The Metabolic Effects of Echinochrome Pigment Extracted from Sea Urchin on Diabetic Rats


Doctoral Thesis / Dissertation, 2018

243 Pages, Grade: 4


Excerpt

ACKNOWLEDGMENT
First and foremost thanks to God
I would like to express my great appreciation and infinite gratitude to
Prof. Dr. Mohamed Assem Said Marie,
Professor of Environmental
Physiology, Zoology Department, Faculty of Science, Cairo University, for his
supervision, scientific guidance, and continuous help throughout the whole
work.
Special gratitude and sincere thanks to Prof. Dr. Amel Mahmoud
Soliman Professor of Physiology, Zoology Department, Faculty of Science,
Cairo University, for her great assistance, supervision, valuable suggestions and
for his kind help throughout this work and during the preparation of the
manuscript.
I wish to express my deep thanks, grateful acknowledgement and
gratitude to Prof. Dr. Sohair Ramadan Fahmy, Professor of Physiology,
Zoology Department, Faculty of Science, Cairo University for her kind help
during this work. Again, special thanks to staff members and my colleagues of
the Zoology Department, Faculty of Science, Cairo University, for their
encouragement.
Finally, I deeply thank my family for their love, support and
encouragement through the work.

ABSTRACT
Student Name: Ayman Saber Mohamed
Title of the thesis: Metabolic effects of Echinochrome pigment extracted from sea
urchin on diabetic rats
Degree: Ph.D. in Zoology (Molecular and Integrated Physiology)
Diabetes mellitus is one of the most public metabolic disorders. It is mainly
classified into type 1 and type 2. Echinochrome (Ech) is a pigment from sea urchins
that has antioxidant, anti-microbial, anti-inflammatory and chelating abilities. The
present study aimed to investigate the anti-diabetic mechanisms of Ech pigment in
streptozotocin-induced diabetic rats. Thirty-six male Wistar albino rats were divided
into two main groups (18 rats/group). Each group was divided into 3 subgroups (6
rats/subgroup); control, diabetic and Ech subgroups. Diabetic models were induced by a
single dose of streptozotocin (60 mg/kg, i.p) for type 1 diabetes and by a high fat diet
for 4 weeks before the injection of streptozotocin (30 mg/kg, i.p) for type 2 diabetes.
Diabetic groups were treated orally with Ech (1 mg/kg body weight in 10% DMSO)
daily for 4 weeks. Ech groups showed a reduction in the concentrations of glucose,
globulins, triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol
(LDL-C), creatinine, urea, uric acid, malondialdehyde (MDA) and the activities of
arginase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline
phosphatase (ALP) and gamma-glutamyltransferase (GGT). While, it caused general
increase in the levels of insulin, total bilirubin (TB), direct bilirubin (DB), indirect
bilirubin (IB), total protein (TP), albumin, nitric oxide (NO) and the activities of
glucose-6-phosphate dehydrogenase (G6PD), hexokinase, glutathione-S-transferase
(GST),
superoxide dismutase (SOD) and glutathione reduced (GSH). The
histopathological investigation showed partial restoration of pancreatic islet cells and
clear improvement in the hepatic and kidney architecture. The results of this study
clearly show that Ech has anti-diabetic potential in both types of diabetes. The possible
anti-diabetic mechanisms of Ech involving improved glucose metabolism, restoration
of cells, improve insulin secretion, improve insulin signaling and antioxidant activity
Key words: Diabetes-Echinochrome-Oxidative stress-Pancreas-Liver-Kidney-
Histopathology.

i
List of contents
Page
Title
1
I. Introduction
15
Aim of work
16
II. Materials and methods
16
II.1. Chemicals and reagents
16
II.2. Sea urchin Collection
17
II.3. Echinochrome (Ech) extraction
17
II.4. Experimental animals
17
II.5. Ethical Consideration
18
II.6. Induction of type 1 diabetes mellitus (T1DM)
18
II.7. Induction of type 2 diabetes mellitus (T2DM)
18
II.8. Experimental design
19
II.9. Determination of the physical parameters
19
II.9.1. Body weight
19
II.9.2. Urine volume
20
II.9.3. Hot plate test
20
II.9.4. Wire suspension
20
II.10. Animal handling and specimen collection
21
II.11. Samples preparation
21
II.11.1. Serum preparation
21
II.11.2. Liver and kidney homogenate preparation
21
II.11.3. Histopathological examination
22
II.12. Biochemical assessment
22
II.12.1. Diabetic markers
22
II.12.1.1. Determination of glucose
23
II.12.1.2. Determination of Insulin
24
II.12.1.3. Determination of arginase

ii
25
II.12.1.4. Determination of Hexokinase (HK)
27
II.12.1.5.
Determination of glucose-6-phosphate
dehydrogenase (G6PDH)
29
II.12. 2. Serum biomarkers for liver function
29
II.12.2.1. Determination of serum aminotransferase
enzymes (ASAT, ALAT)
31
II.12.2.2. Determination of alkaline phosphatase
32
II.12.2.3. Determination of gamma-glutamyltransferase
(GGT)
33
II.12.2.4. Determination of total bilirubin, direct &indirect
35
II.12.2.5. Determination of total protein
36
II.12.2.6. Determination of serum albumin and globulins
38
II.12. 3. Determination of lipid profile
38
II.12.3.1. Determination of serum triglycerides (TG)
39
II.12.3.2. Determination of serum total cholesterol (TC)
41
II.12.3.3. Determination
low
density lipoprotein cholesterol
(LDL-C)
43
II.12.3.4. Determination of High density lipoprotein
cholesterol (HDL-C)
II.12. 4. Determination of kidney Function tests
44
II.12.4.1. Determination of creatinine and creatinine
clearance
45
II.12.4.2. Determination of uric acid
47
II.12.4.3. Determination of urea
49
II.12. 5. Determination of Oxidative Stress parameters
49
II.12.5.1.
Determination of lipid peroxide
(Malandialdehyde)
50
II.12.5.2. Determination
of
glutathione reduced (GSH)
51
II.12.5.3. Determination of catalase (CAT)

iii
53
II.12.5.4. Determination of superoxide dismutase (SOD)
54
II.12.5.5. Determination
of
glutathione-S-transferase (GST)
55
II.12.5.6. Determination of nitric oxide (NO)
56
II.13. Histological
examination
57
II.14. Statistical
analysis
58
III. Results
58
III.1. Physical parameters
58
III.1.1. Body weight
59
III.1.2. Urine volume
60
III.1.3. Hot plate test
62
III.1.4. Wire suspension
64
III.2. Diabetic markers
64
III.2.1. Serum
glucose
65
III.2.2. Insulin
67
III.2.3. Serum
arginase
68
III.2.4. Liver
hexokinase
70
III.2.5. Liver glucose-6-phosphate dehydrogenase
(G6PD)
71
III.3.Serum biomarkers for liver function
71
III.3.1.
Serum aspartate aminotransferase (AST)
73
III.3.2.
Serum alanine aminotransferase (ALT)
74
III.3.3.
Serum alkaline phosphatase (ALP)
76
III.3.4.
Serum gamma glutamyl transferase (GGT)
77
III.3.5.
Serum total bilirubin (TB)
79
III.3.6.
Serum direct bilirubin (DB)
80
III.3.7.
Serum indirect bilirubin (IB)
81
III.3.8.
Serum total protein (TP)
83
III.3.9. Serum
albumin

iv
85
III.3.10. Serum
globulins
86
III.3.11.
Albumin/globulins ratio (A/G)
88
III.4. Lipid profile
88
III.4.1.
Serum triglycerides (TG)
89
III.4.2.
Serum total cholesterol (TC)
91
III.4.3.
Serum low density lipoprotein cholesterol (LDL-
C)
92
III.4.4. Serum
high
density
lipoprotein cholesterol (HDL-
C)
88
III.5. Kidney Function tests
94
III.5.1. Serum
creatinine
95
III.5.2. Urine
creatinine
97
III.5.3. Creatinine
clearance
98
III.5.4.
Serum uric acid
100
III.5.5. Urine
uric
acid
101
III.5.6. Serum
urea
103
III.5.7. Urine
urea
104
III.6. Oxidative stress parameters in liver
104
III.6.1. Liver
malondialdehyde
(MDA)
106
III.6.2.
Liver glutathione reduced (GSH)
107
III.6.3.
Liver catalase (CAT)
109
III.6.4.
Liver superoxide dismutase (SOD)
110
III.6.5. Liver
glutathione-S-transferase
(GST)
112
III.6.6.
Liver nitric oxide (NO)
113
III.7. Oxidative stress parameters in kidney
113
III.7.1.
Kidney malondialdehyde (MDA)
115
III.7.2.
Kidney glutathione reduced (GSH)
116
III.7.3. Kidney
catalase
(CAT)
118
III.7.4.
Kidney superoxide dismutase (SOD)

v
119
III.7.5.
Kidney glutathione-S-transferase (GST)
121
III.7.6.
Kidney nitric oxide (NO)
122
III.8. Histopathological examination
122
III.8.1.
Histopathological examination of pancreas
124
III.8.2.
Histopathological examination of liver
126
III.8.3.
Histopathological examination of kidney
128
IV. Discussion
167
Conclusion
168
V. Summary
171
VI. References

vi
List of tables
Title
Page
Table 1:
The curative potency of echinochrome (Ech) on the final
body weight (gm) of diabetic rats.
58
Table 2:
The curative potency of echinochrome (Ech) on urine
volume (ml/24hr) of diabetic rats.
60
Table 3:
The curative potency of echinochrome (Ech) on hot plate
period (Sec) of diabetic rats.
61
Table 4:
The curative potency of echinochrome (Ech) on wire
suspension period (Sec) of diabetic rats.
63
Table 5:
The curative potency of echinochrome (Ech) on glucose
concentration (mg/dl) of diabetic rats.
64
Table 6:
The curative potency of echinochrome (Ech) on insulin
concentration (µU/ml) of diabetic rats.
66
Table 7:
The curative potency of echinochrome (Ech) on arginase
activity (U/L) of diabetic rats.
67
Table 8:
The curative potency of echinochrome (Ech) on hexokinase
activity (U/min/ gm.tissue) of diabetic rats.
69
Table 9:
The curative potency of echinochrome (Ech) on glucose-6-
phosphate dehydrogenase (G6PD) activity (U/min/
gm.tissue) of diabetic rats.
70
Table 10:
The curative potency of echinochrome (Ech) on aspartate
aminotransferase (AST) (U/ml) of diabetic rats.
72
Table 11:
The curative potency of echinochrome (Ech) on alanine
aminotransferase (ALT) activity (U/ml) of diabetic rats.
73
Table 12:
The curative potency of echinochrome (Ech) on alkaline
phosphatase (ALP) activity (U/L) of diabetic rats.
75
Table 13:
The curative potency of echinochrome (Ech) on -
glutamyltransferase (GGT) activity (U/L) of diabetic rats.
76
Table 14:
The curative potency of echinochrome (Ech) on total
bilirubin concentration (mg/dl) of diabetic rats.
78
Table 15:
The curative potency of echinochrome (Ech) on direct
bilirubin (DB) concentration (mg/dl) of diabetic rats.
79
Table 16:
The curative potency of echinochrome (Ech) on indirect
bilirubin (IB) concentration (mg/dl) of diabetic rats.
81

vii
Table 17:
The curative potency of echinochrome (Ech) on total
protein (TP) concentration (g/dl) of diabetic rats.
82
Table 18:
The curative potency of echinochrome (Ech) on albumin
concentration (g/dl) of diabetic rats.
83
Table 19:
The curative potency of echinochrome (Ech) on globulins
concentration (g/dl) of diabetic rats.
85
Table 20:
The curative potency of echinochrome (Ech) on albumin/
globulins (A/G) ratio of diabetic rats.
87
Table 21:
The curative potency of echinochrome (Ech) on
triglycerides (TG) concentration (mg/dl) of diabetic rats.
88
Table 22:
The curative potency of echinochrome (Ech) on total
cholesterol (TC) concentration (mg/dl) of diabetic rats.
90
Table 23:
The curative potency of echinochrome (Ech) on low density
lipoprotein cholesterol (LDL-C) concentration (mg/dl) of
diabetic rats.
91
Table 24:
The curative potency of echinochrome (Ech) on high
density lipoprotein cholesterol (HDL-C) concentration
(mg/dl) of diabetic rats.
93
Table 25:
The curative potency of echinochrome (Ech) on serum
creatinine concentration (mg/dl) of diabetic rats.
94
Table 26:
The curative potency of echinochrome (Ech) on urine
creatinine concentration (mg/dl) of diabetic rats.
96
Table 27:
The curative potency of echinochrome (Ech) on creatinine
clearance (ml/min) of diabetic rats.
97
Table 28:
The curative potency of echinochrome (Ech) on serum uric
acid concentration (mg/dl) of diabetic rats.
99
Table 29:
The curative potency of echinochrome (Ech) on urine uric
acid concentration (mg/dl) of diabetic rats.
100
Table 30:
The curative potency of echinochrome (Ech) on serum urea
concentration (g/dl) of diabetic rats.
102
Table 31:
The curative potency of echinochrome (Ech) on urine urea
concentration (g/dl) of diabetic rats.
103
Table 32:
The curative potency of echinochrome (Ech) on liver
malondialdehyde (MDA) concentration (nmol/g.tissue) of
diabetic rats.
105

viii
Table 33:
The curative potency of echinochrome (Ech) on liver
glutathione reduced (GSH) concentration (mg/g.protein) of
diabetic rats.
106
Table 34:
The curative potency of echinochrome (Ech) on liver
catalase (CAT) activity (U/g. protein) of diabetic rats.
108
Table 35:
The curative potency of echinochrome (Ech) on liver
superoxide dismutase (SOD) activity (U/g. protein) of
diabetic rats.
109
Table 36:
The curative potency of echinochrome (Ech) on liver
glutathione-S-transferase (GST) activity (U/ g. protein) of
diabetic rats.
111
Table 37:
The curative potency of echinochrome (Ech) on liver nitric
oxide (NO) concentration (mol/L) of diabetic rats.
112
Table 38:
The curative potency of echinochrome (Ech) on kidney
malondialdehyde (MDA) concentration (nmol/g. tissue) of
diabetic rats.
114
Table 39:
The curative potency of echinochrome (Ech) on kidney
glutathione reduced (GSH) concentration (mg/ g. tissue) of
diabetic rats.
115
Table 40:
The curative potency of echinochrome (Ech) on kidney
catalase (CAT) activity (U/ g. tissue) of diabetic rats.
117
Table 41:
The curative potency of echinochrome (Ech) on kidney
superoxide dismutase (SOD) activity (U/ g. tissue) of
diabetic rats.
118
Table 42:
The curative potency of echinochrome (Ech) on kidney
glutathione-S-transferase (GST) activity (U/ g. tissue) of
diabetic rats.
120
Table 43:
The curative potency of echinochrome (Ech) on kidney
nitric oxide (NO) concentration (mol/L) of diabetic rats.
121

ix
List of figures
Title
Page
Figure 1:
Formation of advanced glycation endproducts.
7
Figure 2:
The polyol pathway.
8
Figure 3:
Relation between NADPH and GSH.
10
Figure 4:
Synthesis of NO.
10
Figure 5:
Activation of PKC.
11
Figure 6:
External shape of Paracentrotus lividus.
16
Figure 7:
The curative potency of echinochrome (Ech) on the final
body weight (gm) of diabetic rats.
59
Figure 8:
The curative potency of echinochrome (Ech) on urine
volume (ml/24hr) of diabetic rats.
60
Figure 9:
The curative potency of echinochrome (Ech) on hot plate
period (Sec) of diabetic rats.
62
Figure 10:
The curative potency of echinochrome (Ech) on wire
suspension period (Sec) of diabetic rats.
63
Figure 11:
The curative potency of echinochrome (Ech) on glucose
concentration (mg/dl) of diabetic rats.
65
Figure 12:
The curative potency of echinochrome (Ech) on insulin
concentration (µU/ml) of diabetic rats.
66
Figure 13:
The curative potency of echinochrome (Ech) on arginase
activity (U/L) of diabetic rats.
68
Figure 14:
The curative potency of echinochrome (Ech) on
hexokinase activity (U/min/ gm.tissue) of diabetic rats.
69
Figure 15:
The curative potency of echinochrome (Ech) on glucose-6-
phosphate dehydrogenase (G6PD) activity (U/min/
gm.tissue) of diabetic rats.
71
Figure 16:
The curative potency of echinochrome (Ech) on aspartate
aminotransferase (AST) (U/ml) of diabetic rats.
72
Figue 17:
The curative potency of echinochrome (Ech) on alanine
aminotransferase (ALT) activity (U/ml) of diabetic rats.
74

x
Figure 18:
The curative potency of echinochrome (Ech) on alkaline
phosphatase (ALP) activity (U/L) of diabetic rats.
75
Figure 19:
The curative potency of echinochrome (Ech) on -
glutamyltransferase (GGT) activity (U/L) of diabetic rats.
77
Figure 20:
The curative potency of echinochrome (Ech) on total
bilirubin concentration (mg/dl) of diabetic rats.
78
Figure 21:
The curative potency of echinochrome (Ech) on direct
bilirubin (DB) concentration (mg/dl) of diabetic rats.
80
Figure 22:
The curative potency of echinochrome (Ech) on indirect
bilirubin (IB) concentration (mg/dl) of diabetic rats.
81
Figure 23:
The curative potency of echinochrome (Ech) on total
protein (TP) concentration (g/dl) of diabetic rats.
83
Figure 24:
The curative potency of echinochrome (Ech) on albumin
concentration (g/dl) of diabetic rats.
84
Figure 25:
The curative potency of echinochrome (Ech) on globulins
concentration (g/dl) of diabetic rats.
86
Figure 26:
The curative potency of echinochrome (Ech) on albumin/
globulins (A/G) ratio of diabetic rats.
87
Figure 27:
The curative potency of echinochrome (Ech) on
triglycerides (TG) concentration (mg/dl) of diabetic rats.
89
Figure 28:
The curative potency of echinochrome (Ech) on total
cholesterol (TC) concentration (mg/dl) of diabetic rats.
90
Figure 29:
The curative potency of echinochrome (Ech) on low
density lipoprotein cholesterol (LDL-C) concentration
(mg/dl) of diabetic rats.
92
Figure 30:
The curative potency of echinochrome (Ech) on high
density lipoprotein cholesterol (HDL-C) concentration
(mg/dl) of diabetic rats.
93
Figure 31:
The curative potency of echinochrome (Ech) on serum
creatinine concentration (mg/dl) of diabetic rats.
95
Figure 32:
The curative potency of echinochrome (Ech) on urine
creatinine concentration (mg/dl) of diabetic rats.
96
Figure 33:
The curative potency of echinochrome (Ech) on creatinine
clearance (ml/min) of diabetic rats.
98

xi
Figure 34:
The curative potency of echinochrome (Ech) on serum uric
acid concentration (mg/dl) of diabetic rats.
99
Figure 35:
The curative potency of echinochrome (Ech) on urine uric
acid concentration (mg/dl) of diabetic rats.
101
Figure 36:
The curative potency of echinochrome (Ech) on serum
urea concentration (g/dl) of diabetic rats.
102
Figure 37:
The curative potency of echinochrome (Ech) on urine urea
concentration (g/dl) of diabetic rats.
104
Figure 38:
The curative potency of echinochrome (Ech) on liver
malondialdehyde (MDA) concentration (nmol/g.tissue) of
diabetic rats.
105
Figure 39:
The curative potency of echinochrome (Ech) on liver
glutathione reduced (GSH) concentration (mg/g.protein) of
diabetic rats.
107
Figure 40:
The curative potency of echinochrome (Ech) on liver
catalase (CAT) activity (U/g. protein) of diabetic rats.
108
Figure 41:
The curative potency of echinochrome (Ech) on liver
superoxide dismutase (SOD) activity (U/g. protein) of
diabetic rats.
110
Figure 42:
The curative potency of echinochrome (Ech) on liver
glutathione-S-transferase (GST) activity (U/ g. protein) of
diabetic rats.
111
Figure 43:
The curative potency of echinochrome (Ech) on liver nitric
oxide (NO) concentration (mol/L) of diabetic rats.
113
Figure 44:
The curative potency of echinochrome (Ech) on kidney
malondialdehyde (MDA) concentration (nmol/g. tissue) of
diabetic rats.
114
Figure 45:
The curative potency of echinochrome (Ech) on kidney
glutathione reduced (GSH) concentration (mg/ g. tissue) of
diabetic rats.
116
Figure 46:
The curative potency of echinochrome (Ech) on kidney
catalase (CAT) activity (U/ g. tissue) of diabetic rats.
117
Figure 47:
The curative potency of echinochrome (Ech) on kidney
superoxide dismutase (SOD) activity (U/ g. tissue) of
diabetic rats.
119
Figure 48:
The curative potency of echinochrome (Ech) on kidney
glutathione-S-transferase (GST) activity (U/ g. tissue) of
diabetic rats.
120
Figure 49:
The curative potency of echinochrome (Ech) on kidney
nitric oxide (NO) concentration (mol/L) of diabetic rats.
122
Figure 50:
Photomicrograph of hematoxylin and eosin stained
pancreas sections.
123
Figure 51:
Photomicrograph of hematoxylin and eosin stained liver 125

xii
sections.
Figure 52:
Photomicrograph of hematoxylin and eosin stained kidney
sections.
127
Figure 53:
The hypoglycemic mechanisms of echinochrome.
166

xiii
List of abbreviation
Abbreviation
Meaning
A/G
Albumin/globulins
AAP
Amino-antipyrine
AAP
Aminophenazone
Ach
Acetylcholine
AchE
Acetylcholine esterase
AGEs
Advanced glycation endproducts
ALP
Alkaline phosphatase
ALT
Alanine aminotransferase
anti-GAD
Anti-glutamic acid decarboxylate
AR
Aldose reductase
AST
Aspartate aminotransferase
BCG
Bromocresol green
CAT
Catalase
CE
Cholesterol esterase
CO
Cholesterol oxidase
CVD
Cardiovascular disorders
DAG
Diacylglycerol
DB
Direct bilirubin
DCHB
Dichloro-2-hydroxybenzenesulfonic acid
DHBS
Dichloro-2-hydroxybenzene sulfonic acid
DM
Diabetes mellitus
DMSO
Dimethyl sulfoxide
DTNB
Dithiobis-2-nitrobenzoic acid
Ech
Echinochrome
G6PDH
Glucose-6-phosphate dehydrogenase
GGT
Gamma-glutamyltransferase
GLUT
Glucose transporter
GOD
Glucose oxidase
GSH
Reduced glutathione
GSH
Glutathione reduced
GST
Glutathione-S-transferase
H&E
Hematoxylin and eosin
HDL-C
High density lipoproteins cholesterol
HFD
High fat diet
HK
Hexokinase
HMOX
Heme oxygenase
IA-2
Insulinoma-associated protein-2
IB
Indirect bilirubin
ICA
Islet cell antibody

xiv
LDL-C
Low density lipoprotein cholesterol
MDA
Malondialdehyde
NAD+
Nicotinamide adenine dinucleotide
NEDA
N-(1-naphthyl)-ethylenediamine
NO
Nitric oxide
NOS
Nitric oxide synthase
PI3K
Phosphatidylinositol-3 kinase
PIs
Phosphatidylinositides
PKC
protein kinase C
PLC
Phospholipase C
PLD
Phospholipase D
POD
Peroxidase
PP
Pancreatic polypeptide
ROS
Reactive oxygen species
SDH
Sorbitol dehydrogenase
SOD
Superoxide dismutase
STZ
Streptozotocin
T1DM
Type 1 diabetes mellitus
T2DM
Type 2 diabetes mellitus
TAGEs
Toxic advanced glycation endproducts
TB
Total bilirubin
TBA
Thiobarbituric acid
TC
Total cholesterol
TCA
Trichloroacetic acid
TG
Triglycerides
TP
Total protein
VLDL
Very low density lipoproteins

1
I. Introduction
Glucose is an essential metabolic substrate of all mammalian cells. Most
of the energy needed to sustain life is delivered by oxidation of glucose
(Pischetsrieder, 2000). Although glucose is required by all cells, its main
consumer is the brain in the fasting or postabsorptive phase, which accounts for
approximately 50% of the body's glucose use. Another 25% of glucose disposal
occurs in the splanchnic area (liver and gastrointestinal tissue), and the
remaining 25% takes place in insulin-dependent tissues, including muscles and
adipose tissues (DeFronzo, 2004). Approximately 85% of endogenous glucose
production is derived from the liver, with glycogenolysis and gluconeogenesis
contributing equally to the basal rate of hepatic glucose production. The
remaining ~15% of glucose is produced by the kidneys (Mari et al., 1994;
DeFronzo, 2004).
The pancreas is considered as a doubled-entity organ, with both exocrine
and endocrine components, reciprocally interacting with a composed system
whose function is relevant for digestion, absorption, and homeostasis of
nutrients (Piciucchi et al., 2015). Pancreatic islets composed of many types of
cells, including insulin-producing cells, glucagon-releasing cells,
somatostatin-producing cells, pancreatic polypeptide (PP)-containing cells and
ghrelin containing cells (Damasceno et al., 2014). All of these hormones are
involved in the regulation of nutrient metabolism and glucose homeostasis
(Assmann
et al., 2009).
Normally, following glucose ingestion, the increase in plasma glucose
concentration triggers insulin release, which stimulates splanchnic and
peripheral glucose uptake and suppresses endogenous glucose production. In
healthy adults, blood glucose levels are tightly regulated within a range of 70 to
99 mg/dl, and maintained by specific hormones (e.g., insulin, glucagon,

Introduction
2
incretins) as well as the central and peripheral nervous system, to meet
metabolic requirements (Wardlaw and Hampl, 2007).
Various cells and tissues (within the brain, muscles, gastrointestinal tract,
liver, kidney, and adipose tissue) are also involved in blood glucose regulation
by means of uptake, metabolism, storage, and excretion (DeFronzo, 2004). The
majority of glucose uptake in peripheral tissues occurs in muscles, where
glucose may either be used immediately for energy or stored as glycogen
(Guyton and Hall, 2006). Transport of glucose into muscles is insulin-
dependent, and thus requires insulin for activation of the major enzyme
(glycogen synthase) that regulates production of glycogen (Porte et al., 2003).
While adipose tissue is responsible for a much smaller amount of peripheral
glucose uptake (2%-5%), it plays an important role in the maintenance of total
body glucose homeostasis by regulating the release of free fatty acids (which
increase gluconeogenesis) from stored triglycerides, influencing insulin
sensitivity in the muscles and liver (DeFronzo, 2004). While the liver does not
require insulin to facilitate glucose uptake, it needs insulin to regulate glucose
output (DeFronzo, 2004). So, for example, when insulin concentrations are low,
hepatic glucose output rises (Porte et al., 2003). Additionally, insulin helps the
liver to store most of the absorbed glucose in the form of glycogen (Guyton and
Hall, 2006). The kidneys are increasingly recognized to play an important role
in glucose homeostasis via release of glucose into the circulation
(gluconeogenesis), uptake of glucose from the circulation to meet renal energy
needs, and reabsorption of glucose at the proximal tubule (Wright et al., 2007).
The kidneys also aid in the elimination of excess glucose (when levels exceed
approximately 180 mg/dL, though this threshold may rise during chronic
hyperglycemia by facilitating its excretion in the urine (ADA, 2008).

Introduction
3
Diabetes mellitus (DM) is a metabolic disorder resulting from a defect in
insulin secretion, insulin action, or both (Kumar and Clark, 2002). Insulin
deficiency, in turn, leads to chronic hyperglycemia with disturbances of
carbohydrate, fat and protein metabolism (Lindberg et al., 2004). DM is
considered as one of the most dangerous metabolic disorders in the world
(Sosale
et al., 2015). It is a complex and potentially debilitating disease that
affects an estimated 8.3% of the adult population or 382 million people
worldwide (IDF, 2013). Egypt will have at least 8.6 million adults with diabetes
and will be the tenth largest population of diabetics in the world (Shaw et al.,
2010). The eleventh most important cause of premature mortality in Egypt is
diabetes mellitus (Saad et al., 2013). It's responsible for 2.4% of all years of life
lost. Also, diabetes is the six most important cause of disability burden in Egypt
(NICHP, 2004).
DM generally classified into type 1 (T1DM) and type 2 (T2DM) diabetes
mellitus. Type 1diabetes (T1DM) is an autoimmune disease, which
characterized by loss of insulin producing cells and reliance on exogenous
insulin for survival (Simmons and Michels, 2015). T1DM is characterized by
mononuclear infiltration of the pancreatic islets, followed by the destruction of
insulin-producing cells (Mathis et al., 2001). The two main forms of clinical
type 1 diabetes are type 1a (about 90% of type 1 cases in Europe) which is
thought to be due to immunological destruction of pancreatic cells, resulting in
insulin deficiency; and type 1b (idiopathic, about 10% of type 1 diabetes), in
which there is no evidence of autoimmunity (Bastaki, 2005). Type 1a is
characterized by the presence of islet cell antibody (ICA), anti-glutamic acid
decarboxylate (anti-GAD) and insulinoma-associated protein-2 (IA-2) that
identify the autoimmune process with cells destruction (Zimmet, et al., 2004).
Autoimmune diseases such as Grave's disease, Hashimoto's thyroiditis and
Addison's disease may be associated with T1a (Atkinson and Maclaren, 1994).

Introduction
4
There is no known etiological basis for type 1b diabetes mellitus. Some of these
patients have permanent insulinopaenia and are prone to ketoacidosis, but have
no evidence of autoimmunity (McLarty et al., 1990). This form is more
prevalent among individuals of African and Asian origin (Ahrén and Corrigan,
1984).
Mainly, 90-95% of diabetes are diagnosed as T2DM (Leng et al., 2014).
T2DM is increasing in prevalence worldwide (Unwin et al., 2010), and it is
strongly associated with obesity and insulin resistance (Guilherme et al., 2008),
as well as defects in pancreatic cells function and mass (Butler et al., 2003).
T2DM is a multifactorial disease, where the pathophysiology of which involves
not only the pancreas, but also the liver, gastrointestinal tract, adipose tissue and
brain (Cornell, 2015). The cells normally compensate insulin resistance
through more insulin production to keep the glucose near the normal level
(Riguera, 1997). Reduced sensitivity to insulin in liver, muscle, and fatty tissue
lead to impaired insulin secretion and hyperglycemic condition which consider
one of the most characteristic feature of T2DM (Cornell, 2015). Insulin
resistance may be defined as a condition in which normal insulin concentrations
fail to achieve a normal metabolic response (Campbell et al., 1988). Insulin
resistance is usually characterized by a reduction in insulin-stimulated storage of
glucose as glycogen in skeletal muscle and liver (Nielsen, 2008). Insulin
resistance in skeletal muscles is among the earliest detectable defects in humans
with T2DM (Mauvais-Jarvis and Kahn, 2000). Type 2 diabetic patients are
characterized by a decreased fat oxidative capacity and high levels of circulating
free fatty acid (Black et al., 2000). The latter is known to cause insulin
resistance by reducing stimulated glucose uptake most likely via accumulation
of lipid inside the muscle cells (Boden, 1999).
As the disease progresses tissue or vascular damage ensues, leading to
severe diabetic complications such as retinopathy (Bearse et al., 2004),

Introduction
5
neuropathy (Seki et al., 2004), nephropathy (Shukla et al., 2003),
cardiovascular complications (Saely et al., 2004) and ulceration (Wallace et al.,
2002). Thus, uncontrolled diabetes is implicated in a wide range of
heterogeneous diseases.
Diabetic retinopathy is characterized by a spectrum of lesions within the
retina and is the leading cause of blindness among adults aged 20­74 years
(Frank, 2004). These include changes in vascular permeability, capillary
microaneurysms, capillary degeneration, and excessive formation of new blood
vessels (neovascularization). The neural retina is also dysfunctional with death
of some cells, which alters retinal electrophysiology and results in an inability to
discriminate between colors (Forbes and Cooper, 2011).
More than half of all individuals with diabetes eventually develop
neuropathy (Abbott et al., 2011). Diabetic neuropathy affects all peripheral
nerves, including pain fibers, motor neurons and the autonomic nervous system
(Said, 2007). The pathogenesis of diabetic neuropathy is complicated, and the
mechanism of this disease remains poorly understood. It has been suggested that
hyperglycemia is responsible for changes in the nerve tissue (Vinik et al., 1992).
Changes in the blood vessels supplying the peripheral nerves underlie the
mechanisms involved in microvascular damage and hypoxia. Advanced
neuropathy due to nerve fiber deterioration in diabetes is characterized by
altering sensitivities to vibrations and thermal thresholds, which progress to loss
of sensory perception. Pain is also seen in some diabetic individuals without
clinical evidence of neuropathy (10­20%), which can seriously impede quality
of life (Obrosova, 2009).
Diabetic nephropathy represents the major cause of end stage renal
failure in Western societies (Gilbertson et al., 2005). Clinically, it is

Introduction
6
characterized by the development of proteinuria with a subsequent decline in
glomerular filtration rate, which progresses over a long period of time, often
over 10­20 years. If left untreated, the resulting uremia is fatal (Forbes and
Cooper, 2011). Importantly, kidney disease is also a major risk factor for the
development of macrovascular complications such as heart attacks and strokes
(Matsushita
et al., 2010). Once nephropathy is established, blood pressure is
often seen to rise, but paradoxically in the short term, there can be
improvements in glycemic control as a result of reduced renal insulin clearance
by the kidney (Amico and Klein, 1981).
Cardiovascular disorders (CVD) accounts for more than half of the
mortality seen in the diabetic population (Haffner et al., 1998), and diabetes
equates to an approximately threefold increased risk of myocardial infarction
compared with the general population (Domanski et al., 2002). In T1DM, it is
not common to see the progression of CVD without impairment in kidney
function (Groop et al., 2009). In T2DM, kidney disease remains a major risk
factor for premature CVD, in addition to dyslipidemia, poor glycemic control,
and persistent elevations in blood pressure (Drury et al., 2011). CVD in
diabetes includes premature atherosclerosis, manifest as myocardial infarction,
stroke as impaired cardiac function well as predominantly diastolic dysfunction
(Forbes and Cooper, 2011).
Oxidative stress has been strongly implicated in the development of
diabetes and diabetic complications (Lupachyk et al., 2013). Metabolic
disorders, assist the increased reactive oxygen species (ROS) production in the
physiological system such as obesity, insulin resistance and diabetes mellitus
(Bhattacharya
et al., 2013). Excess production of ROS can overcome the
antioxidant system of the body, leading to oxidative stress (Ashafaq et al.,
2014). Hyperglycemia condition can induce oxidative stress through many
mechanisms such as glucose autoxidation, polyol pathway, advanced glycation

Introduction
7
endproducts, (AGEs) formation and protein kinase C (PKC)
(Tangvarasittichai, 2015).
AGEs are modifications of proteins or lipids that become non-
enzymatically glycated and oxidized after contact with aldose sugars (Singh et
al., 2001). Early glycation and oxidation processes, resulting in the formation of
Schiff bases and Amadori products. Further glycation of proteins and lipids
causes molecular rearrangements that lead to the generation of AGEs (Schmidt,
et al., 1994) (Fig.1).
Figure 1: Formation of advanced glycation endproducts.
AGEs may produce ROS, bind to specific cell surface receptors, and
form cross-links (Brownlee et al., 1985). AGEs form in vivo in hyperglycemic
environments and during aging and contribute to the pathophysiology of
vascular disease in diabetes (Schmidt et al., 1995).
AGEs induce link processes
in the structure of long lifespan proteins, such as collagen, modifying blood
vessel structure (Christiane et al., 2014). By binding to their specific receptors,
they activate intracellular signaling pathways which lead to cytokine production,
responsible for the proinflammatory and prosclerotic effects (Inagaki et al.,

Introduction
8
2003). Toxic advanced glycation endproducts (TAGEs) derived from
glyceraldehydes, are very aggressive compounds and represent the dominant
form of AGEs. The interaction between TAGEs and their receptors in
endothelial and inflammatory cells, leads to intracellular generation of ROS via
the electron transport chain, NADPH oxidase, xanthine oxidase and arachidonic
acid metabolism (Brownlee, 2005).
Aldose reductase (AR) participates in the glucose metabolism by being
the rate-limiting enzyme of the polyol (polyhydric alcohol) pathway. In this
pathway, glucose is reduced to sorbitol by AR, using NADPH as an electron
donor. Sorbitol is subsequently oxidized to fructose by the enzyme sorbitol
dehydrogenase (SDH), using nicotinamide adenine dinucleotide (NAD+) as an
electron acceptor (Forbes and Cooper, 2011) (Fig. 2).
Figure 2: The polyol pathway.
AR acts on many different aldehyde-containing substrates, but sugars
with shorter chains are better substrates than hexoses (Kinoshita, 1990). Due to
its low affinity for glucose, only 3% of all glucose is converted to sorbitol at
normal glucose levels (Ko et al., 1995). Instead, glucose is mainly
phosphorylated into glucose 6-phosphate by hexokinase, which is further
metabolized to pyruvate and lactate via the glycolytic pathway. During
hyperglycemias conditions, however, one third of the glucose is metabolized by
the polyol pathway (Ramana et al., 2003). In addition, the AR activity is

Introduction
9
enhanced by high glucose concentrations (Kubo et al., 2001). This
hyperglycemia-induced an increase in flux through the polyol pathway has been
proposed to play an important role in the pathogenesis of secondary diabetic
complications in the eyes, nerves and kidneys (Ramana et al., 2003). Although
it is unknown how the increased flux through the polyol pathway is involved in
the occurrence of diabetic disorders several possible mechanisms have been
suggested. One of the first mechanisms proposed is based on the development of
osmotic stress due to sorbitol accumulation (Kinoshita, 1990). Glucose can
easily penetrate the cell membrane while the polyol sorbitol is an osmolyte with
low permeability. Thus, the excessive polyol pathway metabolism results in an
accumulation of sorbitol inside the cells. This causes hypertonicity and osmotic
imbalance that in turn leads to typical diabetic complications, due to swelling
and disruption of the intracellular environment (Kinoshita and Nishimura,
1988)
.
The most discussed explanation of the deleterious effects of diabetes
caused by a hyperactive polyol pathway is an increased oxidative stress (Lee
and Chung, 1999). When the flux through the polyol pathway is enhanced the
cofactor NADPH is depleted resulting in decreased regeneration of reduced
glutathione (GSH), which also requires NADPH to be formed. GSH is a key
player in this antioxidative system, with a significant function in ROS
scavenging (Noctor et al., 1998) (Fig. 3).

Introduction
10
Figure 3: Relation between NADPH and GSH.
The depletion of NADPH also affects the production of NO since NO
synthase competes with AR for the same cofactor (Fig.4).
Figure 4: Synthesis of NO.
Decreased levels of NO produce vasoconstriction and slowing of nerve
conduction, which is involved in the pathogenesis of diabetic neuropathy
(Stevens
et al., 1994). It has also been suggested that NOS is inactivated by
enhanced levels of ROS, produced as a result of the hyperglycemia increased
endothelial cell sorbitol concentrations (Gupta et al., 2002).

Introduction
11
Protein kinase C (PKC) comprises a family of at least 12 isoforms of
serine threonine kinases (Mellor and Parker, 1998). Many in vivo (Shiba et al.,
1993) and in vitro (Inoguchi
et al., 1992) studies have suggested that increased
diacylglycerol (DAG) levels in vascular tissues, is related to PKC activation in
diabetes mellitus. The source of DAG that activates PKC can be derived from
the hydrolysis of phosphatidylinositides (PIs) or from the metabolism of
phosphatidylcholine (PC) by phospholipase C (PLC) or phospholipase D (PLD).
Alternatively, DAG can be synthesized by de novo pathway from glycolytic
intermediates (Park et al., 1999). In metabolic labeling studies, it has been
reported that the incorporation of glucose into the glycerol backbone of the
DAG was increased by hyperglycemia, suggesting that increased DAG contents
were partly derived from de novo pathway (Inoguchi et al., 1994). Recent
studies have identified that the activation of PKC and increased DAG levels are
associated with many vascular abnormalities in retinal, renal, and cardiovascular
tissues (Koya and King, 1998) (Fig. 5).
Figure 5: Activation of PKC.

Introduction
12
Streptozotocin (STZ) is an antibiotic that was first isolated from the
bacterium Streptomyces achromogens (Vavra et al., 1959). Streptozotocin is
well known for its selective beta cell cytotoxicity, which induces DM in rats
(Mitra
et al., 1996). STZ induced diabetic rat is a suitable model to investigate
the metabolic changes associated with DM as in humans (Ugarte et al., 2012).
The toxic mechanism of STZ is mediated by ROS (Szkudelski, 2001). A single
dose of STZ can induce T1DM in rats (Yin et al., 2006), while T2DM can be
induced by STZ injection after high fat diet (HFD) feeding (Skovso, 2014). The
animal model of high-fat diet, which combined with low dose STZ induced
diabetes manifests many characteristics of human T2DM, such as
hyperglycemia, hyperlipemia and lack of insulin secretion (Yao et al., 2015). In
addition, during HFD/STZ rat model, HFD induces insulin resistance and low
doses of intraperitoneal STZ induce moderate impairment of insulin secretion.
Most of the modern anti-diabetic drugs, including insulin and oral
hypoglycaemic agents only control blood sugar levels as long as they are
regularly administered and are associated with a number of undesirable effects
(Cheng and Caughey, 2007). This generates the need for better, convenient and
less toxic treatment options.
Traditionally, natural products have played an important role in drug
discovery and were the basis of most early medicines (Newman et al., 2000).
The history of the extraction of natural products dates back to the Mesopotamian
and Egyptian times, where production of perfumes, or pharmaceutically - active
oils and waxes were a major business (Bart, 2011). Besides venoms, toxins, and
antibiotic peptides from animals (frogs, spider, snake, etc.), a new focus is
nowadays on the marine world (Bart, 2011). Countless marine plants and
animals contain biochemical secrets that, if unlocked, can provide new insights
and understanding of human diseases and their treatment (Murti and Agrawal,

Introduction
13
2010).
Marine organisms are a wonderful source of biologically active natural
products (Bhakuni and Rawat, 2005).
Many bioactive compounds have been
extracted from various marine animals (Harvey, 2000). The study of marine
chemical compounds produced by different organisms showed the strategies for
their use for human benefit (Muller et al., 2003). The number of natural
products isolated from marine organisms increases rapidly, and now exceeds
with hundreds of new compounds being discovered every year (Proksch and
Muller, 2006). Chemicals produced by or found in marine organisms have been
shown to have a wide variety of applications as pharmaceuticals for humans and
other animals (Fahmy and Soliman, 2013). These marine chemicals included
antibacterial, analgesic, anti-inflammatory, antimalarial, anticancer, antiparasitic
and antiviral agents (Raval et al., 2013).
A majority of pharmacologically active secondary metabolites have been
isolated from echinoderms (Carballeria et al., 1996). There is much valuable
information for new antibiotic discoveries which give new insights into the
bioactive compounds from the sea urchin (Bragadeeswaran et al., 2013).
Sea urchin (Paracentrotus lividus) is a widespread species in the Atlantic
and the Mediterranean coasts and is subjected to intensive commercial fishing in
several countries (Arafa et al., 2012). Alike many other marine invertebrates,
sea urchins have been considered a source of biologically active compounds
with biomedical applications (Kelly, 2005). However, the potential of echinoids
as a source of biologically active products are largely unexplored
(Bragadeeswaran
et al., 2013).
The sea urchin shells are containing various polyhydroxylated
naphtoquinone pigments, spinochromes (Jeong et al., 2014a) as well as their
analogous compound echinochrome (Ech) which showed bactericidal effect
(Service and Wardlaw, 1984). Ech chemical structure was confirmed by X-ray

Introduction
14
analysis (Gerasimenko et al., 2006). It is water insoluble compound that
possesses strong antioxidant effects and is the active substance in the drug
Histochrome (Jeong et al., 2014b). Histochrome drug is used in ophthalmic
practice to treat intraocular hemorrhage, diabetic retinopathy, dystrophies,
central retinal vein thrombosis, and post-traumatic hemorrhage in Russia
(Mischenko
et al., 2003). It has been suggested that the phenolic hydroxyl
groups in these molecules could participate in antioxidant activity as was
observed in other well-known antioxidant polyphenols such as tea catechins
(Shankarlal
et al., 2011). The similar compounds are also found in the shells of
sea urchins and thus suggesting that they, as well as echinochrome, would act as
antioxidant substances similar to other polyphenolic antioxidants in edible plants
(Chantaro
et al., 2008). Ech can act through many antioxidant mechanisms,
including the scavenging of active oxygen radicals (Lebedev et al., 2005),
interaction with lipoperoxide radicals (Boguslavskaya et al., 1985), chelation of
metal ions (Lebedev et al., 2008), inhibition of lipid peroxidation (Lebedev et
al., 2008), and regulation of the cell redox potential (Mischenko et al., 2009).
Furthermore, a complex of polyhydroxylated naphthoquinone pigments and
minerals from shells of sea urchins decreases the concentration of blood glucose,
stimulates the synthesis of phospholipids in the liver, and has antioxidant
properties (Kovaleva et al., 2013).

Introduction
15
Aim of work
The present study carried out to evaluate the antidiabetic efficacy of
echinochrome pigment representing its mechanism in streptozotocin-induced
types I and II diabetic rat models.
Therefore, the present study aimed to include these parts:
The first one aims for the preparing of an echinochrome (Ech) from
shells and spines of sea urchin "Paracentrotus lividus".
The second part includes investigating the effect of Ech on some serum
constituents of both diabetic types including, diabetic markers (glucose, insulin,
arginase, G6PD, hexokinase), liver function markers (ALT, AST, ALP, GGT,
total protein, albumin, direct bilirubin, indirect bilirubin and total bilirubin),
kidney function markers (urea, uric acid, creatinine) and lipid profile
(triglyceride, total cholesterol, LDL-C, HDL-C). Furthermore, evaluate the in
vivo antioxidant effect of Ech through assessing some liver and kidney
parameters include oxidative stress marker such as malondialdehyde (MDA) and
glutathione reduced (GSH) and some antioxidant enzymes such as catalase
(CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST).
The third part aims to evaluate the effect of Ech on pancreas, liver and
kidney architectures using the light microscope.

16
II.
Materials and methods
II.1. Chemicals and reagents
Streptozotocin, dimethyl sulfoxide (DMSO), insulin and hexokinase kits
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gamma-
glutamyltransferase (GGT) kit was purchased from Spectrum Diagnostics
company (Obour City, EGY), while the other kits were purchased from the
Biodiagnostic Company (El Motor St, Dokki, EGY).
II.2. Sea urchin Collection
Sea urchins (Paracentrotus lividus) were collected from the
Mediterranean coast of Alexandria (Egypt) (Fig. 6) and transported to the
laboratory packed in ice. The samples were thoroughly washed with sea water to
remove sand and overgrowing organisms at the collection site and transported to
the laboratory. The collected specimens were identified by the standard
literature of taxonomic guide (Clark and Rowe, 1971). The collected specimens
were immediately shade dried.
Figure 6: External shape of Paracentrotus lividus.

Materials and methods
17
II.3. Echinochrome (Ech) extraction
Pigments in the shells and spines were isolated by the Amarowicz
method with slight modifications (Amarowicz et al., 1994; Kuwahara et al.,
2009). After removal of the internal organs, the shells and spines were washed
with a stream of cold water, air-dried at 4°C for 2 days in the dark and then were
grounded. The powders (5 g) were dissolved by gradually adding 10 ml of 6 M
HCl. The pigments in the solution were extracted 3 times with the same volume
of diethyl ether. The ether layer collected was washed with 5% NaCl until the
acid was almost removed. The ether solution including the pigments was dried
over anhydrous sodium sulfate and the solvent was evaporated under reduced
pressure. The extract including the polyhydroxylated naphthoquinone pigment
was stored at -30°C in the dark.
II.4. Experimental animals
Male albino Wistar rats (Rattus norvegicus) weighing 140 ± 10 gm for
T1DM and 80 ± 10 gm for T2DM were used in this study. The rats were
obtained from the National Research Center (NRC, Dokki, Giza). They were
grouped and housed in polyacrylic cages (six animals per cage) in the well-
ventilated animal house of the Zoology Department, Faculty of Science, Cairo
University. Rats were given food and water ad libitum. Rats were maintained in
a friendly environment of a 12 hr/12 hr light-dark cycle at room temperature (22
­ 25
0
C). They were acclimatized to laboratory conditions for 7 days before
commencement of the experiment.
II.5. Ethical Consideration
Experimental protocols and procedures used in this study were approved
by the Cairo University, Faculty of Science, Institutional Animal Care and Use
Committee (IACUC) (Egypt) (CUFS/F/33/14). All the experimental procedures

Materials and methods
18
were carried out in accordance with international guidelines for the care and use
of laboratory animals.
II.6. Induction of type 1 diabetes mellitus (T1DM)
All rats were starved for 12 hrs before the experiment, but were allowed
free access to water. T1DM was induced by intraperitoneal injection of 60
mg/kg of streptozotocin (STZ) dissolved in 0.1mol/l sodium citrate buffer at pH
4.5. Blood glucose levels were measured 72 hr after injection of STZ using a
blood glucose meter device (ONE TOUCH Ultra 2, USA). Rats were starved,
but had access to drinking water for 6 hrs before blood glucose measurement.
Fasting plasma glucose concentrations 300 mg/100 ml were considered type 1
diabetic in this experiment (Chen et al., 2014).
II.7. Induction of type 2 diabetes mellitus (T2DM)
The rats were fed a high fat diet with energy of 5.3 kcal/g, comprising
60% calories from fat, 35% from protein and 5% from carbohydrate, according
to a modification of the protocols of Reed et al. (2000). After 4 weeks the rats
injected intraperitoneally with a single dose of prepared solution of STZ (30
mg/kg dissolved in 0.1mol/l sodium citrate buffer at pH 4.5). After 72 hours,
fasting plasma glucose concentrations 300 mg/100 ml were considered
diabetic type 2 in this experiment (Ebaid, 2014).
II.8. Experimental design
After one week of acclimatization, 36 rats were assigned into two main
groups (18 rats/group).
x First group: Eighteen rats were randomly assigned into three subgroups
(6 rats/subgroup):
¾ Control subgroup: After a single dose of citrate buffer (0.1mol/l,
i.p), the rats received 1ml (10% DMSO, orally) daily for 4 weeks.

Materials and methods
19
¾ T1DM subgroup: After a single dose of STZ (60 mg/kg, i.p), the
rats received 1ml (10% DMSO, orally) daily for 4 weeks.
¾ Ech subgroup: After a single dose of STZ (60 mg/kg, i.p), the rats
received 1ml Ech (1mg/kg body weight in 10% DMSO, orally)
(Lennikov
et al., 2014) daily for 4 weeks.
x Second group: Eighteen rats were randomly assigned into three
subgroups (6 rats/subgroup):
¾ Control subgroup: After 4 weeks of normal diets feeding, the rats
injected with a single dose of citrate buffer (0.1mol/l, i.p) then
received 1ml of (10% DMSO, orally) daily for 4 weeks.
¾ T2DM subgroup: After 4 weeks of HFD feeding, the rats injected
with a single dose of STZ (30 mg/kg, i.p) then received 1ml of
(10% DMSO, orally) daily for 4 weeks.
¾ Ech subgroup: After 4 weeks of HFD feeding, the rats injected
with a single dose of STZ (30 mg/kg, i.p) then received 1ml Ech
(1mg/kg in 10% DMSO, orally) daily for 4 weeks.
II.9. Determination of the physical parameters
II.9.1. Body weight
Body weight measured at the beginning and at the end of the
experiments.
II.9.2. Urine volume
Urine collected 24 hrs per-experiment ending using a metabolic cage.
Volume of urine measured, then the urine used for kidney function tests.

Materials and methods
20
II.9.3. Hot plate test
The hot plate latency was measured using a modification of the original
method of Eddy and Leimbach (1953). Briefly, the modified apparatus consists
of an electric cooking plate (Saiso, Japan) with a 1500 Watts stainless steel
heating element connected to a thermostat (0-400
0
C); a thermocouple connects
the thermostat to a chrome plated drip pan. The thermocouple together with the
thermostat control the temperature of the hot plate within the desired range once
set. Pain sensitivity was evaluated by the response latency for paw licking on the
hot place. In order to avoid tissue damage, the maximum time the animal could
spend on the hot plate was pegged at 60 seconds. Response latencies were
measured at 15 minute intervals and the average of the results was taken.
II.9.4. Wire suspension
The wire suspension assay measured muscle strength and the prehensile
reflex, an animal's ability to grasp a taut horizontal wire with its forepaws and to
remain suspended. Rats were held gently by the tail and the forepaws were
placed on a suspended wire 2 mm in diameter and 62 cm above a cushioned
surface. The latency to let go was measured using a stopwatch, with shorter
latency to drop indicating reduced strength and/or reflex ability. If a rat did not
let go of the wire within 60 s, it was removed from the wire and a latency of 60 s
was assigned to that measurement. Each rat was used only once in the wire
suspension assay (Troen et al., 2008).
II.10. Animal handling and specimen collection
After the end of all experiments, the rats were fully anesthetized with 3%
sodium pentobarbital, and the chest was opened. A needle was inserted through
the diaphragm and into the heart. Negative pressure was gently applied once the
heart had been punctured, and the needle was repositioned as required until

Materials and methods
21
blood flowed into the syringe. Blood samples collected in centrifuge tubes.
Liver, pancreas and kidney were removed and immediately blotted using filter
paper to remove traces of blood. Part of the liver and kidney stored at -80
o
C for
biochemical analysis. Another parts of pancreas, liver and kidney were
suspended in 10% formal saline for fixation preparatory to histopathological
examination.
II.11. Samples preparation
II.11.1. Serum preparation
The blood samples were centrifuged at 3000 rpm for 20 minutes. The
collected serum, stored at -20
o
C until used for biochemical assays.
II.11.2. Liver and kidney homogenate preparation
Liver and kidney tissues were homogenized (10% w/v) in ice-cold 0.1 M
Tris-HCl buffers (pH7.4). The homogenate was centrifuged at 3000 rpm for 15
min. at 4
o
C and the resultant supernatant was used for the biochemical analyses.
II.11.3. Histopathological examination
Pancreas, liver and kidney tissues were fixed in 10% neutral-buffered
formalin. The fixed specimens were washed, dehydrated, and embedded in
paraffin wax. The tissues were sectioned at a thickness of 4­5 µm and stained
with hematoxylin and eosin (HE) according to Bancrof and Stevens (1996),
as routine procedures for histopathological examination.

Materials and methods
22
II.12. Biochemical assessment
II.12.1. Diabetic markers
II.12.1.1. Determination of glucose
Glucose was determined by colorimetric method using Biodiagnostic
kits, according to the method described by Freund et al. (1986).
Principle:
The enzymatic method uses glucose oxidase (GOD) to catalyze the
oxidation of glucose to hydrogen peroxide and gluconic acid. Hydrogen
peroxide, when combined with 4-aminoantipyrine and a derivative from phenol,
forms a red dye compound. The intensity of the red color produced is directly
proportional to the glucose quantity in the sample. The color intensity is directly
proportional to the protein concentration. It is determined by measuring the
increase in the absorbance at 546 nm.
Reagents:
R1
Standard (ST)
100mg/dl
R2
Phosphate Buffer
100mmol/L
Glucose oxidase
10000 U/L
Peroxidase
2000 U/L
4-AAP
1mmol/L
Phenol
10mmol/L
Procedures:
Blank
Standard
Sample
Reagent (R2)
1.0 ml
1.0 ml
1.0 ml
Distilled water
10 L
-
-
Standard
-
10 µl
-
Sample
-
-
10 µl
Mix, incubate for 10 minutes at room temperature, measure absorbance
of sample (A
sample
) and standard (A
standard
) against reagent blank within 30
minutes.

Materials and methods
23
Calculation:
Where 100 is the standard concentration.
II.12.1.2. Determination of Insulin
Insulin was determined by colorimetric method using Sigma-Aldrich
kits, according to the method described by Herbert et al. (1965).
Principle:
The Mouse/Rat insulin ELISA Kit based on the direct sandwich
technique in which two monoclonal antibodies are directed against separate
antigenic determinants on the insulin molecule. During incubation insulin in the
sample reacts with enzyme (HRP)-conjugated anti-insulin antibody and anti-
insulin antibody bound to microplate well. A simple washing step removes the
unbound enzyme labeled antibody. The bound HRP complex is detected by
reaction with TMB substrate.The reaction is stopped by adding acid to give a
colorimetric endpoint that is read using an ELISA reader.
Reagent:
Microwell coated with Insulin MAb
Insulin Standard 1: 1 vial (ready to use)
Insulin Standards 2-6: 5 vials (ready touse)
Insulin Enzyme Conjugate: 1 vial
Assay Diluent: 1 bottle (ready to use)
TMB Substrate: 1 bottle (ready to use)
Stop Solution: 1 bottle (ready to use)
20x Wash concentrate: 1 bottle

Materials and methods
24
Procedure:
1. Place the desired number of coated strips into the holder.
2. Pipette 25 µL of Insulin standards, control, and sera into appropriate wells.
3. Add 100 µL of working Insulin Enzyme Conjugate to all wells.
4. Thoroughly mix for 10 seconds, it is important to have a complete mixing in
this step.
5. Incubate for 60 minutes at room temperature (18­26
o
C).
6. Remove liquid from all wells. Wash wells three times with 300 µL of 1x wash
buffer. Blot on absorbent paper towels.
7. Add 100 µL of TMB substrate to all wells.
8. Incubate for 15 minutes at room temperature.
9. Add 50 µL of stop solution to all wells. Shake the plate gently to mix the
solution.
10. Read absorbance on ELISA Reader at 450 nm within 15 minutes after
adding the Stopping Solution.
Calculation:
Determine the insulin concentration by using standard curve.
II.12.1.3. Determination of arginase
Arginase was determined using Biodiagnostic kit according to the
method described by Marsch (1965).
Principle:
The method based upon the colorimetric determination of urea by
condensation with diacetyl monoxime in an acid medium in the presence of
ferric chloride and carbazide.
Excerpt out of 243 pages

Details

Title
The Metabolic Effects of Echinochrome Pigment Extracted from Sea Urchin on Diabetic Rats
College
Cairo University  (Faculty of Science)
Grade
4
Author
Year
2018
Pages
243
Catalog Number
V387206
ISBN (eBook)
9783668612952
ISBN (Book)
9783668612969
File size
7032 KB
Language
English
Tags
metabolic, effects, echinochrome, pigment, extracted, urchin, diabetic, rats
Quote paper
M.Sc Ayman Mohamed (Author), 2018, The Metabolic Effects of Echinochrome Pigment Extracted from Sea Urchin on Diabetic Rats, Munich, GRIN Verlag, https://www.grin.com/document/387206

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