β-cells

proliferation. The β-cells ductile origin from in-situ progenitor cells during acute

pancreatic injury has produced controversial results, as it has been reported in

previous studies. The controversy is that the pancreatic ductile cells whether

50

contribute or not is not proved and is yet controversial hence remains to be

elucidated. (42-46)

3.6. Dipeptidyl Peptidase IV (DPP -IV)

The DPP IV is a 110 KD cell surface glycoprotein. DPP-IV is a serine-

protease which cleaves 2- amino acids from small peptides containing alanine or

proline from N-terminal end. This enzyme, also known as the T-cell antigen

CD26, is found in many locations including intestinal and renal brush-border

membranes, on hepatocytes and capillary endothelial cells and in a soluble form

in plasma. Although its specificity suggests a role of the metabolism of many

endogenous peptides, it seems as DPP IV activity is especially critical for

inactivation of GLP-1. The in vivo N-terminal truncation of both endogenous and

exogenous GLP-1 was likely to have a physiological role in mediating the rapid

degradation of native GLP-1. The peptide has an apparent plasma half-life of

approximately 1–2 minutes and clearance rate is 5–10 L/min, exceeding the

cardiac output by a factor of 2 to 3.

ROUTE OF EXRETION OF GLP. In patients with type 2 diabetes, studies

suggest a correlation between glycemic control and plasma DPP IV activity, but

this does not seem to be correlated to the amount of intact biologically active

hormone in plasma. Targeted disruption of the CD26 gene had increased levels

of intact endogenous GLP-1 supporting the importance of DPP IV in GLP-1

metabolism. DPP IV inhibition was able to completely prevent the N-terminal

degradation of native GLP-1 in vivo and this was also associated with

enhancement of its insulinotropic effects.(38, 47-52)

3.7. Dipeptidyl Peptidase Inhibitor

51

Dipeptidyl peptidase-IV inhibitors completely block the DPP-IV enzyme.

Thus elevate endogenous GLP-1 level and exaggerate the incretin action. DPP-

IV inhibitors are novel blood glucose lowering agents taken orally. DPP-IV

inhibitors may be used as monotherapy or combined with some other antidiabetic

compounds such as sulfonylureas, metformin, and thiazolidinediones. Sitagliptin,

vildagliptin and saxagliptin are already on sale. Other family members such as

linagliptin and alogliptin are in final phases of clinical trials. The proliferative and

anti-apoptotic actions of GLP-1 on islet β-cells have been mirrored by studies

using DPP-IV inhibitors in rodents with induced diabetes mellitus. Mice model on

high fatty diet, treated with streptozotocin (low dose) followed by 2–3 months of

treatment with des-fluoro-sitagliptin showed improvement in fasting and

postprandial blood glucose, increased insulin, and increased numbers of insulin-

positive β-cells in association with normalization of β -cell mass and restoration

toward normal of the β-cell: β-cell ratio. (38, 48-52).

4. RENIN ANGIOTENSIN ALDOSTERONE SYSTEM (RAAS)

4.1. History of Discovery

The story of the discovery of the RAS began more than 100 years ago

(1896). A Finnish Professor of Physiology, Robert A. Tigerstedt (1853–1923), at

the Karolinska Institute in Stockholm, and Per Gustav Bergman, a medical

student, performed a new experiment in rabbit model. Both were inspired by

Charles Dourad Brown Sequard, who was famous for discovering inner

secretions of organs; like injecting extracts of animal organs into experimental

animals. Tigerstedt and Bergman prepared extract of rabbit kidney (donor) and

injected its cold extract into the jugular vein of recipient rabbit model. They

52

observed a consistent rise in systemic blood pressure. It was concluded that the

kidney tissue extract might contained a pressor molecule hence they termed it as

Renin. They further proved that the Renin pressor substance was located in the

cortex of kidney, and that the pressor effect was i ndependent of nervous reflexes.

(Tigerstedt and Bergman 1898). Intriguingly, these observations remain obscured

and forgotten for many years.

Years later, H. Goldblatt (1934) performed experiments on dogs by

clipping one or both kidney arteries. He proved the possibility of pressor

substance is being secreted by kidneys because clipping renal arteries produced

severe hypertension. This was followed by experiments in two laboratories at

Indianapolis and Argentina by scientists. Their experiments proved that the Renin

is produced by kidney but itself is not pressor substance, but rather an enzyme

which converts some other proteins to release pressor peptides which increase

blood pressure. This new agent was termed as angiotensinogen released from

liver. (53-56)

4.2. Synthesis of Circulating Renin

The human genome contains only one copy renin gene called Ren-1c.

However, certain mice strains have two distinct renin genes labelled as Ren-1d

and Ren-2.

Human Ren-1c renin gene expression varies in different organ- systems

of human body. But the kidneys are naturally expressing the gene product, the

renin, in large quantities which is readily releasable into blood capillaries. It is

proved from animal experiments that complete bilateral removal of kidneys leads

to undetectable amounts of renin in the blood plasma. At present, it is known that

the Renin is produced by special cells called juxtaglomerular cells (JG) cells

which are specialized vascular smooth muscle cells located in afferent arteriole

53

near the glomeruli of nephrons. A low salt diet causes increased release of renin

from afferent arterioles. Similar to other hormones, first pre-pro-renin is

synthesized by renin mRNA, which is transported to rough ER. The pre- peptide

is cleaved in rough ER, pro- peptide is cleaved in Golgi apparatus, where renin is

glycosylated with mannose 6 phosphate and deposited in granules. Upon

stimulation, active renin, which is a 40,000-Da single-chain polypeptide enzyme,

is secreted into blood circulation. However, small amounts are secreted into

renal interstitium also.

The JG apparatus plays a major role in regulation and secretion of renin

release. The JG apparatus comprises of glomerular mesangial cells, JG granular

cells of vascular smooth muscles of afferent arterioles and macula densa of distal

part of renal tubules respectively. (28, 29, 54-59)

Three classical stimuli, all elicited by a decline in systemic blood pressure

or alteration in blood volume, which are known to increase renin synthesis and

release as follows:

4.2.1. Decreased stretch of the afferent arteriole:

The granular JG cells of afferent arteriole are very sensitive to vascular

stretch. It is a local effect which is not driven by any neural reflex input.

An increase in JG cell stretch occurs in response to increased systemic

blood pressure, which in turn, increases intracellular calcium. The calcium in turn

increases vascular tone and result is reduced renin release. On the contrary, as

the systemic blood pressure declines, it reduces vascular stretch and hence JG

cells, which become stimulated resulting in release of large quantities of renin into

circulation. (28, 29, 54-59)

54

4.2.2. Decreased delivery of salt (sodium chloride)

to the macula densa:

Macula densa are specialized chemo

sensitive cells located in the distal convoluted tubule (DCT) lying in the

angle of glomerulus of same nephrons. The macula densa functions through the

tubuloglomerular feedback. The tubuloglomerular feedback helps in maintaining

GFR at a steady state. A reduced load of salt in DCT is sensed by macula densa.

This message is conveyed to afferent arteriole and mesangial cells of glomerulus.

The messengers are the ATP, adenosine, NO and increased synthesis of PGE2.

This local chemical modulation stimulates the JG cells of afferent arteriole, to

synthesize and release renin in the circulation. (28, 29, 54-59)

4.2.3. Adrenergic stimulation.

The JG cells are innervated by sympathetic nerve fibers. The sympathetic

nerve endings release neurotransmitter which act on JG cells through β1-

adrenergic receptors. The β1-adrenergic receptors are expressed on the surface

of the JG cells. Stimulated β1-adrenergic receptors increase intracellular cyclic

adenosine monophosphate (cAMP) which stimulates renin release. Sympathetic

nerve fibers of kidney are very potent in action and in stimulating release of renin

and this occurs in response to acute sodium changes and diameters of renal

blood vessels i.e., renal vasoconstriction and vasodilatation. (28, 29, 54-59)

4.3. Physiology of Renin angiotensin aldosterone system (RAAS)

The RAAS was slowly and gradually elucidated as shown in Figure II-6, II-

7 and II-8. In response to stimuli as mentioned above, renin is released from

kidney, and is a proteolytic enzyme. It acts on its substrate, angiotensiongen

55

(AGT). The AGT is a circulating protein (α-2 globulins fraction). The renin cleaves

angiotensiongen into Angiotensin-I (Ang I), which is a decapeptide, containing 10

amino acids. Angiotensin I is biologically inert and is converted into Angiotensin-II

(Ang II) by the action of enzyme. The conversion of angiotensin I to II is mediated

through catalytic action of enzymes called Angiotensin Converting Enzyme

(ACE). Angiotensin-II is an octapeptide containing 8 amino acids. The ACE is

present on the vascular endothelial cell surfaces on whole body, but in particular

the pulmonary blood vessels. (Figure II-6, II-7 and II-8). Angiotensin-II is

biologically active hormones which mediates its physiological actions through

angiotensin receptors. (28, 29, 54-58)

Details are given as under:

4.3.1. Alternative enzymatic pathways are present and are able to convert Ang I and Ang

II (Figure II-6, II-7 and II-8).

Tonin and Cathepsin D are alternative enzymatic agents of renin capable

of converting Ang I into Ang II. Similarly alternative of ACE do exists, and include

trypsin, cathepsins G and heart chymase, which are capable of performing

function similar to ACE. However, the contribution of these alternative pathways

in Ang II production in humans remains ambiguous and needs to be elucidated.

4.3.2. ACE can act on substrates other than Ang I.

The ACE is also capable of degrading substance P, bradykinin and other

small peptides. The physiological role of this enzymatic conversion is not

understood clearly, but it is proved that the ACE inhibition leads to the

accumulation of these substances;

Accumulation of substance P, bradykinin and other small peptides may be

responsible for some of the beneficial effects such as antihypertensive, but may

56

exert adverse effects like angioedema, cough etc similar to ACE inhibitors. (28,

29, 54-58)

4.3.3. Angiotensin II can be converted to

angiotensin III and IV

There are several angiotensin peptide0s with biological effects (Figure II-6,

II-7 and II-8). Angiotensin II may be converted to Ang III [angiotensin-(2-8)] and

Ang IV [angiotensin-(3-8)], respectively. Ang III and IV is suggested of playing

role in the brain, whereas angiotensin-(1-7) has vasodepressor properties.

Angiotensin-(1-7) may be formed directly from Ang I. Vasodepressor activity of

Angiotensin-(1-7) may augment the antihypertensive activity of ACE inhibitor

drugs. (28, 29, 54-58)

4.3.4. Components of the RAAS also reside

within local tissues.

As discussed, most of renin comes from kidney and angiotensiongen

(AGT) comes from liver, but it is also well understood that Renin, AGT and ACE

are expressed in various tissues locally. The local Renin, AGT and ACE system is

known as Tissue (local) RAAS.

Tissue RAAS has been found in heart, brain, gut, pancreas, adrenals,

reproductive organs, blood vessels, renal interstitium and adipose tissue, etc.

Brain and Intrarenal RAAS contribute in salt, water and systemic blood pressure

regulation, whereas, vascular and cardiac RAAS are involved in the

cardiovascular pathophysiology. (28, 29, 54-58)

57

4.3.5. Angiotensin (AT) Receptors

AT-1 and AT-2 are the two main receptor isoforms as detected in rodent

models. AT1 and the AT2 receptors have been cloned and belong to G protein

coupled receptor super family. The AT-1 receptor, in turn, is divided into AT1a

and AT1b. AT4R and AT1-7R and intracellular receptors are some other forms of

receptors.

AT1 receptor: mediates classical physiological functions of Ang II, such as,

vasoconstriction, salt and water retention, cell growth & proliferation. AT1

receptors can be selectively sartans drugs.

AT2 receptors: Fetal tissue expresses AT2 receptors, however, AT2 receptors

are observed at the site of tissue injury. AT2 receptors mediate apoptosis, cell

differentiation, inhibition of cell growth and vasodilatation. (28, 29, 54-58)

58

Figure. II-6. Release of renin and stimulation angiotensinogen into Antgiotenis I and II. Adapted from: SHRAYYEF, et al. (24)

59

Figure. II-7. Release of renin and stimulation angiotensinogen into

antgiotenis I and II. Adapted from SHRAYYEF, et al (24)

60

Figure. II-8. Physiological effects of Renin angiotensin aldosterone

system. Adapted from SHRAYYEF, et al. (24)

61

4.4. Direct Renin Inhibitors- Aliskerin

4.4.1. Aliskerin

Aliskerin is a new class of anti hypertensive drugs. It is first in a class of

drugs labelled as Direct Renin Inhibitors (DRI). Currently, it is licensed for

Primary/Essential Hypertension. In the market, it is available with the trade name

of Rasilez, UK and Tekturna, US. (60)

Aliskerin

Figure. II-9. Physiological effects of Renin angiotensin aldosterone

system.

Adapted from: GRADMAN, et al. (60)

4.4.2. Mechanism of action of Aliskerin

Aliskerin binds to renin at the S3bp site. This site is essential for

physiological activity of renin. Binding to this pocket, S3bp site, makes the renin

action blocked. (60).

http://en.wikipedia.org/wiki/File:Aliskiren_Structural_Formulae_V.1.svg

62

4.4.3. Aliskerin was co-developed By Swiss

Pharmaceuticals

Novartis and Speedel are two Swiss Pharmaceutical companies which co-

developed a direct renin inhibitor, called aliskerin. US-FDA approved the

Aliskerin for the primary hypertension in 2007. (60)

In a clinical trial by Novartis, an increased incidence of hypotension,

hyperkalemia, renal problems and non-fatal stroke was observed in diabetic

patients. Then a new contraindication was added to A liskerin, that it must not be

used with angiotensin receptor blockers (ARBs) and angiotensin converting

enzyme inhibitors (ACEIs) in diabetics. (60-62) A new warning to avoid Aliskerin

with ARBs and ACEIs in patients with moderate to severe renal impairment with

glomerular filtration rate less than 60 ml/min. (63-64)

5. CINNAMOMUM CASSIA

5.1. Overview

Cassia is a member of plant family known as Cinnamon. It is also known

as "Java cinnamon", Saigon cinnamon”, "Padang cassia" or as the "Chinese

cinnamon". (Figure II-10 and II-11)

In botanical taxonomy, the Cassia is termed as C. cassia blume, C. cassia,

C. aromaticum (nees) syn, C. burmannii, C burmannii blume, C. obtusifolia, C.

cassia (nees) ex blume, C. tora and C. loureini nees. (65, 66)

It is publicly known fact that the Cinnamon is primarily used as herbal

spice, taste enhancer, and food additive, and to some extent in liquors

preparation. Coumarins are a natural component of cassia which belongs to

63

benzopyrene family. The coumarins are present in sufficient quantities in cassia

which may be health hazardous. But it is well known fact that the Ceylon

cinnamon contains little amounts of coumarins and is thus free of health hazards.

Cassia and cinnamon vary too much in their biochemical composition. Ceylon

cinnamon is rich in benzyl-benzoate and eugenol but lacks coumarin. Coumarins

are present in Bark of cassia considerably. However, amount depends upon

species, sub-species of cinnamon and more over climate conditions. (67-69)

64

Figure. II-10. Cinnamomum cassia. Adapted from; www.wikipedia.com

/accessed/15thmarch2014

65

Figure. II-11. Cinnamomum cassia. Adapted from; www.wikipedia.com

/accessed/15thmarch2014

66

5.2. Historical Perspectives of C. cassia as medicine

The use of C. cassia as medicine by human beings dates back since

centuries. In the Ancient Egypt, the embalming fluids were mixed with C. cassia

as spicy ingredients. In the Ayuverdic medicine, the C. cassia had been used as

anti-nauseating, anti flatulent and anti diarrheal agent.

In the early 16th century, Portuguese found the Sri-Lankan cinnamon trees.

They started exporting C.Cassia to Europe for nearly two centuries. Later on,

Dutch occupied Sri Lanka in mid of 17th century and also exported C. Cassia.

Subsequently, the British (1796) made East India Company in the Indian

subcontinent. The East India Company became the major exporter of C. cassia to

Europe. Undoubtedly, Sri Lanka is the sole supplier of C. cassia bark and oils

throughout the World till present. (65, 69, 70)

The oil and bark of both Ceylon and Chinese cinnamon are used by

Pharma industry. However the food industry prefers use of Ceylon cinnamon.

China has already become one of the main cinnamons exporting country in the

World next to Sri Lanka. (65, 69, 70)

From historical perspective, cinnamon is mentioned by Greeks,

Dioskurides, Plinius, Galenu, Romans, Herodotus and Theophrastus. It is also

quoted in Bible. Dioskurides mentioned 5 cassia and 7 species of cinnamon, it

was used as digestive and diuretic agent.

In Europe, cinnamon was introduced in 8th century. It was most expensive

spice herb. It was used by Kings and Popes only. The first written manuscript

detailed from an Arabian source as medicinal remedy. (65, 69, 70)

67

Cinnamon trees do existed in Cyelon as earlier as 1310 A.D as mentioned

by Johannes of Montevino. Nicolo Conit reported a real description of cinnamon

tree about one century later. A more authentic real description of cinnamon tree is

reported by Nicolo Conit about one century later. Mr Holland (1756) was the first

to cultivate cinnamon trees.

Mr. Holland was first who cultivated the cinnamon trees in the cultivation

areas in 1765. As the production was increased by Holland, similar was its export

to Europe at that time. After capture of Sri Lanka (1796) by East India Company,

then trading and export was monopolized by that company. Mr. Holland also

started cultivating cinnamon in Java and Sumatra, since then cinnamon was used

as cardiac tonic (1833).

Cinnamon oil was first extracted by St Amando of Doornyk by the end of

15th century. Cinnamon oil was used as a herbal medicine in nerve sensitivity

diseases. Higher doses of oil were used as nerve, uterus, muscle and cardiac

stimulant. Side effects of higher doses of Oil were also reported at that time as it

might cause methaemoglobinaemia and renal disease. It was also reported that

oil exhibited anti-bacterial effects against typhus ailment. (65, 69, 70)

5.3. Cinnamomum cassia used as Traditional Medicine

In traditional herbal medicine, the cinnamon was used as stomachic agent

and nerve tonic. Cinnamon was used for treatment of vomiting, gastro

esophageal reflux, hyperacidity, diarrhea, dyspepsia and abdominal bloating. (73)

The cinnamon was used as a germicidal agent, antispasmodic and astringent

agent. The chronic bronchitis, rheumatic pain, impotence, injuries, eye disease,

gynecological problems, leucorrhoea, toothache, and dyspnea etc, were treated

with cinnamon. (74-76)

68

Cinnamon relieved dyspepsia and its symptoms as that flatulence, gut

spasms, and gut bloating. Similarly it was used as appetite stimulant and treating

diarrheal disease. (77-79)

In folk medicine, the cinnamon is used as a topical agent for wound

healing. And also used for treating diarrhea, dyspepsia, cold and flu. Use for

treating diarrhea and dyspepsia is well documented. (78-79). Drops of cinnamon

oils were considered remedial for painful menses and for bleeding cessation. The

essential oil as cinnamon drops was good remedy for dysmenorrhoea and

menorrhagia. (67, 78, 79)

5.4. Adult Posology and Mode of administration

Spirit: is prepared as 1:10 parts of oil in 90% ethanol, and administered 0.3 to

1.2 ml per day in 3 divided doses (80)

Tincture: is prepared as 1:5 parts of oil in 70% ethanol as solvent V/V, and given

as 2-4 ml per day in 3 divided doses. (81, 82)

Dried bark: is given orally as 1.5-4 grams per day or as infusion 0.5-1.0 gram in

3 divided doses per day. (78, 79)

Fluid extract: is prepared as 1:1 fluid extract in 70% ethanol as solvent V/V, and

given 3 times per day. (67, 79, 81)

Aetheroleum: is given orally in 3 divided doses, amounting about 50-200 mg per

day. (67, 79,83)

69

5.5. Non-clinical Pharmacological Data

In vitro tests

Bactericidal activity:

Essential oil of cinnamon was administered in in-vitro experiments to

bacteria and its effect was observed. Bactericidal activity was observed against

gram +ve bacteria (Bacillus subtilis and Staphylococcus aureus) and gram –ve

bacteria (K.pneumoniae, P. aeroginosa, P. vulgaris and E.coli). Very low MICs

(minimal inhibitory activity) concentrations were reported for bactericidal activity.

(70, 84)

The bactericidal activity was observed against Listeria monocytogenes.

The lowest minimal inhibitory activity (MIC) for cinnamon bark essential oil was

500 ppm. Such low MICs is an excellent bactericidal activity. (70, 84)

Fungicidal activity:

The cinnamaldehyde compounds of cinnamon essential oils showed

fungicidal activity. Cinnamaldehyde causes inhibition of fungi proliferation. It also

neutralizes its mycotoxins. The fungi included Aspergillus clavus, Aspergillus

parasiticus, Aspergillus niger, and Candida albicans. Dermatophytes are the skin

diseases related to fungi, are all inhibited by cinnamon oil extract. Fungicidal

inhibitory zone was 28 millimeter, which was less as compared to ketoconazole,

but the fungicidal activity was comparable. Aflatoxin synthesis is also inhibited by

Cinnamaldehyde. (72, 76).

70

Spermicidal activity:

The cinnamon oil component cinnamaldehyde exhibits sperm killing

potential. Sperm immobility and sperm cell death was observed at a minimum

concentration of 1:400 V/V. (85)

Inflammation soothing activity:

Inflammatory process is hampered by essential cinnamon oils, including

bark oils. The inflammation in rheumatoid disorders was also alleviated by

cinnamon oils; hence they were used for this purpose. The soothing effect was

attributed to irritant and pungent ingredients. Cinnamon oil also inhibits

prostaglandin synthesis. Inhibition of cyclo-oxygenase enzyme by eugenol of

cinnamon oils is already documented. (86)

Cytotoxic activity:

The hepatotoxicity of cinnamaldehyde is proved in rat models. The

underlying mechanism is the depletion of glutathione stores. The cinnamon

extracts of Petroleum ether (25:1) and chloroform (68:1) showed severe

cytotoxicity. ED50 values were found at 60 and 58 μg/ml in human cancer (KB)

cells. The ED50 values for mouse leukemia and L1210 cells were observed at 24

and 20 μg/ml respectively. (86, 87)

Anti-tumor activity:

Potent anti tumor activity is proved in in-vitro animal models. The

components of cinnamon include; Cinnamaldehyde (CA) and its two analogues;

the 2-hydroxycinnamaldehyde and 2-benzoyloxycinnamaldehyde. The analogues

are reported as chemotherapeutic and chemo-preventive agents. (69- 72)

71

Spasmolytic activity:

The Spasmolytic activity is observed in the ileum and tracheal smooth

muscles of guinea pig model. This effect was produced by the cinnamon bark oil

(EC50 41 mg/litre) and cinnamaldehyde which resembled to Papaverie like

Spasmolytic effects. The Spasmolytic effects were comparable to other agents.

Relaxing effects on ileal smooth muscle were more pronounced compared to

tracheal smooth muscle. The Cinnamaldehyde, Eugenol-acetate, and Eugenol

show Spasmolytic activity. Spasmolytic activity was comparable to isoprenaline

and phospho-di-esterase (PDE) inhibitors. (88)

Flatulence:

The effects of cinnamon oils were assessed on stomach and intestine

regarding relief of flatulence. The reduction in flatulence was comparable to

phenoxyethanol, p-hydroxybenzaldehyde, isobutanol, menthol, m-Cresol,

dimethicone, and silica. (78, 79)

In vivo tests

Anti-inflammatory activity:

The anti-inflammatory activity of cinnamaldehyde was observed on cotton

pellet induced chronic inflammation. In this experiment, dry ethanolic extract was

given orally at 400 mg/kg. An inflammation soothing effect was observed. It also

revealed anti-proliferative activity. (86)

72

Analgesic activity:

Dry ethanolic extract of Cinnamomum zeylanicum is reported in rats

exhibited a potent analgesic activity. Acetic acid induced and hot plate thermal

stimulation induced writhing tests revealed analgesic activity. A dose of 200-

400mg/kg was sufficient to produce analgesic activity. (71, 72)

Spasmolytic activity:

The cinnamaldehyde exerts a papaverine-like muscle relaxing activity,

which is mediated through vasodilating activity. Movement of mouse intestine

and stomach of rats were moderately inhibited by intravenous dose of 5-10

mg/kg. Cinnamaldehyde speeded up the healing of stress induced stomach

erosions. (70)

Cinnamaldehyde administered in the peritoneum of mice model reduced

gut peristalsis at a dose of 250mg/kg body weight. While in anesthetized rats, 5-

20 mg/kg given intravenously reduced the stomach motility. (69, 71, 72)

Bile Secretion:

Experimental rats showed increased bile flow when given 500 mg/kg body

weight cinnamaldehyde. The observed effect was excellent. (71, 72)

Nervous System:

Motor activity was inhibited by cinnamaldehyde at a dose of 250-

1000mg/kg when administered orally. Paradoxically, stimulation of central

nervous system has been reported in rabbits at dose of 10-20 mg/kg intra-

arterially. (71, 72).

73

Cardiovascular activity:

Dogs revealed stimulatory effect on cardiovascular system at intravenous

dose of 5 and 10 mg/kg body weight of cinnamaldehyde. A dose dependent blood

pressure lowering effect was also reported. (76)

5.6. Toxicology

5.6.1. Teratogenicity:

Teratogenicity of methanol extract of Cinnamon in rat models has not been

proved when administered orally. While in the chick embryos, cinnamaldehyde

showed 49% lethality and 58.2% malformations. Teratogenicity of chick embryo

was observed at its toxic dose of 0.5 mmol/ embryo. (65, 66, 71, 72, 76)

5.6.2. Reproductive organs:

The mice were fed on ethanolic extract of C. zeylanicum at doses of 100

mg/kg/day orally, it produced astonishing results. The weight of reproductive

organs was increased. Sperm count and motility was also increased. Sperm

toxicity has not been reported in earlier in-vitro studies. (85)

5.6.3. Carcinogenicity:

Inhibition of thioredoxin reductase has been observed by Cinnamaldehyde;

hence it can be used as chemotherapeutic and chemopreventive purpose. (71,

72)

5.6.4. Genotoxicity and Mutagenecity

Reports on Genotoxicity and Mutagenecity are conflicting. Some of

previous studies used Ames testing to detect mutagenic effects and it was

74

reported positively. Experiment on Chinese hamster using Cinnamaldehyde and

cinnamon bark oil produced chromosomal aberrations. Mutagenecity of ethanolic

extract of cinnamon by Ames test has also been reported. Drosophila testing

showed Genotoxicity of cinnamaldehyde, while aqueous extract of cinnamon

produced no such effect. Other previous studies have not reported of any

Mutagenecity with Cinnamaldehyde, cinnamon bark oil and cinnamon extracts

using Ames testing. Both rat and hamster liver microsomal preparations were

used for mutagenicity detection, but the trans-cinnamic acid and trans-

cinnamaldehyde produced no any mutagenic effect in salmonella typhimurium

strains. (71, 72)

5.6.5. Acute toxicity:

Severe skin irritation of rabbits was observed with undiluted cinnamon bark

oil, however, very mild irritation was observed on hairless skin of mice model.

Cinnamon oil mixed with petroleum was applied to skin of 25 volunteer human

beings, revealed no skin irritation. The dermal lethal dose in rats is determined at

0.59 mg per kg, while oral lethal dose of cinnamaldehyde in rats is estimated at

2.22 g/kg. Oral lethal dose of cinnamon bark oil is estimated at 4.16 g/kg while

dermal lethal dose was produced at 0.69 ml/kg. (65, 66)

5.6.6. Sub-acute and chronic toxicity:

Upper limit of daily intake of cinnamon is set out at 700 μg/kg per day in

human beings. This upper limit is recommended by the Joint FAO/WHO Expert

Committee on Food Additives (JECFA). The stomach irritation and liver toxicity

has been reported with cinnamaldehyde. Maximum dose eugenol dose is up to

2.5 mg/kg.

75

Animal studies have reported both gastric irritation and severe

hepatotoxicity in Sprague Dawly rats at a dose of 10g/kg body weight (for 16

weeks). Pronounced hepatoxicity was reported as indicated by histological

findings of hepatocyte swelling (65, 66).

76

CHAPTER III

MATERIALS AND METHODS

3.1. Study Design: Experimental – Analytical study

3.2. Study Settings: Isra University, Al-Tibri Medical College and

Diagnostic and Research Laboratory, LMUHS, Hyderabad.

3.3. Duration of Study: One year from the date of synopsis approval-

January 2014- December 2014.

3.4. Sample Size: Sixty Wistar Albino rats of either sex were selected

from Animal House of AL-Tibri Medical College, Karachi.

3.5. Sampling technique

Phase I. Purposive sampling was done to select healthy rats

Phase II. Induction of diabetes mellitus with alloxan (Sigma Aldrich,

stored at 40C) – rats which achieved blood glucose levels of >250

mg/dl were selected (after duration of 72 hours).

Phase III. Simple random sampling was done to divide the selected

animals into subgroups amongst the groups.

3.6. Grouping of Animals:

Total 60 Albino rats of either sex

Rats were divided into six groups comprising of 10 rats in each

group.

77

Each Rat group was sub divided into 2 sub-groups containing 5 rats

each sub group.

Group A (n = 10)

Further sub divided into 2 groups

Group A1- Simple control (n = 5) ---- This group was given normal saline only.

Group A2- Diabetic Control (n = 5) --This group was treated with Alloxan only.

Group B (n = 10) ----------------- Extract - treated group

Further sub divided into 2 groups:

Group B1 (n = 5) -----------------------It was treated with low dose extract

Group B2 (n = 5) -----------------------It was treated with high dose extract

Group C (n = 10) ------------------ Aliskiren treated group

Further sub divided into 2 groups:

Group C1 (n = 5) ----------------------It was treated with low dose of drug

Group C2 (n = 5) --------------------- It was treated with high dose of drug

Group D (n = 10) ---------------- sitagliptin treated group

Group D1 (n = 5)--------------------- It was treated with low dose of drug

Group D2 (n = 5) ---------------------It was treated with high dose of drug

Combination of low & high dose of Extract + Aliskiren & Sitagliptin:

Group E (n = 10) –---------------Low dose of combination (Ext + drug)

E1(n = 5) = LExt + LDrug 1 (Aliskiren)

E2 (n = 5) = LExt + LDrug 2 (sitagliptin)

Group F (n = 10) -----------------High dose of combination (Ext + drug)

F1(n = 5) = HExt + HDrug 1 (Aliskiren)

F2(n = 5) = HExt + HDrug 2 (sitagliptin )

78

Animal Groups (n=60)

Groups Subgroups Dose

Group A (n=10)

A1 (Normal saline) -

Alloxan Induced

Diabetes mellitus (120mg i.p.)

A2 (Alloxan induced)

120 mg/kg i.p.

Group B (n=10)

B1 – CCBE low

dose

CCBE

0.3g/day

B2 – CCBE high dose

CCBE 0.6g/day

Group C (n=10)

C1 – Aliskerin low

dose

Aliskerin

100mg/day

C2 – Aliskerin high dose

Aliskerin 200mg/day

Group D (n=10)

D1 - Sitagliptin low dose

Sitagliptin 100mg/day

D2 - Sitagliptin

high dose

Sitagliptin

200mg/day

Group E (n=10)

E1 - CCBE+ Aliskerin low dose

CCBE 0.3g + Aliskerin 100mg

E2 – CCBE+

Sitagliptin low dose

CCBE 0.3g +

Sitagliptin 100mg

Group F (n=10)

F1 – CCBE+ Aliskerin high dose

CCBE 0.6g + Aliskerin 200mg

F2 - CCBEs+ Sitagliptin high dose

CCBE 0.6g + Sitagliptin

200mg

79

3.7. Inclusion Criteria

200-250 grams

Rats – both genders

Alloxan induced diabetic rats with minimum blood sugar level of

250mg/dl.

3.8. Exclusion criteria

Animals which fail to achieve required blood sugar level mentioned in

inclusion criteria

Diseased rats under weight

3.9. Data collection Procedure

Animal were handled under close supervision during experimentation. The

blood samples and pancreatic tissue were stored and processed in a systemic at

Diagnostic and Research Laboratory, LUMHS, Hyderabad.

3.10. Animal Housing and Experimental Details

Albino rats were housed in animal cages with a 12 hour light & dark cycle.

They were given standard diet and water ad-libitum.

To induce the experimental diabetes alloxan was dissolved in normal

saline. The rats achieving the level of blood glucose more than >250mg/dl were

considered as diabetic. The blood samples for measurements of glucose levels of

all rats were taken at 0hrs, 6hrs, 12hrs, 24hrs, and 72 hrs for diabetic experiment

for confirmation. The day on which blood glucose level >250mg/dl was confirmed,

that day was counted as first day of experimentation.

80

3.11. Preparation of ethanol based Cinnamomum cassia 129

3.11.1. Plant Material

The fresh stem bark of Cinnamomum cassia was selected for the present

study purpose.

3.11.2. Collection of Plant Material

The stem bark of Cinnamomum cassia (cinnamon) was collected from the

local market. The approval for conducting research on animals was taken from

ethical committee of the Isra University, Hyderabad, Sindh, Pakistan. The

taxonomy of Plant material was identified and authenticated by International

Pharmaceutical Laboratory of Baqai University, Karachi, and Sindh.

3.11.3. Preparation of Plant extract

Ethanol based Cinnamomum cassia (cinnamon) plant extract was

prepared as under:

3.11.4. Dryness of Cinnamon Stem Bark

The plant materials (stem bark) were washed thoroughly in running tap

water. The material were placed and spread properly on cotton cloth and were

fully covered from all sides by net cloth to minimize the entry of dust and other

foreign particles on the stem bark of Cinnamon. These materials were kept in a

well cleaned and airy room having ceiling and exhaust fan to regulate the air flow

in the room. The room temperature was maintained between 35-40oC during day

time and 25-30oC during night time. The humidity was minimized by keeping

81

exhaust fan open in the morning and switch off at night. This procedure was

performed continuously for 6 weeks, till the plant materials were completely dried.

3.11.5. Checking level of Cinnamon Stem Bark Dryness

To check the amount of dryness in cinnamon stem bark, one of the bark

pieced was picked and broken up from the center into two halves on weekly

basis. As this began to dry the stem bark became hard and their fibers lost the

elasticity, and were easily broken up in smaller pieces showing their dryness

completely. After drying, the cinnamon stem barks were then grinded in grinder

machine to get coarse powder.

3.11.6. Sifting of Cinnamon Bark Powder

The sifting procedure was done by using sifting net having the holes of 0.5

mm in size. The size of the hole was checked by vernier caliper. The cinnamon

stem bark powder was sifted thoroughly. The granules of powder lesser than 0.5

mm were kept in the container for further procedure, while particles of more than

0.5 mm size were again grinded to obtain smaller size of the particles. The

materials were again sifted and this was done repeatedly to achieve the required

size of particles which is 0.4 mm or lesser during sifting and required amount to

be used for study purpose.

3.11.7. Preparation of Cinnamon Stem Bark Extract

The cinnamon stem bark powder obtained after complete sifting were

transferred to Buchner flask with help of thistle funnel through continuous shaking

as the mouth Buchner flask is very narrow. The extract was prepared by taking

sufficient amount of 98% ethanol in the flask, so that whole powder material was

82

completely dipped into the ethanol. The mixture was kept for one week and

during this period daily shaking was required to separate out all the chemicals

and then the mixture was mixed in the ethanol. This was considered as fraction-1.

This fraction was decant off and kept in a well cleaned and sterilized flask. This

procedure was repeated again to obtain fraction-2 from the material. At the end of

second fraction and after one week, a glass tube of 1 mm diameter was then

inserted to the bottom of flask and a negative pressure was applied through pump

to obtain maximum extract from the liquid powder mixture. The equal amount of

98% ethanol was then added into the mixture after obtaining fraction-1 and

fraction-2 of extract.

3.11.8. Filtration of Mixture

After obtaining the solution from the mixture, another conical flask along

with funnel was taken and the filter paper was placed on the inner surface of the

funnel to filter the Cinnamomum cassia solution.

3.11.9. Separation of Extract from Ethanol

The filtered solution was then transferred in the round flask of the rotary

evaporator for separating the alcohol form plant materials. The solution was

continuously heated below 50oC and also under negative pressure of 2 bar to

minimize the boiling point. This separates the ethanol from the plant extract.

3.11.10. Re-separation of Ethanol from Extract

The extract obtained still contained traceable amounts of ethanol. The

extracted material was shifted in a beaker covered by a coverlid. A glass tube

was passed from the hole of the cover inside the beaker. Air was then entered

83

into the beaker through the glass tube connected with air pump. The inlet of the

pump was fixed with Whatman filter paper of 0.22µ. An air pressure was then

introduced into the material to enhance the complete evaporation of ethanol from

the extract.

3.11.11. Storage of Extract

The extracted solution of Cinnamomum stem bark extract was kept in a

well cleaned and air tight bottles. The extract bottles were then placed in the

refrigerator at 80C for further process. The material obtained was dark chocolate

in color and gummy in appearance having a pH of 7.0. 129

3.11.12. Animal Sacrifice and Blood sampling

Fasting rats were sacrificed after thirty days of treatment.

Blood will be collected into heparinized tubes.

Serum was separated out.

Following laboratory tests were conducted.

1. Estimation of blood glucose level – by glucose oxidase method

2. Insulin was measured by ELISA method fully automatic MEIA analyzer.

3. Superoxide dismutase (Misra and Fridovich14).

4. Glutathione peroxidase (Rotruck et al.15)

5. Histo-pathological studies

The animals were sacrificed by neck dislocation. The sections of

pancreatic tissue were examined microscopically for histological changes.

Each sample of pancreatic tissue obtained was washed in normal saline

and tissues were fixed in previously marked containers, containing 10%

84

formaldehyde as preservative. The tissues were embedded in paraffin, cut into 5

µm thick sections and stained with Hematoxylin-Eosin (H & E) staining for

histological examination.

3.11.13. Determination of Glutathione Peroxidase

(GPX)

o GPX Randox Diagnostics Assay Kit

o Assay Protocol

GPX activity was determined at 250C by measuring the rate of NADPH

oxidation as a decrease in absorbance at 340 nm (ε = 6.2 mM cm-1) according to

the method of Halliwell and Foyer (1978). The reaction mixture (1.0ml) consisted

of 100 mM Tris-HCl buffer (pH 7.8), 21 mM EDTA, 0.005 mM NADPH, 0.5 mM

oxidized glutathione (GSSG), and the enzymes extract. NADPH was added to

start the reaction. Unit activity is defined as “one unit will reduce 1.0 mole of

oxidized glutathione per minute under standard assay conditions”.

3.11.14. Determination of superoxide dismutase

(SOD)

o SOD Randox Diagnostics Assay Kit

o Assay Protocol

The assay for SOD activity was performed by following the method of

Beyer and Fridovich (1987). The assay mixture consisted of 27.0ml of 0.05 M

potassium phosphate buffer (pH 7.8), 1.5ml of L-methionine (300 mg/10 ml),

1.0ml of nitrobule tetrazolium salt (NBT) (14.4mg/10ml), and 0.75ml of Triton X-

100. Aliquots (1.0ml) of this mixture were delivered into small glass tubes,

85

Followed by the addition of 20µl enzyme extract and 10 µl of riboflavin (4.4 mg/

100ml). The cocktail was mixed and the illuminated for 15 minutes in an

aluminum foil-lined box, containing 25W fluorescent tube. In a control tube

sample was replaced by 20µl of buffer and the absorbance was measured 560

nm. The reaction stopped by switching off the light and placing the tubes in dark.

Increase in absorbance due to formation of formazan was measured at 560 nm.

Under the described conditions, the increase in absorbance in control was taken

as 100% and the enzyme activity in the sample was calculated by determining

the percentage inhibition per minute. One unit of SOD is that amount of enzyme

which causes a 50% inhibition of the rate for reduction of nitrobule tetrazolium

(NBT) under the conditions of the assay.

3.11.15. Insulin Detection

o MSD Metabolic Assays Mouse/Rat Insulin Kit

o Assay Protocol

150 µL of “Blocker A solution” was dispensed into a well. Plate was sealed,

and incubated at 300–1000 rpm at room temperature. Shook it vigorously. Plates

were washed 3 times with a Phosphate buffered saline plus 0.05% Tween-20

(PBS-T). 40 µL of 1x antibody solution was put into Meso scale discovery (MSD)

plate. Added 10µL of sample in the wells appropriately and immediately. Plates

were sealed with adhesive seal, incubated for 2 hours and shook vigorously.

Following, the plate were washed three times with PBS-T. 150µL of Read Buffer

T was added to each of MSD plate. Plates were read immediately following

addition of the Read Buffer.

86

3.12. Data Analysis

The normality of data was checked with Shapiro-Wilk test and was

analyzed by analysis of variance. The variables which yielded significant F-ratio

and p-value were treated with post-Hoc Fischer`s LSD testing for multiple

comparisons. The data collected was analyzed on SPSS version 21.0. A p-value

of ≤0.05 was taken statistically significant.

87

CHAPTER IV

RESULTS

The present study was an experimental study conducted at animal house

of Al-Tibri Medical College, Karachi, a campus of Isra University. The primary

goal of study was to evaluate effects of Cinnamomum cassia bark extract (CCBE)

on blood glucose homeostasis. And secondary objectives were to observe effects

on the insulin secretion, superoxide dismutase and glutathione peroxidase activity

and histology of pancreas. The study observed significantly positive effects of

CCBE on glucose homeostasis, insulin secretion, superoxide dismutase and

glutathione peroxidase activity compared to DPP-IV inhibitor (sitagliptin) and

direct renin inhibitor (aliskerin) as alone and in combination at different doses.

Significant F-ratio and p-value (shown in table IV-1) of variables were

further analyzed by multiple comparison post host tests.

Cumulative results for blood glucose, insulin secretion, superoxide

dismutase and glutathione peroxidase are shown in table IV -1 to IV -5 and

Graphs IV -1, IV -2, IV -3 and IV -4. Blood glucose lowering activity of CCBE was

observed at both low and high doses of 0.3g/day and 0.6g/day respectively.

Glucose homeostasis potential of CCBE was comparable to sitagliptin. CCBE in

combination with sitagliptin revealed more synergistic effect on blood glucose

levels. The blood glucose lowering effect of Aliskerin was not observed in present

study.

Insulin levels were found elevated in low dose and high dose CCBE

compared to sitagliptin, aliskerin and controls. The synergistic insulin secretory

effect of CCBE combined with sitagliptin was observed as shown in graph IV-2.

The animals of CCBE groups showed elevated antioxidant enzyme activities as

88

shown in graphs IV-3 and IV-4 and table IV -1 to IV -5. The superoxide dismutase

(SOD) and glutathione peroxidase (GPX) were found elevated in CCBE

compared to all groups except controls. SOD and GPX activity of CCBE animals

was higher than Sitagliptin-alone treated animals.

Blood glucose, insulin, SOD and GPX were analyzed in individual

subgroups and results are shown in table IV-6 to IV-11 and graphs IV-5 to IV-28.

Blood glucose in subgroups A1 and A2 was found highly different

(p<0.0001) as shown in table IV-6 and graph VI-5. Similar are the observations

for the insulin, SOD and GPX activity.

Groups B1 and B2 were treated with low and high dose CCBE (0.3g/dl and

0.6g/dl respectively) and statistically significant results were observed for all of

the four variables. Blood glucose, insulin, SOD and GPX activity were noted as

359.0±64.48 and 251.4±70.24 mg/dl (p=0.036), 6.40±1.34 and 10.4±4.61 µU/L

(0.015), 126.2±14.73 and 178.6±97.02 U/ml (0.001) and 7651.0±3133.10 and

13628.4±1358.12 U/L (0.004) respectively (Table IV-7 and Graphs IV-9 to 12).

Group C1 and C2 were treated with Aliskerin 100 and 200 mg/day. The

present study revealed no significant results for any of variables. The blood

glucose lowering effect, insulin, SOD and GPX activities were found non-

significant (Table IV-8, Graphs IV-13 to 16).

The sitagliptin showed significant differences as shown for groups D1 and

D2 in table IV-9 and Graphs IV-17 to 20.

Blood glucose, insulin, SOD and GPX levels for CCBE low and high dose

combined with Aliskerin or sitagliptin are shown in table IV-10 and 11

respectively. The CCBE combined with sitagliptin revealed better results than

alone or in combination with Aliskerin. An optimal blood glucose lowering effects

of CCBE was observed with high doses of 0.6g/day alone or in combination with

89

sitagliptin and similar are the observations for the insulin. CCBE and sitagliptin

groups showed elevated SOD and GPX activity as shown in tables IV-10 and 11

and Graphs IV-23, 24, 27 and 28 respectively.

Histology of Pancreas: Histological findings of pancreas were interpreted and

compared to controls. Alloxan induced diabetic rats revealed complete loss of

microscopic architecture of tissue with loss of microscopic details of Islets of

Langerhans (Photomicrographs IV.2). CCBE treated animals showed improved

tissue architecture, pancreatic acini, and visible Islets of Langerhans low doses.

But at high doses more pronounced histological improvement was observed. This

shows cytoprotective effects of CCBE on free radical mediated pancreatic injury.

Similarly, sitagliptin showed a cytoprotective effects as shown in

Photomicrograph IV-8. Aliskerin group did not show any change in histology of

pancreas compared to Alloxan animal’s pancreas (Photomicrographs IV-6 and 7).

90

Table IV.1. Analysis of variance (ANOVA) results of animal groups (n=60)

F-value p-value

Blood Glucose (mgdl-1) 115.50 0.023

Insulin (µUL-1) 13.72 0.006

Superoxide Dismutase (Uml-1) 13.03 0.014

Glutathione Peroxidase (UL-1) 11.35 0.024

91

Table IV.2. Blood Glucose level (mg/dl) in animal groups (n=60)

Mean Std.

Deviation

95% Confidence

Interval for Mean

Lower

Bound

Upper

Bound

Group A. Controls 271.6 163.1 154.8 388.30

Group B. CCBE 335.20 71.1 284.3 386.06

Group C. Aliskerin 403.0 54.8 364.03 442.56

Group D. Sitagliptin 351.8 68.8 302.5 401.06

Group E.

CCBE+Aliskerin+Sitagliptin

328.0 79.1 271.3 384.6

Group F. High Dose

CCBE+Aliskerin or Sitagliptin

280.4 63.54 234.9 325.85

p≤0.01

92

Table IV.3. Serum Insulin levels (µU/ml) in animal groups (n=60)

Mean Std.

Deviation

95% Confidence Interval

for Mean

Lower

Bound

Upper

Bound

Group A. Controls 13.2 10.5 5.6 20.7

Group B. CCBE 6.1 1.52 5.0 7.19



Group E.

CCBE+Aliskerin+Sitagliptin

8.0 3.62 5.40 10.59

Group F. High Dose

CCBE+Aliskerin or Sitagliptin

10.60 3.43 8.14 13.05

p≤0.05

93

Table IV.4. Superoxide Dismutase (U/ml) in animal groups (n=60)

Mean Std.

Deviation


for Mean

Lower

Bound

Upper

Bound

Group A. Controls 191.4 39.20 163.35 219.44

Group B. CCBE 123.4 39.50 95.13 151.60

Group C. Aliskerin Ϯ 77.9 25.64 59.55 96.20


Group E. CCBE+ Aliskerin+

Sitagliptin

142.70 60.67 99.29 186.10

Group F. High Dose CCBE+

Aliskerin or Sitagliptin

149.30 54.33 110.43 188.16

p≤0.02 Ϯ p=Non-significant

94

Table IV.5. Glutathione Peroxidase (U/L) in animal groups (n=60)

Mean Std.

Deviation


for Mean

Lower

Bound

Upper Bound

Group A. Controls 8475.80 3261.8 6142.39 10809.20

Group B. CCBE 5739.80 3255.7 3410.0 8086.1



Group E. CCBE+ Aliskerin+

Sitagliptin

6291.80 2992.5 4151.08 8432.5

Group F. High Dose CCBE+

Aliskerin or Sitagliptin

7214.40 3229.5 4904.1 9524.69

p≤0.03

95

Table IV.6. Blood Glucose, Serum Insulin, Superoxide Dismutase and

Glutathione Peroxidase in Group A animals

Parameters

Group A1 (Control)

Normal Saline

Group A2 (Alloxan)

120mg/kg i.p

p-value

Mean S.D Mean S.D

Blood Glucose (mgdl-1) 122.0 9.33 420.1 66.92 0.005

Insulin (µUL-1) 23.0 2.73 3.40 1.14 0.0001

Superoxide Dismutase

(Uml-1)

196.0 18.38 71.4 10.59 0.004

Glutathione

Peroxidase (UL-1)

10832.2 943.9 2745.1 1367.46 0.006

96


Glutathione Peroxidase in Group B animals

Parameters

Group B1

(CCBE 0.3gdl-1)

Group B2

( CCBE 0.6gdl-1)

p-

value

Mean S.D Mean S.D

Blood Glucose

(mgdl-1)

359.0 64.48 251.4 70.24 0.036

Insulin (µUL-1) 6.40 1.34 10.4 4.61 0.015

Superoxide

Dismutase (Uml-1)

126.2 14.73 178.6 97.02 0.001

Glutathione

Peroxidase (UL-1)

7651.0 3133.10 13628.4 1358.12 0.004

CCBE- Cinnamomum cassia bark extract

97


Glutathione Peroxidase in Group C animals

Parameters

Group C1 (Aliskerin 100mgday-1)

Group C2 (Aliskerin

200mgday-1)

p-

value

Mean S.D Mean S.D


Insulin (µUL-1) 2.40 1.14 3.20 0.83 0.91


(Uml-1)

69.0 32.17 86.80 15.7 0.58

Glutathione Peroxidase

(UL-1)

3469.4 908.5 2956.6 653.1 0.47

98


Glutathione Peroxidase in Group D animals

Parameters

Group D1 (Sitagliptin

100mgday-1)

Group D2 (Sitagliptin

200mgday-1)

p-

value

Mean S.D Mean S.D


Insulin (µUL-1) 7.0 1.58 9.8 4.20 0.06


(Uml-1)

102.60 18.88 175.6 99.7 0.032

Glutathione

Peroxidase (UL-1)

3869.40 1410.1 8756.6 3960.1 0.048

99


Glutathione Peroxidase in Group E animals

Parameters

Group E1 (CCBE 0.3gdl-1+

Aliskerin 100mgday-1)

Group E2 (CCBE 0.3gdl-1 +

Sitagliptin 100mgday-1)

p-

value

Mean S.D Mean S.D

Blood Glucose

(mgdl-1)

331.6 108.45 206.40 66.30 0.016

Insulin (µUL-1) 7.0 2.7 10.4 1.14 0.033

Superoxide

Dismutase (Uml-1)

126.0 29.7 159.4 32.20 0.012

Glutathione

Peroxidase (UL-1)

6925.1 2895.1 8858.1 1911.1 0.025

CCBE- Cinnamomum cassia Bark Extract

100


Glutathione Peroxidase in Group F animals

Parameters

Group F1

(CCBE 0.6gdl-1+ Aliskerin 200mgday-1)

Group F2

(CCBE 0.6gdl-1 + Sitagliptin 200mgday-

1)

p-value

Mean S.D Mean S.D

Blood Glucose

(mgdl-1)

286.4 50.79 180.4 50.47 0.01

Insulin (µUL-1) 7.40 2.07 12.60 3.91 0.039

Superoxide

Dismutase (Uml-1)

153.6 58.9 297.6 70.36 0.008

Glutathione

Peroxidase (UL-1)

7131.0 2841.9 11497.4 2622.90 0.036

CCBE- Cinnamomum cassia Bark Extract

101

Graph IV-1. Bar graphs showing blood glucose distribution among different

groups.

102

Graph IV-2. Bar graphs showing insulin secretion among different groups

103

Graph IV-3. Bar graphs showing Superoxide dismutase activity among different

groups

104

Graph IV -4. Bar graphs showing Glutathione peroxidase activity among different

groups

105

Graph IV -5. Bar graphs showing blood glucose level in subgroups A1 and A 2. A

highly significant p-value was observed (p=0.0001).

106

Graph IV -6. Bar graphs showing insulin levels in subgroups A1 and A 2. A highly

significant p-value was observed (p=0.0001).

107

Graph IV -7. Bar graphs showing Superoxide dismutase activity in subgroups A1

and A 2. A highly significant p-value was observed (p=0.0001).

108

Graph IV -8. Bar graphs showing Glutathione peroxidase activity in subgroups A1

and A 2. A highly significant p-value was observed (p=0.0001).

109

Graph IV -9. Bar graphs showing blood glucose level in subgroups B1 and B 2. A


110

Graph IV -10. Bar graphs showing insulin level in subgroups B1 and B 2. A


111

Graph IV -11. Bar graphs showing superoxide dismutase activity in subgroups B1

and B 2. A significant p-value was observed (p=0.001).

112

Graph IV -12. Bar graphs showing Glutathione peroxidase activity in subgroups

B1 and B 2. A significant p-value was observed (p=0.001).

113

Graph IV -13. Bar graphs showing blood glucose levels in subgroups C1 and C

2. Non-significant p-value was observed (p>0.05).

114

Graph IV -14. Bar graphs showing blood glucose levels in subgroups C1 and C

2. Non-significant p-value was observed (p>0.05).

115

Graph IV -15. Bar graphs showing superoxide dismutase activity in subgroups

C1 and C 2. Non-significant p-value was observed (p>0.05).

116


C1 and C 2. Non-significant p-value was observed (p>0.05).

117

Graph IV -17. Bar graphs showing blood glucose levels in subgroups D1 and D

2. Significant p-value was observed (p=0.001).

118

Graph IV -18. Bar graphs showing insulin levels in subgroups D1 and D 2.

Significant p-value was observed (p=0.001).

119

Graph IV -19. Bar graphs showing superoxide dismutase activity in subgroups

D1 and D 2. Significant p-value was observed (p=0.0001).

120


D1 and D 2. Significant p-value was observed (p=0.001).

121

Graph IV -21. Bar graphs showing blood glucose levels in subgroups E1 and E 2.


122

Graph IV -22. Bar graphs showing insulin levels in subgroups E1 and E 2.


123

Graph IV -23. Bar graphs showing superoxide dismutase activity in subgroups E1

and E 2. Significant p-value was observed (p=0.001).

124

Graph IV -24. Bar graphs showing glutathione peroxidase activity in subgroups

E1 and E 2. Significant p-value was observed (p=0.001).

125

Graph IV -25. Bar graphs showing blood glucose levels in subgroups F1 and F 2.


126

Graph IV -26. Bar graphs showing insulin levels in subgroups F1 and F 2.


127

Graph IV -27. Bar graphs showing superoxide dismutase activity in subgroups F1

and F 2. A highly significant p-value was observed (p=0.001).

128


F1 and F 2. Significant p-value was observed (p=0.001).

129

Photomicrograph IV – 1 Group A1. (Control). Normal Architecture or Pancreas,

is visible and islet of Langerhans are also visible. X100 (H & E)

Photomicrograph IV – 2. Group A2 (Alloxan induced Rats) completely disturbed

Architecture of pancreas, islet of Langerhans is not properly visible. X100 (H&E)

130

Photomicrograph IV – 3 Group B1 (CCBE 0.3g/dl). Islets of Langerhans are partially

visible but smaller in size X100 (H&E)

Photomicrograph IV-4.Group B2 (CCBE 0.6g/dl) Anatomical architecture of pancreas is

visible, with islets of Langerhans showing moderate recovery. X400 (H&E)

131

Photomicrograph IV-5 Group C1 (Aliskerin) Anatomical Architecture of pancreas is

disturbed; islets of Langerhans are also invisible. X100 (H&E)

Photomicrograph IV-6. Group C2 (Aliskerin) Anatomical architecture of pancreas is

disturbed; islets of Langerhans are also invisible. X100 (H & E)

132

Photomicrograph IV-7. Group D1 (Sitagliptin) Anatomical architecture of pancreas is

partially disturbed; with mild recovery of islets of Langerhans. X400 (H & E)

Photomicrograph IV-8. Group D2 (Sitagliptin) Anatomical architecture of pancreas is

partially disturbed; with mild recovery of islets of Langerhans. X400 (H & E)

133

Photomicrograph IV-9. Group E1 (Low dose CCBE+Aliskerin) Anatomical architecture

of pancreas is mildly distorted with partial recovery of islets of Langerhans. X200 (H&E)

Photomicrograph IV-10. Group E2 (Low dose CCBE+Sitagliptin) Anatomical

architecture of pancreas is nearly well defined; islets of Langerhans are moderately

visible. X100 (H & E)

134

Photomicrograph IV-11. Group F1 (High Dose CCBE+Aliskerin) Anatomical

architecture of pancreas is mildly disturbed with partial recovery of islets of Langerhans.

X200 (H & E)

Photomicrograph IV-12. Group F2 (High Dose CCBE+Sitagliptin) Anatomical

architecture of pancreas is well defined; islets of Langerhans are almost recovered. X200

(H & E)

135

CHAPTER V

DISCUSSION

Type 2 diabetics are increasing in number throughout the World because

of obesity, sedentary life style, population growth, aging and urbanization. The

prevalent diabetic epidemic is particularly relevant to Pakistan. According to WHO

estimates that half of 333 million diabetic people will be from Asia in 2030. While

International Diabetes Federation (IDF) estimates that the number of diabetics

older than twenty is projected to increase from 285 million in 2010 to 439 million

in 2030 (6, 7).

Currently, Pakistan is catering a large number of diabetics and ranks sixth

position in World. Shera et al reported that 15% of Pakistan population is

diagnosed of having DM, and more millions are undiagnosed. They are unaware

about DM because of lack of health facilities and present late with diabetic

complications. (6, 7)

Although type 2 DM is rigorously treated with a number of available anti

diabetic pills but diabetics still feel miserable because of no significant

improvement in quality of life. Hence there is need for alternative herbal

molecules to be investigated and made available to minimize hundreds of

physical sicknesses of DM which are not treatable effectively by current allopathic

drug therapy. The present study was an experimental study conducted on rat

model DDP-IV inhibitor (sitagliptin), DRI (aliskerin) and herbal medicine

(Cinnamomum cassia).

Optimal control of blood glucose is essential for quality of life in patients

with type 2 diabetes mellitus (89). Prolonged uncontrolled diabetics are prone to

136

development of retinal damage, blindness, renal damage, renal failure, end stage

renal disease and nerve damage. All these complications are now well

established (90). Likewise, similar is the incidence of cardiovascular heart

diseases in diabetics. (91, 92).

Oral antidiabetic drugs being used include, sulfonylureas, biguanides,

glucosidase inhibitors, DDP-IV inhibitors and meglitinides are well known for

reducing hemoglobin A1C. Undoubtedly, antidiabetic oral agents and insulin are

effective at reducing plasma glucose levels, but side effects like weight gains, risk

of lactic acidosis and hypoglycemia, contraindications of some agents in heart

failure, gastric upset and diarrhea are problematic. Thus it necessitates for

alternative options like herbal agents, dietary and lifestyle modifications. Insulin

use is awkward in that the diabetics have to use multiple times, with risk of

hypoglycemia and local side effects at the site of injection. (74)

Cinnamomum cassia is a culinary spicy herbal agent used since centuries

back. Cinnamon use for lowering plasma glucose has been known since ancient

days. Use of Cinnamon as medicine dates back 5000 years approximately, when

it was primarily used for stomach and digestive problems, as appetizer, as anti -

nauseating, anti-gas, anti-spasmodic, anti-flatulent, and for diarrhea. (74)

It is reported that the Cinnamon significantly helps type 2 diabetics to

manage their glycaemic status. Type 2 diabetics having problems in controlling

glucose levels even with insulin therapy are normalized with concomitant use of

cinnamon. (93- 96)

Active compound of Cinnamon capable of exerting insulin like activity is

debatable. The original active compound regulating blood glucose level was

137

recognized as “methylhydroxy chalcone polymer (MHCP)”. The MHCP is

proposed of exerting glucose lowering effect through molecular mimicry. It is

demonstrated in in-vitro experiments that the MHCP activates a number of cell

receptors including the insulin receptors (96). Anderson investigated MHCPs

ability to function as insulin mimetic in 3T3-L1 adipocytes (94). Recent reports

had claimed of glycaemic regulatory effects as exerted by polyphenols, in

particular “Polyphenol A”. (93- 96) A search of Pubmed, Medlip, Google scholars

and a number of websites revealed limited number of literature on the effects of

Cinnamomum cassia on blood glucose levels in diabetes mellitus in both animal

and human studies. Cinnamomum cassia is reported in a few clinical trials and

animal studies, of possessing glucose regulating effects and anti-oxidant effects.

1. ANIMAL STUDIES - CINNAMOMUM CASSIA

STUDY OF KAMBLE et al (97)

KAMBLE, et al (2013) recently performed an experimental study in alloxan

induced albino diabetic rats at Al-Ameen Medical College, Karnataka, India to

investigate the effects of C. cassia on glucose homeostasis. Study compared

effects of aqueous extract of C. cassia (60 mg/kg), glibenclamide (5 mg/kg) and

Metformin (0.5gm/kg) in diabetic rats. All the agents under study were given

orally as single morning dose. Fasting blood glucose was checked with

glucometer on days 0, 10 and 15.

KAMBLE, et al (97) reported significant results of C. cassia (60 mg/kg)

compared to antidiabetic agents. Study reproted that the aqueous extract of C.

cassia (60 mg/kg) alone produced significant effects on glucose homeostasis

compared to glibenclamide and Metformin (p<0.05). It was further reported that

effects of C. cassia on glucose homeostasis were more pronounced when it was

138

combined with glibenclamide, this indicated a synergistic potentiating effect of C.

cassia. However, synergistic effect of C. cassia was not observed with Metformin.

Kamble et al concluded that the C. cassia showed independent effect on glucose

homeostasis and it was potentiated when combined with glibenclamide. (97)

Similar are the results of present study, as it is confirmed that CCBE exerts

glucose lowering effect in rat model as alone and synergistic effect with

sitagliptin.

STUDY OF QIN et al (98)

An animal study by QIN et al (98) with euglycemic clamps had shown a

dose dependent enhanced glucose utilization in rat model when an aqueous

extract of C. cassia was given at 30-300 mg/kg body weight to animals.

Enhanced effects on insulin receptors in skeletal muscle were observed as under;

o Increase in “insulin receptor (IR)-β” by insulin administration

o Increase in “IR substrate (IRS)-1 tyrosine phosphorylation”

o Increase in “IRS-1-phosphatidylinositol (PI) 3-kinase”.

It was concluded by researcher that in-vivo effects of C.cassia are similarly

a result of potentiation of insulin signaling pathways (98).

QIN, et al (98) had reported that the fructose induced “insulin resistance” in

male Wistar albino rats was ameliorated by C.cassia extract ingestion. The

insulin resistance was improved and insulin dependent glucose uptake was

enhanced in C.cassia extract fed animals. It was concluded that the C.cassia

extract overcomes insulin resistance and improves glucose uptake through

insulin mediated signaling pathways. (98) The present study witnessed similar

effects on glucose homeostasis which are similar to above study.

139

STUDY OF KANNAPPAN et al (99)

KANNAPPAN et al (99) conducted a study on effects of cinnamon bark

extract (CBEt) on insulin sensitivity and glucose tolerance. It was reported by

researcher that the CBEt ingested rats revealed enhanced expression of

hexokinase. And also the skeletal muscle and liver glycogen content was

elevated. This indicated that the CBEt increases glucose transport in insulin-

dependent cells and improves glucose homeostasis (99). The findings are also in

agreement with present study in regards of glucose homeostasis.

STUDY OF KIM et al (100)

KIM et al had reported similar effects of CBBE on glucose homeostasis in

type 2 diabetic db/db mice model. The blood glucose was measured on weeks 2,

4 and 6. Significantly positive effects on glucose homeostasis were observed and

it was concluded that the CCBE exerts its blood glucose lowering effects through

insulin-mediated glucose regulation hence glucose homeostasis was maintained

(100). The findings support the results of present study.

STUDY OF BABU (101)

BABU (101) conducted a study with “Cinnamaldehyde” derived from true

Ceylon Cinnamomum zeylanicum. Cinnamaldehyde was administered at different

doses (5, 10 and 25 mg/kg) for 45 days to streptozocin-induced diabetic male

Wistar rats. The results were highly yielding regarding glucose and blood lipids

homeostasis, and yielding effects were significantly dose dependent. After 6

weeks of experimental period a significant reduction was observed in;

Total cholesterol

Triglycerides and

140

Enteric α-glycosidase activity.

Overall, it was concluded that the cinnamaldehyde exerts hypoglycemic

and hypolipidemic activity (101). The results of above study are in agreement to

present study in terms of glucose lowering effect.

STUDY OF JIA, et al (102) and TALPUR, et al. (103)

Several recent studies reported that the “Cinnamon oil” and “Polyphenolic

oligomers” extracts had shown promising hypoglycemic, hypolipidemic and anti-

oxidant activities in streptozocin induced diabetic rat models (102) (103). The

results are in full agreement with present study.

STUDY OF VERSPOHL et al. (104)

VERSPOHL et al. (104) evaluated effects of CCBE on blood glucose

homeostasis and serum insulin release in rats. He used extracts of common and

cassia cinnamon bark extract. It was concluded that the C. cassia extract was

more superior compared to common cinnamon extract regarding insulin secretion

(104).

Mechanisms of action of CCBE on glucose homeostasis by different

researchers have been proposed as;

1. Increase in serum insulin levels, (101)

2. Increase in hepatic glycogen storage (101)

3. Insulin-receptor signaling (98)

4. An insulino-mimetic effect (105)

5. Reduction in intestinal α-glycosidase activity (106)

6. Increased insulin secretion (104)

141

The results of present study are consistent with above mechanisms of

actions regarding glucose homeostasis and insulin. Hence, it may be claimed by

present study that the CCBE increases insulin from β-cells surviving in Islets of

Langerhans of Pancreas in experimental rats. On the basis of these studies

cinnamon is well known for pharmacological properties in the treatment of type 2

diabetes.

STUDY OF BABU et al (107)

A most recent study has been reported by BABU et al (107) from Tamil

Nadu, India. The study was conducted on streptozocin induced diabetic Wistar

rats. The end points of study were to evaluate in-vivo effects of cinnamaldehyde,

an active ingredient of C.cassia, as a blood glucose regulator, insulin stimulator,

anti-oxidant and anti-peroxidant agent. Effects on pancreatic β-cell were

evaluated. Following results were reported;

o Study of Babu et al - In vitro effects of cinnamaldehyde on plasma

glucose levels (107)

Babu et al in an experimental study evaluated the free radical scavenger

activity of cinnamaldehyde i.e. Superoxide (O-2) and nitric oxide (NO) radicals. On

comparative analysis, it was reported that the cinnamaldehyde exerted better O-2

and NO scavenging potential compared to controls and ascorbic acid fed

animals, thus cinnamaldehyde proved of its antioxidant property.

o Study of Babu et al- Effects of cinnamaldehyde on plasma glucose levels

(107)

In streptozocin inducted diabetic Wistar rats, it was reported that the active

ingredient of C. cassia, the cinnamaldehyde, shows significant effect on glucose

142

homeostasis compared to diabetic controls. Cinnamaldehyde was used at doses

of 5, 10 and 20 mg/kg. Cinnamaldehyde showed significant effect on glucose

homeostasis in a dose dependent fashion. Maximum effect on glucose

homeostasis was observed at 20 mg/kg doe of cinnamaldehyde (p<0.05).

Keeping in view, beneficial effects on glucose homeostasis, further studies were

conducted to observe combined effects on insulin secretion and anti-oxidant

activities of cinnamaldehyde. However, regulatory effects on glucose

homeostasis of Babu et al (107) are in agreement with present study. “CCBE

ameliorates glucose homeostasis” has been observed in our present animal

model study.

o Study of Babu et al- Effect of cinnamaldehyde on plasma insulin levels

(107)

Effects of cinnamaldehyde on plasma insulin levels were further preceded

in the same study. Plasma insulin levels were measured in normal controls,

diabetic and experimental animals of cinnamaldehyde and glibenclamide.

Cinnamaldehyde significantly increased plasma insulin from pancreatic tissue

compared to diabetic controls and glibenclamide taken as standard reference

(p<0.05). It was concluded that the cinnamaldehyde enhances insulin secretion

from β-cells of pancreas

The findings of present study agree very much. Insulin levels were found

elevated in low dose and high dose CCBE compared to sitagliptin and controls.

Again synergistic insulin secretory effects were observed for CCBE combined

with sitagliptin as shown in graph IV-2. The animals of CCBE groups showed

elevated antioxidant enzyme activities as shown in graphs IV-3 and IV-4.

143

o Study of Babu et al- In vivo antioxidant effect of cinnamaldehyde (107)

Indicators of lipid peroxidation were studied as malonaldehyde (TBARS)

and hydroperoxides (HP). Non-enzymatic antioxidants were also studied and

included ascorbic acid, α-tocopherol, ceruloplasmin and reduced glutathione

(GSH) in normal controls, diabetics, cinnamaldehyde and glibenclamide groups.

(107)

Markers of lipid peroxidation were highly elevated in streptozocin induced

diabetic rats compared to other groups. Cinnamaldehyde treated animals showed

elevated markers of lipid peroxidation compared to normal rats, however,

difference was statistically non-significant (p>0.05).

Diabetic rats revealed a significant (p<0.05) increase in peroxides and

hydroperoxides (TBARS), decreased levels of anti-oxidants, and decreased

vitamin C compared with normal rats.

After administration of cinnamaldehyde and glibenclamide, a significant

reduction was observed in lipid peroxides (TBARS) and levels were very close to

normal. GPX, SOD and CAT were all increased in cinnamaldehyde and

glibenclamide animals compared to diabetic rats (p<0.05) and were

approximating to normal rats. It was concluded that the cinnamaldehyde and

glibenclamide reduced markers of lipid peroxidation and increased GPX, SOD

and CAT activity (107)

The findings are consistent with present study as elevated activity of anti-

oxidant enzymes was confirmed. The superoxide dismutase (SOD) and

glutathione peroxidase (GPX) were found elevated in CCBE compared to all

other groups except controls. SOD and GPX activity of CCBE animals was higher

than sitagliptin-alone treated animals.

144

o Study of Babu et al- Histological findings of pancreas (107)

Diabetic group: Histological examination of diabetic rats showed

complete destruction of pancreatic tissue and loss of normal tissue architecture in

STZ-induced diabetic rats compared to normal (control) rats. Diabetic rats

revealed distorted and feebly visible pancreatic acini and islets of Langerhans

were ill defined and shrunken. (107)

Cinnamaldehyde and Glibenclamide groups: STZ-induced diabetic rats,

in which cinnamaldehyde and glibenclamide were administered, showed a normal

architecture, pancreatic acini and islets of Langerhans. Pancreatic islets were

visibly regenerating with newly sprouting capillaries. (107)

The findings of present study showed that the CCBE protected pancreatic

tissue as examined by histological details of various group animals. The CCBE

protected pancreatic tissue against free radical mediated injury. The details of

histological findings are shown in IV-1 to IV-13. CCBE animals showed

comparatively intact islets of Langerhans, pancreatic acini and organ architecture.

STUDY OF SHEN, et al (108)

SHEN et al repoted experience of cinnamon extract (CE) on regulation of

glucose homeostasis in type 2 diabetic rat model.

o Study of SHEN et al (2014)- in-vitro experiment on 3T3-L1 adipocytes and

C2C12 myotubes in type 1 diabetic rats

Shen et al repoted that the CE regulates glucose homeostasis through its

effects on glucose transporte-4 (GLUT4) translocation in adipocytes and

myocytes both. This effect was mediated through insulin-signaling and AMP-

145

activated protein kinase (AMPK) pathways. Phosphorylation of AMPK and Acetyl-

CoA carboxylase was enhanced by CE.

The findings of glucose lowering effects of Shen et al are similar to present

study. Present study confirmed the effects of CCBE on glucose homeostasis.

(108)

o Study of SHEN et al (2014)- in-vivo experiment on type 2 diabetic rats

To elucidate effects of CE on glucose homeostasis and uptake by cells, an

in-vivo study investigated the possible mechanisms. Effects of CE were

investigated by performing OGTT and insulin-glucose tolerance tests in type 2

diabetic rats. Shen et al reported that one of the mechanisms of CE mediated

glucose homeostasis is by GLUT-4 translocation linked by AMPK signaling

pathways. Insulin was found to antagonize the activation of AMPK. Hence, it was

concluded that the GLUT-4 translocation is enhanced by CE and regulates blood

glucose levels (108). The findings of present study are in agreement with Shen et

al. (108).

STUDY OF MORGAN et al (109)

MORGAN et al reported a study on effects of Cinnamon aqueous extract

(CAE) on the natural antioxidant enzymes of kidney, brain and testicular tissue in

Bisphenol A (BPA) and Octyphenol (OP) induced injury in male albino rats. (109)

A significant rise in blood urea and serum creatinine was observed.

Similarly, malonaldehyde (MDA) concentrations were eleva ted in kidney, brain

and testes of BPA and OP treated rats. BPA and OP treated rats showed a

severe reduction in GSH, SOD and CAT activity compared to control rats. (109)

146

On the contrary, the findings were highly significant in animals that were

pre-treated with CAE, approximately 2 hours before BPA or OP was instituted.

Pre-administration of CAE protected organ histology (kidney, brain and testes)

and also increased natural anti-oxidant systems. GSH, SOD and CAT levels of

kidney, brain and testes of CAE rats were elevated compared to diabetic rats and

were in normal range compared to non-diabetic control rats. (109)

The findings of present study are consistent with MORGAN et al. The

superoxide dismutase (SOD) and glutathione peroxidase (GPX) were found

elevated in CCBE (p=0.0001). SOD and GPX activity of CCBE animals was

higher than sitagliptin-alone treated animals.

2. CLINICAL STUDIES/TRIALS- CINNAMOMUM CASSIA

STUDY OF KHAN et al (110)

A small clinical trial was reported by KHAN et al (110) from Department of

Human Nutrition, NWFP Agriculture University Pakistan. This clinical trial included

sixty type 2 diabetics. The inclusion criteria were type 2 diabetic patients of > 40

years of age, not taking insulin therapy, or a drug for major systemic disease and

fasting blood glucose of 140-400 mg/dl were selected and studied. Sixty type 2

diabetics were divided into study and placebo groups. C. cassia was

administered orally in doses of 1, 3 and 6 grams per day for total 40 days. C.

cassia was filled in capsules as an add-on therapy to anti-diabetic drug. The part

of C. cassia is not specified. A 20 days washout period was also allowed. Blood

samples were collected for measurement of blood glucose, triacylglycerol, total

cholesterol, LDL-c and HDL-c at day 0, and after C. cassia administration at 20

and 40th day and day 60 (post-intervention day 20) of washout period. It was

reported that the C. cassia reduced fasting blood glucose and reduced all sub-

fractions of lipids except HDL-c which was similar in pre- and post-intervention

147

periods. The glucose lowering effects of C. cassia was also noted on post-

intervention day 20th of washout time period. The glucose lowering effect was

observed at doses of 1 gram per day and was pronounced at 6 grams per day. C.

cassia exerted sustained glucose lowering effects which was persistent in high

dose (6 grams) for many days after C. cassia intake was stopped. It was

concluded that the C. cassia exerts glucose lowering effect in a time dependent

fashion. The authors were of opinion that the C. cassia may even show effects at

doses may be less than 1 gram per day. It was also suggested that the C. cassia

exerts a prolonged & sustained glucose regulating effects hence it is not

necessary to use C. cassia on daily basis as its effects persisted for many days

(110). The findings of present animal study are consistent regarding the blood

glucose lowering effects of CCBE.

STUDY OF ANURADHA AND DEVI (111)

ANURADHA AND DEVI (111) also reported significant reduction in both

fasting and post prandial blood glucose after 4 g cinnamon supplementation for

90 days. Findings of present study support above mentioned results of CCBE on

glucose homeostasis.

STUDY OF MANG, et al (112)

MANG, et al (112) reported a double blind clinical study from Hannover,

Germany. The study collected a sample of 79 type 2 diabetics according to well

delineated inclusion and exclusion criteria. Eventually, 14 diabetics were dropped

because of various reasons, and finally 65 subjects were reported. C. cassia was

given orally as powder filled capsules of amount 3 grams approximately for four

months. Variables studied were the fasting plasma glucose hemoglobin A1c and

blood lipids at end of four month. It was reported that the mean fasting plasma

148

glucose was reduced by 10.3% compared to baseline while placebo groups

revealed a 3.4% reduction. The findings were statistically significant (p=0.0001).

While A1C and blood lipids showed non-significant reduction when compared

pre- and post-intervention values. Plasma glucose reduction was significantly

correlated with baseline concentrations, indicating the C. cassia exerts more

effects in diabetics with very high baseline blood glucose levels. Finally it was

concluded that the C. cassia exerts moderate blood glucose lowering effects and

may be used in diabetics as add on therapy (MANG et al., 2006). The findings

are supported by present study.

STUDY OF VANSCHOONBEEK et al (113)

A previous double blind placebo controlled clinical trial by

VANSCHOONBEEK; et al (113) had reported contrary results regarding effect of

C. cassia on glucose homeostasis from Netherlands. The study comprised of 25

postmenopausal diabetic women who ingested 1.5 grams of C. cassia for 6

weeks to investigate effects on blood glucose, insulin sensitivity and blood lipids.

Before and after second and sixth week of C. cassia supplementation, blood

samples were investigated for research variables as mentioned above. C. cassia

failed to show any effects on the glucose homeostasis, insulin sensitivity and

blood lipids. The results of previous clinical trials were strongly negated, and it

was concluded that the C. cassia (1.5 grams per day) had no beneficial effects on

study parameters. It was recommended that the C. cassia supplementations

should be re-visited before health claims were made. (113) The findings of above

study are inconsistent with most of studies mentioned in literature (74, 110, 114,

115).

149

STUDY OF SUPPAPITIPORN et al (116)

SUPPAPITIPORN et al (116) recruited 60 type 2 diabetics at King

Chulalongkorn Memorial Hospital in Thailand. The study was proposed to study

and report effects of C. cassia on glucose homeostasis in terms of fasting

glucose levels and A1C, liver aminotransferases, BUN, creatinine, blood pressure

and BMI. C. cassia 1.5 grams per day were given orally for 12 weeks. The final

results were mixed, as two variables (SGOT and HbA1c) were improved to some

extent. Non significant effects were observed for fasting glucose levels, SGPT,

BUN, creatinine, blood pressure and BMI. However, A1C improvement was

significant in terms of proportion of patients achieving HbA1c ≤7% was greater in

patients receiving C. cassia compared to placebo controls. SGOT improvement

was found statistically significant (p = 0.001) (116). Findings of above study are

also inconsistent with present study with regard to blood glucose lowering effect.

STUDY OF ZIEGENFUSS (117)

ZIEGENFUSS (117) conducted a double-blind, placebo-controlled clinical

trial on 22 subjects with pre-diabetes with metabolic syndrome. The study used a

commercially available Cinnamon extract (Cinnulin PF®). The study subjects

were divided into two groups a control group (placebo therapy) and experimental

group (500 mg Cinnulin PF® daily).

The Cinnamon extract was given for 12 weeks and objective was to

explore the effects on fasting blood glucose levels and body composition.

Following results were declared in cinnamon group;

Fasting blood glucose- a reduction by -8.4%.

Systolic blood pressure- a reduction by -3.8%.

Lean body mass – an increase by +1.1%.

Body fat- an increase by 0.7%. (117)

150

The finding of glucose homeostasis of present study is consistent with

above.

STUDY OF WANG, et al (118)

Polycystic ovary syndrome (PCOS) is a common female condition

associated with fertility problems, insulin resistance and compensatory hyper-

insulinemia which are reported present in 50-70% of normal weight and up to

95% in over-weight female (118).

WANG et al. conducted a study to investigate if any of the beneficial effect

of cinnamon on the PCOS female. It was postulated that the water soluble

“Polyphenols” compounds possess insulin potentiating effects which were to be

examined. Fifteen female with PCOS were randomized to study and placebo

groups. Study group received cinnamon for 8 weeks orally. Study reported

following results;

A significant lowering of “insulin-resistance” and “fasting blood glucose”

21% lowering of “Mean blood glucose” level with OGTT

Increased insulin sensitivity as measured by “Matsuda`s insulin sensitivity

index”

It was concluded that the cinnamon extract reduces insulin resistance,

thereby improves glucose homeostasis in female with PCOS compared to

placebo group, (118) the findings are consistent with present study.

STUDY OF HLEBOWICZ et al. (119)

HLEBOWICZ et al. (119) conducted a cross over trial in 14 healthy diabetic

subjects by using 6 grams of cinnamon extract with meals. Finally, result was a

significant reduction in “post-prandial blood glucose level” and “delayed gastric

emptying”. However, no effect was observed on satiety by cinnamon in study

151

population. The glucose lowering effects of above study are consistent with

present study.

STUDY OF SOLOMON et al (120)

SOLOMON et al (120), in a clinical trial, examined effect of C.cassia on

seven lean healthy male subjects, aged 26 ± 1 years, BMI of 24.5 ± 0.3 kg/m2,

with better glycaemic tolerance. The volunteers were invited to ingest glucose in

OGTT supplemented with 5 grams C.cassia powder or placebo. The C.cassia

bark extract was used. C.cassia reduced plasma glucose in 75 gram ingested

OGTT subjects up to 13% when ingested with glucose compared to 10% when

C.cassia was ingested prior to glucose swallowing. (p=0.05).

Moreover, the effects of C.cassia on glucose homeostasis and insulin

sensitivity persisted for 12 h after cinnamon ingestion suggesting that cinnamon

may have prolonged effects on events associated with increased insulin

sensitivity. The findings are in agreement with present study in terms of glucose

lowering and insulin activity.

STUDY OF BLEVINS et al (121)

BLEVINS et al (121) conducted study at the University of Oklahoma USA.

C. cassia supplementation was analyzed regarding any effects on fasting blood

glucose, A1C, insulin, BMI and blood cholesterol. A sample of 77 type 2 diabetics

was recruited for investigating the effects of C. cassia, but many subjected were

dropped or withdrew from study protocol because of various reasons. Finally 58

participants completed the study. Experimental group (n=30) received 500 mg of

C. cassia filled capsules while controls (n=28) received wheat flour filled capsules

as placebo. C. cassia 500 mg was given orally twice a day for 3 months. Findings

were negative regarding effects of C. cassia as no improvement in fasting blood

152

glucose, A1C, insulin, BMI and blood cholesterol. It was concluded that the C.

cassia must be further studied under strict experimental designs regarding

ethnicity, dose, BMI, baseline glucose levels, A1C and concurrent drug intake.

Further studies were recommended. The findings of above study are inconsistent

with present and most of the studies mentioned in literature (74, 110, 114, 115)

STUDY OF CRAWFORD (122)

CRAWFORD (122) reported his experience of C. cassia supplementation

on glycated hemoglobin A1. The study published in Journal of American Board of

Family Medicine. The study was a randomized controlled clinical trial comprising

of 109 type 2 diabetics which were supplemented with C. cassia (1 gram per day)

capsules for 90 days. Baseline A1C was noted and re -noted after 90 days for

comparison. Positive findings were reported regarding amelioration of A1C at pre-

and post-intervention periods and this indicated that the C. cassia has effects on

glucose homeostasis. It was reported that the C. cassia lowered HbA1C by

0.83% (95% CI, 0.46 –1.20) versus 0.37% routine antidiabetic drug therapy (95%

CI, 0.15– 0.59). It was concluded that the C. cassia possessed positive effects on

glucose homeostasis as indicated by improvement in A1C which is a best

available indicator of glycemic control in diabetics (122).

STUDY OF SONI et al (115)

SONI et al (115) studied effects of C. cassia on 30 non-insulin dependent

diabetics from Rajasthan, India. Experimental groups (n=15) received 2 grams of

C. cassia per day while controls (n=15) received placebo therapy. The study

reported significant effects of C. cassia on glucose homeostasis in terms of

fasting and postprandial blood glucose levels. The author has strongly

recommended supplementing type 2 diabetics with C. cassia intervention as it

153

exerts positive effects on glucose homeostasis (115). The findings of present

study conducted on animals also support the recommendation of above study.

STUDY OF ROUSSEL et al., (114)

ROUSSEL et al., (114) reported positive findings in his clinical study.

Baseline age and BMI of subjects and C.cassia was 45.8+/-3.6 years and 45.6+/-

2.7 years and 34.2+/-4.2 and 32.3+/-2.7 kg/m2 respectively. Placebo groups did

not showed any improvement in research variable studied while C.cassia showed

fasting plasma glucose reduction from 114+/- 2.2 to 102+/-4.3 mg/dl (p=0.0001).

Plasma stress markers were significantly improved (p<0.05). Ferric reducing

activity of plasma (FRAP) and plasma thiol (SH) radicals were increased, while

plasma MDA levels decreased in subjects receiving the C.cassia extract. The

effects were more pronounced after 12 than 6 weeks. There was also a positive

correlation (r = 0.74; p = 0.014) between MDA and plasma glucose. In contrast,

RBC antioxidant enzymes SOD and GPX were not altered by supplementation at

6 weeks, but were found significant at 12 weeks (p<0.05). This study supports the

hypothesis that the inclusion of water soluble compounds of C. cassia reduces

risk factors and complications associated with diabetes and cardiovascular

disease (114).

STUDY OF SOLOMON et al (123)

In another clinical trial by SOLOMON (2009) was conducted on eight male

volunteers (aged 25 ± 1 year, body mass index 24.0 ± 0.7 kg/m2). Participants

underwent two 14-day interventions. Participants were divided into C.cassia

(3g/day) or placebo groups [69]. C.cassia reduced the plasma glucose responses

to OGTT and improved insulin sensitivity. However, the effects disappeared

154

rapidly following cessation of C.cassia intake. The results showed an

improvement in glucose homeostasis and insulin sensitivity, but the effects were

reversed quickly afterwards. (123) Ultimate results are consistent with our study.

WICKENBERG et al. 2014 (128)

21 subjects with impaired glucose tolerance (IGT) were included in the

study. 6 g of cinnamon was given twice a day for 12 weeks. No significant change

in the insulin sensitivity was observed in subjects with IGT. The findings of above

study are inconsistent to present and previous studies.

STUDY OF HOEHAN et al (74)

A recent clinical trial has been reported by HOEHAN, et al (74) Clinical trial

was conducted at Raabe College of Pharmacy, Ohio, Northern University, USA.

The study included 18 type 2 diabetics, 9 male and 9 female patients. Clinical trial

was divided into 3 weeks control phase and a 9 week experimental phase. 50%

of patients received C. cassia (1000mg per day) and other 50% received placebo

pills. Objective of study was to compare effect of C. cassia on blood glucose

compared to dietary changes alone. The subjects were educated for intake of

appropriate diabetic diets during study period. The study reported a significant

reduction in blood glucose in experimental groups compared to placebo. A 30

mg/dl reduction was noted in blood glucose in experimental groups (C. cassia

group) (p=0.000000003915). It was concluded that the C. cassia exerts effects on

glucose homeostasis very much comparable to available anti-diabetic drugs (74),

despite change in methodology.

Dipeptidyl peptidase-IV inhibitors (Sitagliptin, vildagliptin and saxagliptin)

which are capable of blocking the DPP-IV enzyme may increase the endogenous

GLP-1 level which in turn exerts incretin effect. DPP-IV inhibitors are novel oral

155

glucose-lowering agents, which may be used as monotherapy or in combination

with other antidiabetic compounds, metformin, thiazolidinediones or even

sulfonylureas. Sitagliptin, vildagliptin and saxagliptin are already on the market,

either as single agents or in fixed-dose combined formulations with metformin.

Other compounds, such as alogliptin and linagliptin, are in a late phase of

development. The proliferative and anti-apoptotic actions of GLP-1 on islet β-cells

have been mirrored by studies using DPP-IV inhibitors in diabetic rodents. High-

fat–fed mice treated with low-dose streptozocin followed by 2–3 months of

treatment with des-fluoro-sitagliptin showed improvement in fasting and

postprandial hyperglycemia, increased pancreatic insulin content, and increased

numbers of insulin-positive β-cells in association with normalization of β -cell

mass and restoration toward normal of the β-cell: β-cell ratio. (33, 48, 49, 50-52)

In present study, effects of sitagliptin were proved in diabetic rats. When

combined with C.cassia the effects on glucose homeostasis were striking.

The In-vivo models have demonstrated that the ductile cells are capable of

giving rise to new β-cells and also the interferon gamma (IFN-γ) over expression.

Wrapping of pancreatic duct with plastic sheaths in order to induce inflammation

with concomitant administration of EGF and gastrin is reported to induce β-cells

proliferation. The β-cells ductile origin from in-situ progenitor cells during acute

pancreatic injury has produced controversial results, as it has been reported in

previous studies. The controversy is that the pancreatic ductile cells contribute or

not is not proved or is controversial and remains to be elucidated. (42-46)

In our study, capillary neogenesis, intact tissue architecture mediated by

sitagliptin in favored by above studies. Effects on serum insulin and augmentation

of enzymes, SOD and GPX, were also significantly present in sitagliptin treated

156

animals. In a most recent study reported by MEGA, (124), it was reproted that six

weeks administration of sitagliptin in Zuker diabetic fatty rat, improved β-cell

functions, and improved pancreatic islet structure. It was reported that the

sitagliptin might mediate its effects through an increase in endogenous incretins

(GLP-1 and GIP). Sitagliptin may exert pancreatic protective effects through its

cytoprotective effects like anti-inflammatory effect, anti-apoptotic effect and pro-

proliferative effects (124). However, our study hypothesizes that the sitagliptin

may partially exert its cytoprotective and islet protective effects through

augmentation of anti-oxidant enzymes which minimize the tissue destruction by

free radicals.

Aliskerin is a newly launched direct rennin inhibitor approved for essential

hypertension. In present study, we did not observed any effect of aliskerin either

on glucose homeostasis or pancreatic cytoprotective effects. As previous studies

reported controversial results. GANDHI (125) reported that the aliskerin improves

glucose homeostasis through expression of GLUT-2 and GLUT-4 transporters in

liver and muscles. The study of KANG (126) did not observe any effect on blood

glucose levels in db/db mice. KANG (126) reported that the aliskerin (25 mg/kg),

infused through an osmotic mini pump showed no significant changes in glucose

homeostasis, serum creatinine, electrolytes and systolic blood pressure in db/db

diabetic mice model. Another study by Al-LAFI (127) had reported no significant

effect of aliskerin on blood glucose levels. Finding of no ef fect of aliskerin of

present study on glucose homeostasis is also in parallel to KANG (126) and AL-

LAFI (127). Present study did not observe any cytoprotective effect of aliskerin or

any effect on anti-oxidant enzyme systems in alloxan induced diabetic in rat

model.

157

CHAPTER VI

CONCLUSION

On the basis of evidence and facts of present study, comparable to both

animal and human studies, it is concluded that the Cinnamomum cassia exerts

glucose lowering effects primarily by stimulation of insulin release from β-cells of

the islets of Langerhans. Cinnamomum cassia augmented the secretion of insulin

many folds probably by protecting β-cells from free radicals due to its anti-oxidant

activity and stimulation of β-cells. C.cassia exerts a direct insulin releasing effect

through incretins.

Microscopic picture of pancreatic histology reveals intact tissue in

Cinnamomum cassia treated animals. Regenerating pancreatic tissue and most

probably the pancreatic β-cells mass is visibly observed. The present study

suggests that the Cinnamomum cassia undoubtedly maintains glucose

homeostasis through release of insulin and anti-oxidant activity which protects

pancreatic tissue.

158

CHAPTER VII

RECOMMENDATION

Further studies are recommended to elucidate various mechanisms of

glucose homeostasis on animal models.

The chemical analysis of Cinnamomum cassia is recommended to find out the

active component which enhances insulin secretion and free radical

scavenging activity alone and components present in its extract.

Animal and clinical trials are recommended to be conducted under strict

experimental conditions with sophisticated instrumentation to prove C.cassia

further for making it available to grassroots as herbal remedy for diabetes

mellitus. This will help to minimize economical burden, improve health and

prevent complication of diabetes mellitus.

Cinnamomum cassia may be used as add- on therapy with anti-diabetic

drugs.

159

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