COMPARATIVE ASSESSMENT OF THE EEFFECT OF ARGINASE INHIBITOR L-NORVALINE AND MELATONIN RECEPTOR AGONIST AGOMELATINE ON METABOLIC SYNDROME IN HYPERLIPIDEMIC DIABETIC RATS

COMPARATIVE ASSESSMENT OF THE EFFECT
OFARGINASE INHIBITOR L-NORVALINE AND
MELATONIN AGONIST AGOMELATINE ON
METABOLIC SYNDROME IN HYPERLIPIDEMIC
DIABETIC RATS
int-templates-design.com
PRESENTED BY- SHUBHAM BALIYAN
M.PHARM
( PHARMACOLOGY) 2ND YEAR
Under the Supervision of Dr . Mamta F. Singh
SARDAR BHAGWAN SINGH POST
GRADUATE INSTITUTE OF
BIOMEDICAL SCIENCES, DEHRADUN

2
CONTENTS
CONTENTS
1- Introduction
2- Review of literature
3- Research Envisaged
4- Aim and objectives
5- Plan of work
6- Material and methods
7- Results and disscussions
8- References

31-12-2019 4
Metabolic Syndrome(MS)
Metabolic syndrome a disorder generally characterized by
a clusters of health problems that includes accumulation
of excessive fats around the waist area that is called
abdominal obesity, excessive parcels of cholesterol in the
blood stream, extreme level of biological substance known
as tri-glycerides, including very low level of HDLC,
Cardiovascular disease, elevated blood pressure and blood
sugar level in the body and elevated levels of
adipocytokines, hormonal imbalance, chronic stress and
consumption of levels of alcohol, wine, smoking and
including sedentary lifestyle which is common occurrence
these days majorly lead to the risk factors that may rise
the chances of risks of metabolic syndrome. (Mascia D et
al., 2012).

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Figure 1.1: Pathophysiology of Metabolic Syndrome

31-12-2019 7
S.NO. YEAR AUTHOR NAME RESEARCH WORK
1. 2003 Assumpaco CR et al., Effect of the L-arginine-Nitric Oxide Pathway and Oxidative stress on the pathogenesis
of the metabolic syndrome.
2. 2004 Fishman EZ et al., Melatonin a tool for effective treatment in metabolic syndrome low melatonin
generation in coronary disease independent of beta –
adrenoceptor blockade.
3. 2006 Teddy Bagnost et al., Treatment of 25-week-old SHR with the arginase inhibitor N-hydroxy-nor-L-norvaline
sustainably reduced systolic blood pressure. Result showed that a long-term
treatment with an arginase inhibitor diminished blood pressure, improved vascular
function in SHR with completely developed HTN.
4. 2007 Jun zhou et al., The melatonin receptor agonist piromelatine enhances impaired glucose metabolism
in chronically stressed rats fed a high-fat diet.
5. 2008 Robert Segal et al., Arginase inhibition by chronic administration of the arginase inhibitor has been
shown to improve endothelial dysfunction in aged rats.
6. 2010 Milano W et al., Successful treatment with agomelatine in NES: a series of five classes.
7. 2010 Kulikovskaya VA et al., Cardio and endothelioprotective impact of arginase inhibitors L-norvaline at modeling
deficiency of nitric oxide at a portion of 10 mg/kg.
8. 2012 Roy et al., Melatonin and metabolic syndrome: a tool for effective therapy in obesity related
problems.
9. 2014 Daniel
Michealmedigan et al.,
Therapeutic capability of nitrite generated NO
pathway in vascular brokenness.
10. 2014 Ezinyeka Lawrence et al., Impact of nitric oxide and insulin resistance on the pathophysiology of metabolic
syndrome: possibly role of L-Arginine and glutamate.
11. 2016 Abhijit De et al., L-norvaline and Alpha-Tocopherol treatment protect against Diabetes-Induced
Oxidative stress in testes of male Rats.
12. 2016 She M. et al., Piromelatine, a novel Melatonin receptor agonist, settles metabolic
profiles and enhances

31-12-2019 8
.insulin resistance in chronic sleep restricted rats.
13. 2017 Hatzis G et al., Melatonin, given at the time of reperfusion, kept ventricular
arrhythmias in confined hearts from fructose- fed diet-induced fatty
liver illness in rats.
14. 2017 Favero G et al., Melatoni reduced obesity and restored adipokines patterns and
metabolism in obese (ob/ob) mice.
15. 2017 Rios-Lugo MJ et al., In high-fat-fed rats, melatonin attenuated body weight increase, the
increase in plasma glucose, insulin, adiponectin, leptin, TGs and
cholesterol levels, and counteracted disrupted 24-hour
patterns.
16. 2018 EL-Bassossy HM et al., Arginase inhibition alleviates HTN in the metabolic syndrome.
17. 2018 Peyton KJ et al., Arginase inhibition prevents the development of hypertension and
improves insulin resistance in obese rats.
18. 2018 John Pernow et al., Arginase as a potential objective in the treatment of cardiovascular
disease.

31-12-2019 9
Arginase inhibitor
Arginase inhibitors are the class of drugs which competitively or non-
competitively inhibit the activity of arginase (subtype I and II) enzyme. arginase
inhibitors used here are proposed to treat DM and vascular endothelial
dysfunction. These inhibitors are also used in hypertension, atherosclerosis,
neurodegenerative disease and renal disease. (Stephan J et al., 2013)
Mechanism of action of Arginase

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Examples of different classes of Arginase inhibitors
Arginase inhibitors may be competitive and non-competitive; few are specific or non-
specific the following are the examples
S.NO Arginase Inhibitor Molecuar Weight and formula Type of Inhibition
1. (S)-(2-BORONOETHYL- C5H13BCINO4S Slow binding competitive
CYSTEINE, HCL) (BEC) 229.5
inhibitor of Arg-I and II
2. BEC AMMONIUM C5H11BNO4S.NH4 Slow Binding Competitive
SALT 210 Inhibitor or Arg-I and II
3. (2S)-AMINO5- C7H13N2O3I Slow Binding Irreversible
IODOACETAMIDOPEN 300.09 Inhibitor of Arginase
TANOIC ACID
4. NG-HYDROXYL-L- C6H12N4O3.C2H4O2 Non-Specific Inhibitors
ARGININE, 250.25
MONOACETATE SALT
5. L-NORVALINE C5H11NO2 Potent Inhibitor
117.15
6. SALVIALONIC ACID C26H22O1 Non-Selective
494.45
7. DL-ALPHA- C6H12F2N2O2.HCL Specific Irreversible
DIFLUROMETHYL 218.7
ORNITHINE HCL

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L-NORVALINE
L-Norvaline is an arginase inhibitor that keeps your body from creating nitric oxide. At the point when the body's nitrous oxide generation
is repressed our body encounters a diminishing in vasodilation that outcomes enlarging of veins.
L-Norvaline Molecular equation is C5H11NO2 having Molecular weight-117.15 L-norvaline is white crystalline powder with dissolving
indicate 303-degree C 307-degree C and well solvent in water, solvent in high temp water and weaken hydrochloric corrosive. Past research
suggested that L-Norvaline is an entrenched arginase inhibitor when managed in diabetic male rodents. Arginase inhibitor L-Norvaline
additionally improves endothelial dysfunctions which may take parts in Type 2 Diabetes and cardiovascular disease.
(Stephanie L et al., 2017)
Structure of L-Norvaline

31-12-2019 12
Melatonin
Melatonin (5-methoxy-N-acetyltryptamine) (MLT) is a hormone basically passed on and
created by the pineal organ that goes about as a significant synchronizer of endocrine rhythms
(Detanico et al., 2009) and is a homeostatic controller or pharmacological help of the impacts
of weight (Kopp et al., 1999). Studies have suggested that MLT treatment reduced body mass
index , blood pressure and LDL-C levels and melatonin levels are reduced in disease associated
with insulin resistance.(Konakchieva et al.,1998; Kitagawa et al.,2012; Sartori et al.,2009; Shieh
et al.,2009)
Melatonin Receptor Agonist
( Agomelatine)
Agomelatine is a melatonin receptor-1/melatonin receptor-2 agonist with high
partiality for MLT receptors and higher instrument of activity when contrasted with
melatonin. Agomelatine anticipated the concealment of body weight addition and
vitality admission and furthermore counteracted dysregulation of qualities engaged
with lipid and glucose digestion, including proinflammatory cytokines. Agomelatine
has been demonstrated to be gainful in improving metabolic disorder through its
enemy of hyperlipidemic activity and modulatory activity on insulin's blend and its
discharge. (Pehrson et al., 2015)
Structure of Agomelatine

13
Research Envisaged
 Previous studies suggested that melatonin agonist treatment not only improves body weight and blood pressure
but also improves glucose homeostasis and dyslipidemia. M-S (Metabolic syndrome) is worldwide public-health
issues for both genders now a days. Clinical challenges in the societies, more energy intake, increasing visceral
obesity, and some sedentary habits of life. M-S*, also known as (IRS)-(e.g. Insulin-resistance-syndrome) is a
condition consisting of Dyslipidemia, obesity, hyperglycemia, hyperinsulinemia, hyperglycemia, and hypertension.
Abdominal adiposity, Insulin resistance, genetic susceptibility, endothelial dysfunction, high blood pressure,
increased level of adipocytokines as well as chronic stress are the quite a few risk factors which comprise the
metabolic syndrome.
 The main criteria shall be to prevent vascular difficulties in hypertensive/insulin resistance as well as affect
carbohydrate metabolism. Inhibition of Arginase by, norvaline, citrulline reduces hypertension related with
metabolic syndrome by prolongation of endothelial- dependent relaxation and NO generation as well as by insulin
resistance inhibition and increased level of triglycerides.
 Relative to current literature and studies, it is surely expected that use of melatonin agonist and arginase inhibitor
may be very beneficial in altering the conditions related with MS & may be clinically benefit for the prevention of
MS as well as associated problems.

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Aim and Objectives
Comparative Assessment of the effect of Arginase inhibitor L-Norvaline and Melatonin agonist
Agomelatine on Metabolic syndrome in Hyperlipidemic Diabetic Rats
AIM
Objectives
1. Induction of metabolic syndrome in rats by hyperlipidemic diet
2. Treatment of rats with arginase inhibitor , L-Norvaline and melatonin agonist Agomelatine for 30
days
3. Investigation of L-Norvaline and Agomelatine effects on metabolic syndrome by estimating the
physical , biochemical and oxidative stress parameters and histopathological studies
4. Evaluation of body weight and BMI
5. Estimation of biochemical parameters like fasting blood glucose, Total Cholesterol, HDL, LDL, VLDL,
Triglycerides level
6. Estimation of Biochemical parameters leptin, Adiponectin and CRP in the serum of treated rats
7. The status of pro-oxidant and the level of antioxidant enzymes like LPO, SOD was estimated in the
serum of treated rats.
8. Histopathology of pancreas of treated rats were done to observe the effect of the treatment

31-12-2019 15
Plan of Work
3rd
semester
4th
Semester
Literature Survey
Procurement of experimental
materials and drugs
Selection of drugs
Animal were housed in standard cages
and kept under physiological
environmental circumstances
After acclimatization of animals in lab
conditions hyperlipidemic diet along with
fructose was used for induction of MS in
rats
The hyperlipidaemic diabetic animals was
divided into 8 groups ( each group contain 6
animals
Treatment of hyperlipidaemic diabetic rats by the Arginase
inhibitor L-Norvaline ( 8 mg/kg and 16 mg/kg p.o.) and
Melatonin receptor agonist Agomelatine ( 10 mg/kg and 20
mg/kg p.o) for a period of 30 days
After the treatment period animals was
anaesthetized with di ethyl ether and blood was
collected from the retro orbital plexus
Serum was separated and used for the estimated
of different biochemical and hormonal parameters
Histopathology of pancreas was performed

31-12-2019 16
Materials and Methods
Experimental work was conducted under the following headings:-
1- Selection of drug
2- Procurement of experimental materials
3- Procurement of animals and division of animals into different
groups
4-Induction of metabolic syndrome with high caloric diet along with
fructose
5-Treatment of the animals with different doses of L-Norvaline and
Agomelatine
6-Evaluation of various parameters from the treated and untreated
groups of rats before and after treatment
7- Estimation of biochemical parameters, and Oxidative stress in
various groups of animals.
8- Histopathological studies of pancreas.
Materials
Drug
S.NO Drug name Company
1. Agomelatine Sun
Pharma
2. L-Norvaline Sigma

31-12-2019 17
Preparation of hyperlipidemic
diet(Ghose B et al., 2014)
Composition of hyperlipidemic diet
S.NO Ingredients Quantity in
g/Kg and
ml/kg
1
L-Cysteine 200/kg
2
Peanut butter 400/kg
3
Fructose 100/kg
4
olive oil 25ml
5
corn oil 25ml
6
Mineral mixture 70gm
Induction of Obesity , Diabetes and
Hyperlipidemia
Obesity and hyperlipidemia were induced by
feeding the animals with hyperlipidemic diet for a
period of 45 days. Diabetes was induced by
fructose which was given in the diet. After the
period of 45 days body weight was checked and
Fasting blood sugar was estimated. Animals
having body weight more than 350 g and FBGL
more than 200 mg/dl was well thought-out obese
and diabetic and was selected for the experiment.
Selection of Doses
The dose were selected on
the basis of earlier
experiments done on L-
Norvaline and Agomelatine
so two doses of L-Norvaline
(8mg/kg and 16mg/kg) and
two doses of Agomelatine
(10 mg/kg and 20 mg/kg)
were selected for the
pharmacological activity
(Sung HA et al., 2010 and
Milano W et al., 2013).

31-12-2019 18
Preparation of the doses
and drug administration
Drug was directed in suspension
form by using DW using 1% CMC as
a suspending agent. Treatment was
given by oral route for 30 days in
the hyperlipidemic diabetic rats.
Treatment protocol
The animals were divided into 8 groups each group having 6 animals and 1 group of normal animals. The
animals were receiving the following treatment for a period of 30 days.
Group 1: Normal group receive distilled water (1 ml/kg, p.o.)
Group 2: Hyperlipidemic diabetic animals were received distilled water 1 ml/kg, p.o
Group 3: Hyperlipidemic diabetic animals were received 10mg/kg, p.o. Agomelatine
Group 4: Hyperlipidemic diabetic animals were received 20mg/kg, p.o. Agomelatine
Group 5: Hyperlipidemic diabetic animals were received 8 mg/kg, p.o. L-Norvaline
Group 6: Hyperlipidemic diabetic animals were received 16 mg/kg, p.o. L-Norvaline
Group 7: Hyperlipidemic diabetic animals were received Orlistat 30 mg/kg, p.o
Group 8: Hyperlipidemic diabetic animals were received Melatonin 20 mg/kg, p.o.

31-12-2019 19
Body weight was checked at regular intervals of 7 days and
BMI was calculated. Fasting blood sugar level was checked at
a regular interval of 15 days. After the treatment period of 30
days , animals were anaesthetized with di-ethyl ether and
blood was collected from the retro-orbital plexus and serum
was separated and various biochemical parameters were
estimated, again body weight was checked for the treated
animals. BMI was calculated. Average feed intake was
estimated for all the treated animals and various physical
parameters was estimated

31-12-2019 20
Evaluation of parameters
Physical parameters
Determination of body weight: Body mass index (BMI):
After HFD and treatment body
weight of all animals in each group
was checked and the weight
differences were calculated.
Obesity is defined by BMI after and
further evaluated by both body fat
percentage and total body length
(Gupta P et al., 2011)
BMI = Mass (kg)/height (m2)

31-12-2019 21
Estimation of Various Biochemical
Parameters from Serum of treated rats
 Fasting serum
glucose(GOD/PAP Method)
 Estimation of lipid profile
TC(CHOD-
PAP Method
Estimation of HDL by
PEG/CHOD-PAP Method
(Miller et al., 1977)
TG (triglycerides)
GPO/PAP Method
(Slateret al., 1971)
Estimation of LDL and
VLDL

31-12-2019 22
Estimation of the Level of Oxidative Stress
Lipid Peroxidation (LPO) was
estimated by the method of
Slater and Sawer (1971)
Reduced
Glutathione
Superoxide
Dismutase
Estimation of Hormonal Parameters from Serum of treated rats
Leptin Adiponectin CRP

31-12-2019 24
Effect of high fat diet and fructose on the body weight of
rats at different time intervals
Groups 0 day 14thday 21stday 28thday 35th day 45th day
Group 1
1ml/kg
normal
saline
203±0.02 205±0.03 206±0.06 195±0.05 210±0.07 205±0.03
Group 2
HFD +
fructose
221±0.13 251±0.53 273±9.54 288±9.80 305.±1.19 360±2.32
Group 3
HFD +
fructose
213±0.02 245±1.63 273±1.02 298±2.54 330±1.83 365±2.70***
Group 4
HFD +
fructose
208±0.01 233±0.57 260±1.32 288±2.42 335±1.70 381±2.49***
Group 5
HFD+
fructose
196±0.33 226±0.66 251±1.54 281±2.47 325±1.59 381±2.49***
Group 6
HFD +
fructose
206±1.94 231±0.54 260±1.32 295±2.02 325±1.63 365±2.10***
Group 7
HFD +
fructose
201±0.07 230±0.47 256±4.95 286±1.21 316±0.60 360±2.32***
Group 8
HFD +
fructose
196±0.33 218±1.66 246±3.33 275±1.90 305±0.23 350±0.00***
Effect of high fat diet and fructose on the Body Mass
Index of rats at different time intervals
Groups 0 day 14thday 21stday 28thday 35th day 45th day
Group
1
1ml/kg
normal
saline
1.86±0.0
2
1.90±0.0
6
1.91±0.0
4
1.94±0.0
5
1.91±0.04 1.95±0.06
Group
2
HFD+
fructose
1.8±0.02 1.9±0.02
1
2.1±0.03
3
2.3±0.06 2.5±0.04 3.1±0.03
Group
3 HFD
+fructose
1.8±0.05 1.9±0.02
2
2.1±0.03
3
2.3±1.23 2.7±0.04 3.0±5.56**
Group
4 HFD
+fructose
1.8±0.07 2.1±0.01
7
2.2±0.03
5
2.3±0.33 2.8±0.07 3.3±0.07***
Group
5 HFD
+fructose
1.7±0.01 1.9±0.02
2
2.0±0.02
1
2.1±0.03 2.5±0.06 3.2±0.04***
Group
6 HFD+
fructose
1.8±3.43 2.0±0.03
4
2.3±0.04
2
2.5±0.04 3.1±0.03 3.3±0.06***
Group
7 HFD+
fructose
1.6± 0.02 1.9±0.02
2
2.1±0.03
3
2.2±0.04 2.6±0.04 2.9±0.08***
Group
8 HFD+
fructose
1.9±0.02 2.1±0.03
3
2.3±0.04
5
2.4±0.07 2.8±0.07 3.1±0.03***

31-12-2019 25
Effect of high fat diet and fructose on Blood glucose
level of rats at different time intervals
Groups 0 day 15th day 30th day 45th day
Group 1
1ml/kg normal
saline
84.4±0.68 85.1±0.89 84.0±1.18 83.31±0.35
Group2 HFD +
fructose
83.9±0.95 124.8±0.65 162.4±0.52 212.7±2.60
Group 3 HFD +
fructose
81.3±0.43 119.9±1.09 157±1.40 198.2±3.31
Group 4 HFD +
fructose
83.5±1.26 126.5±1.39 146.1±0.91 204.4±1.10***
Group 5 HFD +
fructose
87.3±0.72 123.3±1.47 162.1±0.88 223.2±1.57***
Group 6 HFD +
fructose
81.7±1.09 114.4±0.95 133.1±2.56 187.1±1.47**
Group 7 HFD+
fructose
84.8±0.94 144.5±0.91 168.3±1.77 216.3±1.00***
Group 8 HFD+
fructose
82.5±0.76 131±0.84 162±1.01 197±2.31**
Effect of high fat diet and fructose on Total
Cholesterol in hyperlipidemic diabetic rats
Groups 0 Days 15 days 30 days 45 days
Group
1(control
)
54.0±0.85 56.6±0.98 52.5±0.66 54.2±1.12
Group 2 HFD
+ Fructose
58.6±0.85 98.3±1.12 138.6±0.30 204.3±0.95
Group 3 HFD
+ Fructose
42.1±1.64 94.6±1.80 142.6±1.12 210.1±0.74**
Group 4 HFD
+ Fructose
54.4±1.26 96.6±2.62 132.8±1.15 193.5±0.84*
Group 5 HFD
+ Fructose
46.7±1.74 102.7±0.09 148.2±1.93 228.4±1.85***
Group 6 HFD
+ Fructose
56.1±0.75 108.8±0.95 139.6±1.09 208.3±1.88**
Group 7 HFD
+ fructose
52.6±2.02 104.5±3.97 142.0±1.08 212.2±2.24***
Group 8 HFD
+ Fructose
46.6±1.84 96.9±2.62 140.6±1.02 216.3±2.30***

31-12-2019 26
Effect of high fat diet and fructose on
Triglycerides in hyperlipidemic diabetic rats
Group 0 Days 15 days 30 days 45 days
Group
1(control)
82.6±0.61 81.6±0.56 81.6±0.56 80.8±0.95
Group 2 HFD +
Fructose
88.3±1.40 122.3±0.84 135.6±1.19 156.2±1.64***
Group 3 HFD +
Fructose
74.1±0.97 116.6±0.67 131.2±0.50 152.4±0.51***
Group 4 HFD +
Fructose
82.9±0.53 112.6±0.63 129.8±0.47 145.5±1.29**
Group 5 HFD +
Fructose
86.7±0.86 124.7±0.51 138.2±0.84 150.7±1.20**
Group 6 HFD +
Fructose
70.8±0.62 106.8±0.51 128.4±0.82 142.3±0.62**
Group 7 HFD +
Fructose
84.4±0.83 117.5±0.81 131.0±0.61 154.7±0.85***
Group 8 HFD +
Fructose
80.2±0.80 113.3±2.27 124.6±0.86 147.6±1.17***
Effect of high fat diet and fructose on LDL in
hyperlipidemic diabetic rats
Groups 0 day 15 day 30 day 45 day
Group
1(contr
ol)
90.6±1.70 92.6±1.54 90.2±1.43 90.8±1.62
Group 2 HFD
+ Fructose
94.3±1.26 138.4±3.52 152.6±5.47 186.2±1.32***
Group 3 HFD
+ Fructose
88.1±0.05 133.6±3.22 147.2±1.88 173.4±4.86**
Group 4 HFD
+ Fructose
80.9±0.01 139.6±2.86 151.8±2.21 184.5±3.21***
Group 5 HFD
+ Fructose
96.7±1.21 121.6±4.76 147.4±1.76 166.8±0.06**
Group 6 HFD +
Fructose
82.8±0.04 131.8±3.25 156.2±2.32 188.4±0.03***
Group 7 HFD
+ Fructose
90.4±1.04 137.5±2.01 150.6±4.82 177.2±1.51**
Group 8 HFD +
Fructose
87.2±0.06 123.4±3.20 148.6±0.43 170.0±0.04**

31-12-2019 27
Effect of high fat diet and fructose on VLDL in
Groups 0 day 15 day 30 day 45 day
Group
1(control)
18.66±1.25 18.24±1.34 18.66±1.25 18.62±1.35
Group 2 HFD +
Fructose
16.32±2.55 24.22±3.59 36.32±2.98 46.22±3.56
Group 3
HFD + Fructose
14.92±1.45 22.76±1.71 34.92±3.06 42.92±2.12**
Group 4 HFD +
Fructose
18.66±1.23 24.22±0.43 35.66±3.21 44.66±4.63
Group 5 HFD +
Fructose
16.74±2.65 24.64±2.41 36.57±3.90 48.42±2.76***
Group 6 HFD +
Fructose
18.82±1.43 26.24±2.56 38.42±1.76 52.57±1.87***
Group 7 HFD +
Fructose
14.44±1.65 18.66±1.60 32.74±2.93 43.74±1.06
Group 8 HFD +
Fructose
16.26±2.68 26.54±3.26 38.66±1.70 56.62±0.573***
Effect of high fat diet and fructose on HDL
Group 0 day 15 day 30 day 45 day
Group
1(contro
l)
54.6±1.05 54.2±1.03 54.4±1.06 54.2±1.04
Group 2
HFD
+ Fructose
48.5±2.65 34.6±1.95 28±1.57 21.2±2.54
Group 3
HFD
+ Fructose
54.7±1.56 42.4±1.56 38.2±3.65 26.0±1.96
**
Group 4
HFD
+ Fructose
46.3±1.86 38.6±3.40 24.4±4.67 18.4±1.65
***
Group 5
HFD
+ Fructose
58.6±1.78 44.8±1.32 32.2±5.89 24.4±2.72
**
Group 6
HFD
+ Fructose
42.2±1.86 31.6±1.96 22.8±3.76 18.1±2.65
***
Group 7
HFD
+ Fructose
45.7±1.67 36.0±1.86 30.2±2.31 25.8±1.74
**
Group 8
HFD
+ Fructose
52.8±1.87 34.4±2.54 26.4±4.65 20.0±1.03
***

31-12-2019 28
Table 7.1 : Effect of L-Norvaline and Agomelatine on body weight in hyperlipidemic rats after treatment
Groups 0 day (46th
day of
study)
15 day 30 day
( 75the day of
study)
Normal (1ml/kg normal
saline, p.o.)
205±3.26 195±3.32 200±3.28
HDR received distilled
water (1ml/kg, p.o)
360±1.32 356±1.96 356±2.96
HDR received Agomelatine (10
mg/kg, p.o)
365±1.33 300±1.43 280±2.03**
HDR Received Agomelatine
(20mg/kg, p.o)
381±1.49 300 ±1.84 230±2.10***
HDR received L-Norvaline
(8mg/kg, p.o)
381±1.49 280±0.14 260±1.88**
HDR received L-Norvaline (16
mg/kg, p.o)
365±2.70 310±1.62 225±0.28***
HDR received Orlistat 30
mg/kg, p.o)
360±1.32 270±3.84 220±1.12***
HDR received Melatonin 20
mg/kg, p.o)
350±0.92 300±2.89 230±1.98***

31-12-2019 29
Table 7.2 :Effect of L-Norvaline and Agomelatine on Body Mass Index in hyperlipidemic rats after treatment
Groups 0 day (46th
day of study)
15 day 30 day
(75th day of study)
Normal (1ml/kg
normal saline, p.o.)
1.96±0.06 1.95±0.04 1.94±0.04
HDR received
distilled water
(1ml/kg, p.o)
3.1±0.03 3.05±3.46 3.10±1.36
HDR received
Agomelatine (10
mg/kg, p.o)
3.0±0.36 3.1±1.43 2.78±0.47**
HDR Received
Agomelatine
(20mg/kg, p.o)
3.3±0.07 2.70 ±0.84 2.30±0.33***
HDR received L-
Norvaline (8mg/kg,
p.o)
3.2±0.04 2.70±0.14 2.70±0.28**
HDR received L-
Norvaline (16 mg/kg,
p.o)
3.3±0.06 3.00±1.62 2.34±0.13***
HDR received Orlistat 30
mg/kg, p.o)
2.9±0.08 2.78±1.19 2.26±3.72***
HDR received Melatonin
20 mg/kg, p.o)
3.1±0.03 2.60±0.09 2.30±0.33***

31-12-2019 30
Table 7.3: Effect of L-Norvaline and Agomelatine on Blood glucose level of rats at different time intervals
Groups 0 Days
(46th day of study)
15 days 30 days
(75th day of study)
Group 1(control) 84.4±0.68 85.2±0.89 84.0±1.18
Group 2 HDR received
distilled water
(1ml/kg, p.o)
212.7±2.60 210.3±1.58 208.6±1.64
Agomelatine
(10mg/kg p.o)
198.1±3.32 142.6±1.04 121.6±0.98**
Agomelatine
(20mg/kg p.o)
204.4±1.10 144.6±0.08 103.8±0.89**
L-Norvaline 8 mg/kg,
p.o)
223.7±1.57 148.7±1.22 106.2±0.44**
L-Norvaline 16
mg/kg, p.o)
187.1±1.47 138.8±1.03 100.6±0.11**
Orlistat (30 mg/kg,
p.o)
216.6±1.01 212.5±2.58 209.0±1.84*
Melatonin 20 mg/kg,
p.o)
197.6±2.31 120.4±1.46 128.6±0.62**

31-12-2019 31
Table 7.4:Effect of L-Norvaline and Agomelatine on Total Cholesterol in hyperlipidemic diabetic rats
Groups 0 Days
(46th day of study)
15 days 30 days
(75th day of study)
Group 1(control) 54.0±0.33 56.6±0.61 52.5±0.51
distilled water (1ml/kg,
p.o)
204.6±0.42 132.3±1.00 130.6±0.64
Agomelatine (10mg/kg
p.o)
210.1±1.78 120.6±0.51 96.6±0.98**
p.o)
193.4±0.52 104.6±0.60 64.8±0.89***
Group 5 HDR received L-
Norvaline 8 mg/kg, p.o)
228.7±1.68 94.7±0.82 72.2±2.44**
Group 6 HDR received L-
Norvaline 16 mg/kg, p.o)
208.1±1.84 88.8±2.56 66.6±1.42***
Orlistat (30 mg/kg, p.o)
212.6±0.61 134.5±1.41 122.0±0.84*
Melatonin 20 mg/kg, p.o)
216.6±2.20 96.4±1.46 62.6±0.62***

31-12-2019 32
Table 7.5: Effect of L-Norvaline and Agomelatine on Triglycerides in hyperlipidemic diabetic rats
Groups 0 Days
(46th day of study)
15 days 30 days
(75th day of study)
Group 1(control) 82.6±0.61 81.6±0.56 81.6±0.56
distilled water
(1ml/kg, p.o)
156.3±1.40 154.3±0.84 153.6±1.19
p.o)
151.1±0.97 122.6±0.67 88.2±0.50***
p.o)
145.5±0.53 116.6±0.63 74.8±0.47***
p.o)
150.7±0.86 123.7±0.51 82.2±0.84***
p.o)
142.8±0.62 110.8±0.51 68.4±0.82***
154.6±0.83 125.5±0.81 76.6±0.61***
Melatonin 20 mg/kg,
p.o)
147.2±0.80 119.3±2.27 84.6±0.86***

31-12-2019 33
Table 7.6: Effect of L-Norvaline and Agomelatine on LDL in hyperlipidemic diabetic rats
Groups 0 day
(46th day of study)
15 day 30 day (75th
day of
study)
Group 1(control) 90.6±1.70 92.6±1.54 90.2±1.43
p.o)
186.3±1.26 184.4±3.52 182.6±1.47
p.o)
173.1±0.05 144.6±3.22 92.2±2.88**
p.o)
184.9±0.01 131.6±2.86 86.8±0.21***
p.o)
166.7±1.21 129.6±2.76 82.4±0.76***
p.o)
186.8±0.04 121.2±3.25 76.2±1.32***
177.4±1.04 124.5±2.01 74.6±1.82***
Melatonin 20 mg/kg,p.o)
170.2±0.06 143.4±3.20 92.6±1.43**

31-12-2019 34
Table 7.7: Effect of L-Norvaline and Agomelatine on VLDL in hyperlipidemic diabetic rats
Groups 0 day
(46th day of study)
15 day 30 day
(75th day of study)
Group 1(control) 18.66±1.25 18.24±1.34 18.66±1.25
distilled water
(1ml/kg, p.o)
46.32±2.55 44.22±2.59 43.32±2.98
Agomelatine
(10mg/kg
p.o)
42.92±1.45 32.76±1.71 24.92±1.06*
Group4 HDR received
Agomelatine
(20mg/kg p.o)
44.66±1.23 34.22±0.43 25.66±1.21*
Group 5 HDR
received L-Norvaline
8 mg/kg, p.o)
48.74±2.65 24.64±2.41 21.8 ±0.90**
Group6 HDR received
L-Norvaline16 mg/kg,
p.o)
52.82±1.43 26.24±2.56 14.42±1.76***
44.44±1.65 18.66±0.60 11.74±2.93***
Melatonin 20 mg/kg,
p.o)
56.26±2.68 26.54±3.26 18.66±1.70***

31-12-2019 35
Table 7.8 : Effect of L-Norvaline and Agomelatine on HDL in hyperlipidemic diabetic rats
Groups 0 day
(46th day of study)
15 day 30 day
(75th day
of study)
Group 1(control) 54.6±1.05 54.2±1.03 54.4±1.06
p.o)
21.5±2.95 21.0±2.95 20.2±2.94
p.o)
26.7±1.56 42.4±1.56 58.2±3.65***
p.o)
18.3±1.86 38.6±3.40 50.4±4.67***
p.o)
24.6±1.78 44.8±1.32 54.6±5.89***
p.o)
18.2±1.86 31.6±1.96 48.8±3.76***
25.7±1.67 36.0±1.86 52.2±2.31***
Melatonin 20 mg/kg,
p.o)
20.8±1.87 34.4±2.54 58.4±4.65***

31-12-2019 36
Table 7.9: Effect of L-Norvaline and Agomelatine on the levels of Oxidative stress in hyperlipidemic diabetic rats
Group + Treatment Superoxide
Dismutase
(EU/dl)
Reduced
Glutathione
(microgram/ml)
Lipid
peroxidation (n
mol/L)
Group 1-Normal (1ml/kg
normal saline, p.o)
25.7±1.42 42.4±1.40 45.5±1.80
Group 2 -HDR received
p.o)
13.3±1.31 13.4±1.35 79.8±1.25
Group 3- HDR received
Agomelatine (10mg/kg,
p.0)
16.8±0.84*** 34.2±1.81** 65.7±1.13
Agomelatine (20mg/kg,
p.o)
19.3±0.57** 30.4±1.65** 54.2±2.06***
Group 5-HDR received L-
Norvaline (8mg/kg, p.o)
13.2±0,74** 19.4±1.32**
*
64.7±1.22**
Group 6- HDR received L-
Norvaline (16 mg/kg,
p.o)
18.7±1.06 16.6±2.54**
*
60.0±1.18**
Orlistat 30mg/kg, p.o)
19.4±0.75*** 27.4±3.22 52.5±1.88***
Melatonin 20 mg/kg,
p.o)
21.7±0.57*** 30.0±2.12** 46.5±1.65***
A B C A
B
C

31-12-2019 37
Table 7.10:Effect of L-Norvaline and Agomelatine on leptin (Ng/mL) in hyperlipidemic diabetic rats
Groups + Treatment 0 day 30 day
Group 1-Normal (1mg/kg
normal saline, p.o)
10.5±0.72 10.4±0.72
distilled water 1ml/kg, p.
o
12.4±0.26 12.2±0.25
Group 3 received 10 mg/kg
agomelatine
10.7±1.57 9.3±0.61**
Group 4 received 20 mg /kg
agomelatine
12.6±0.07 8.1±1.56**
Group 5 received 8 ml/kg L-
Norvaline
11.2±0.39 10.3±1.02**
L-Norvaline
9.7±0.07 8.6±0.82**
Group 7 received orlistat 30
mg/kg
9.3±0.10 3.4±0.68***
Group 8 received 20mg/kg
Melatonin
6.8±0.04 5.8±0.76**

31-12-2019 38
Table 7.11: Effect of L-Norvaline and Agomelatine on the Adiponectin (Mg/ml) in hyperlipidemic diabetic rats
Group 1-Normal (1mg/kg normal saline, p.o) 2.40.±0.96 2.44±0.92
Group 2 HDR received distilled water 1ml/kg, p. o 1.26±3.22 1.28±3.26
agomelatine
2.58±2.03 8.6.3±0.87*
Group 4 received 20 mg /kg agomelatine 2.72±2.26 12.1±1.48**
Group 5 received 8 ml/kg L- Norvaline 1.89±0.95 9.3±0.42*
Group 6 received 16 mg/kg L-Norvaline 1.55±2.86 10.6±0.82***
Group 7 received orlistat 30 mg/kg 1.21±3.12 18.4±1.21***
Melatonin
2.24±0.94 12.8±1.76**

31-12-2019 39
Table 7.12:Effect of L-Norvaline and Agomelatine on CRP (Mg/L) in hyperlipidemic diabetic rats
Group 1-Normal (1mg/kg
normal saline, p.o)
1.5±0.01 1.7±0.03
distilled water 1ml/kg, p. o
3.2±0.58 3.4±0.55
agomelatine
9.1±0.90 2.3±0.28***
Group 4 received 20 mg /kg
agomelatine
4.4±0.04 1.6±0.08**
Group 5 received 8 ml/kg L-
Norvaline
6.2±0.08 2.8±0.28***
L-Norvaline
8.5±0.72 1.9±0.01***
Group 7 received orlistat 30
mg/kg
4.6±0.42 1.4±1.21**
Melatonin
8.2±1.05 2.8±1.76***

31-12-2019 40
The present study was aimed to evaluate the role of L- norvaline and melatonin receptor agonist agomelatine on metabolic syndrome
in hyperlipidemic diabetic rats. The study was further design to check out the effect of different doses of arginase inhibitor and
melatonin receptor agonist in hyperlipidemic diabetic rats.
Administration of L- norvaline at a dose of 8 mg/kg and 10 mg/kg and agomelatine at a dose of 10mg/kg and 20 mg/kg to the
hyperlipidemic rats for 30 days improved the physical parameters. However, higher dose of both more significantly improved physical,
biochemical and hormonal parameters after treatment.
Treatment with L- norvaline and Agomelatine caused decrease in the level of glucose, decrease in body weight, triglycerides and
increased in HDL level.
The level of leptin and CRP also decreased and adiponectin level increased after treatment with agomelatine and L- Norvaline.
The level of oxidative stress also increased where as the level of GSH and SOD also improved after treatment with agomelatine and L-
Norvaline.
The improvement in lipid metabolism by these compounds supports their antidiabetic and anti-obesity activity along with
antihyperlipidemic activity. Improvement in lipid profile decreases the chances of diabetes and obesity induced cardiovascular disease.
Antioxidant activity helps in decreasing the macro and micro vascular problems and their harmful effects in diabetes.

31-12-2019 41
Both of these compounds also reduced the oxidative stress induced damage of pancreatic Beta- cells and thus increasing
the number of insulin secreting function of Beta- cells present in pancreas.
L-norvaline as a potent arginase inhibitor show a promising improvement in nitrates level by inhibiting the arginase
pathway and increasing the availability of L-Arginine as substrate whereas the Agomelatine as a potent melatonin agonist
show the potential effect on glucose and lipid metabolism as well as prevented the suppression of body weight gain and
energy intake. So, both of the drugs by acting through different mechanisms significantly protect the obese and diabetic
rats from metabolic complication with minimal toxicity.
However, further clinical and preclinical studies are required along with histopathological studies of pancreas for
improvement of tissue damage with structural changes.

31-12-2019 42
Future Prospective
In modern era disease structures have changed along with the changes in the social environment.
The incidence of obesity, Diabetes mellitus, atherosclerotic or cardiovascular disease like stroke
and heart failure has been increasing rapidly now a days. For improving these metabolic
complications, we need to improved diagnostic criteria, individual risk factors, pathophysiology,
changes in lifestyle, decreasing corpulence, smoking and liquor use, improving eating routine and
for acknowledgment of significant pathways that will take into consideration better treatment
options in future.
In terms of obesity and T2DM, the use of anorexigenic gut hormone analogues such as liraglutide
is effective not only for glycaemic control but also for weight loss. These agents have acceptable
safety profile having no cognitive and physiological side effects. At the same time a number of
centrally acting medications have been approved around the world they are loriciferan, the
combination of Topiramate with pheniramine and the combination of naltrexone and bupropion.
These combination therapies appear to be effective results in weight loss and Type 2 Diabetes
Mellitus.

31-12-2019 43
The principle future planned to diminishing the odds of metabolic disorder is change in
way of life then again, a few general wellbeing methodologies, for example, dietary
changes alongside utilization of prescriptions, for example, diuretics, statins and beta
blockers may affect future patterns in metabolic disorder related entanglement.
Some encouraging new medications that monitored glucose levels including amylin
analogues, glucagon-like peptide 1 agonists, dipeptidyl peptidase IV inhibitors have been
reads and endorsed for use in metabolic disorder related issues

31-12-2019 44
Greenberg AS, Wang P, Chen H, Zhu Y. Obesity and the Role of Adipose tissue in Metabolic Syndrome
Associated Problems, 2016, 419-21.
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Harvey BH, Kim J, Montagnani M, Koh KK, A high-fat diet exacerbates depressive like behaviour in rats,
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Zimmet P Global and Societal implications of the Diabetes epidemic, 2001, 782-784.
Eapen D Verma S, Bhanot S, Yao L, McNeill JH, Metabolic syndrome and cardiovascular disease in south
Asians, 2009, 731-43.
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Chau CK, Takagawa Y, Berger ME, Hori MT Association of diet, exercise and smoking modification with
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Hegele EM, Saad MF, Rewers M, Selby J, Howard G, Genetic and physiological insights into the metabolic
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Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome among US adults: ﬁndings from the third
national health and nutrition examination survey. JAMA, 2002, 287: 356–359.
El-Bassossy HM, Fahmy A, Badawy Cinnamaldehyde protects from the hypertension associated with
diabetes. Food Chem Toxicol 2011,3007–3012.
Brown RJ, Fossati P, Prencipe L, Berti G, A Systemic Review of Metabolic Syndrome: finding effects in youth,
2010, 305-12
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COMPARATIVE ASSESSMENT OF THE EEFFECT OF ARGINASE INHIBITOR L-NORVALINE AND MELATONIN RECEPTOR AGONIST AGOMELATINE ON METABOLIC SYNDROME IN HYPERLIPIDEMIC DIABETIC RATS

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COMPARATIVE ASSESSMENT OF THE EEFFECT OF ARGINASE INHIBITOR L-NORVALINE AND MELATONIN RECEPTOR AGONIST AGOMELATINE ON METABOLIC SYNDROME IN HYPERLIPIDEMIC DIABETIC RATS