synthesis, characterization, in vivo anti-diabetic
TRANSCRIPT
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SYNTHESIS, CHARACTERIZATION, IN VIVO ANTI-DIABETIC
ACTIVITY A COUMARIN DERIVATIVES
Fawzia Z. El-Ablack1*, Faten Z. Mohamed
2, Usama B. Elgazzar
3 and Mahmoud M.
Ramzy2
1Chemistry Department, Faculty of Science, Damietta University, new Damietta 34517,
Egypt.
2Chemistry Department (Biochemistry), Faculty of Science, Zagazig University, Zagazig,
Egypt.
3Medical Biochemistry Dep., Faculty of Medicine, Al-Azhar University,
New Damietta, Egypt.
ABSTRACT
Background: Diabetes mellitus (DM) is a group of metabolic
disorders characterized by chronic hyperglycemia resulting from
relative or absolute insulin deficiency with or without insulin
resistance. Aim: This study was aimed to evaluate the role of new
coumarin derivative (cromen) in treatment of alloxan induced diabetes
in rats by. Methods: 50 adult male albino rats were divided into 5
groups. Group I (control group, rats were orally administered with 1 ml
saline daily). Group II (DMSO group, rats were orally administered
with 0.2 % DMSO for 40 days). Group III (standard group, diabetic
animals treated with 100 mg/kg b.wt of metformin for 40 days orally).
Group IV (therapeutic group, diabetic rats treated orally with 50 mg
/kg b.wt of cromen for 40 days). Group V (positive control, animals were injected
intraperitoneal with 150 mg/kg b. wt. alloxan). At the end of experimental period blood
serum, liver, kidney and pancreatic tissues were collected. Results: diabetic rats showed
significant increase in the mean level of blood Glucose, serum ALT, AST, Total bilirubin,
Urea, Creatinine, Cholesterol and triglyceride. Also, significant decrease in ALB level in
compared to control group. In group treated with cromen there was significant decrease in
elevated biochemical parameters. Also, significant increase ALB level when compared with
diabetic rats. Our results were confirmed by histopathological examination of different
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.632
Volume 10, Issue 3, 2044-2067 Research Article ISSN 2278 – 4357
*Corresponding Author
Fawzia Z. El-Ablack
Chemistry Department,
Faculty of Science, Damietta
University, new Damietta
34517, Egypt.
Article Received on
20 Jan. 2021,
Revised on 10 Feb. 2021,
Accepted on 02 March 2021
DOI: 10.20959/wjpps20213-18564
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Fawzia et al. World Journal of Pharmacy and Pharmaceutical Sciences
tissues. Conclusion: this study suggests that cromen exhibits anti-hyperglycemic and anti-
lipidimic in alloxan induced diabetic rats.
INTRODUCTION
Diabetes or diabetes mellitus is a complex or polygenic disorder, which is characterized by
increased levels of glucose (hyperglycemia) and deficiency in insulin secretion or resistance
to insulin over an elongated period in the liver and peripheral tissues.[1]
Both the insulin
resistance and deficiency lead to high blood glucose levels. Evidences show that T2DM
progression is rapid because of complications such as heart, renal, nerve injury, retinopathy,
developing in early stages of the disease.[2]
Identifying newly biomarkers for T2D and its
complications remains challenging due to the heterogeneous nature of T2D. The
heterogeneity relates not only to glycemic control or treatment response.[3]
Alloxan(2,4,5,6tetraoxypyrimidine;2,4,5,6-pyrimidinetetrone) is anoxygenated pyrimidine
derivative which is present as alloxan hydrate in aqueous solution.[4]
Alloxan is a urea
derivative that causes selective necrosis of β-cells of pancreatic islets. In addition, it has been
widely used to produce experimental diabetes in animals such as rabbits, rats, mice and dogs
with different grades of disease severity by varying the dose of alloxan used.[5,6]
The toxic
action of alloxan on pancreatic beta cells involve oxidation of essential sulphydryl (-SH
groups), inhibition of glucokinase enzyme, generation of free radicals and disturbances in
intra cellular calcium homeostasis.[7]
Nitrogen and sulfur containing heterocycles play a critical role in medicinal chemistry and in
the development of potent drug molecules.[8]
Among them, thiazoles and imidazoles have
been well documented in medicinal chemistry with respect to their promising biological
activities. Thiazole derivatives are considered as important class of heterocyclic compounds,
which displayed a wide range of pharmacological activities such as anti-inflammatory,[9]
anticancer,[10]
anticonvulsant,[11]
antibacterial,[12]
and antidiabetic.
MATERIALS AND METHODS
Materials and Apparatus
All reagents were purchased from Aldrich, Fluka and Merck and were used without any
further purification. All melting points are uncorrected. Elemental analyses were obtained
from Microanalysis unit, Cairo University.
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Synthesis of the new coumarin derivative ‘’cromen’’
Ethyl 2-amino-4-phenyl-4H-benzo(h)chromene-3-carboxylate „‟Cromen‟‟ was synthesized
by an efficient, solvent-free one-pot three-component cyclocondensation of
ethylcyanoacetate, benzaldehyde and α-naphthol then Na2CO3 was added. All components
were put in fusion oil path for 15-25 min at 100-120 oC. Then precipitated with ice-water
bath, filtered, then after recrystallized by ethanol.[13]
Characterization of the new coumarin derivative ‘’cromen’’
Synthesized cromen was chemically characterized using infrared (IR), nuclear magnetic
resonance (NMR) and mass spectroscopy. IR spectroscopy analysis was done using FT-IR
spectra (KBr discs, 4000–400 cm-1) by Jasco-4100 spectrophotometer, at the (IR) unit at
Faculty of Science, Damietta University, Egypt. Total organic carbon (TOC) analyzer:
analysis was done at Faculty of Science, Kafr El-Sheikh University, Egypt. Mass spectrum
analysis was done at Faculty of Science, El-Azhar University, Egypt. Molecular docking
analyses were done using Perkin Elmer Chem Bio 3D software by HF method.
Animals
Adult male albino rats, weighing 200-280 g, were obtained from the Experimental Animal
Care Center from Cairo university and were kept in cages at experimental animal house of
faculty of Science, Zagazig University under regulated environmental conditions (25°C and a
12 h light/dark cycle) 7 days before starting the experiment.
Toxicity study
Median lethal dose (LD 50) of the cromen was determined according to method.[14]
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Diabetic model
Diabetes was induced by Monohydrated alloxan (Researsh-Lab Fine Chem Industruies
Mumbai 400 002, India).
Diabetes mellitus (DM) was induced in overnight-fasted rats by a single i.p. injection of
freshly-prepared alloxan monohydrate, dissolved in a cold physiological saline (0.9% NaCl)
solution at the dose rate of 150 mg/kg body weight.[15]
The animals were given free access to
5% glucose solution in order to overcome the alloxan-induced hypoglycaemia for the first
one hour post-treatment with alloxan. Blood glucose level in all animals were measured after
72 hours of drug administration and rats of fasting blood glucose levels higher than 250
mg/dl were considered to be diabetic and used for the further study.[16]
Experimental design
To accomplish the ultimate goal of this study, after the acclimatization period of 7 days with
standard basal diet, a total of 50 adult male albino rats were classified into five groups with
10 rats in each group.
Group I (Control group): Rats were administrated orally with 1 ml saline single dose.
Group II (DMSO group): Rats were administrated by gaveling 1 ml of 0.2% DMSO for 40
days.
Group III (Standard therapeutic): were induced for DM. After 1 week of DM induction,
animals were post treated with metformin (100 mg/kg daily for 40 days orally).
Group IV (Therapeutic group): Rats were induced for DM. After 1 week of DM induction,
animals were post treated with cromen (50 mg/kg daily for 40 days orally)
Group V (Positive control): Rats were injected intraperitoneal (I. p) with alloxan (150
mg/kg b. wt.).
Doses of cromen and metformin were adjusted every week according to any change in body
weight to maintain the same dose per each kg body weight of rat during the entire period of
study for each group.
Collection and Sampling of blood
At the end of the study and after last treatment, rats were fasted for 12 hours; blood samples
were collected from the retro-orbital venous plexus under light ether anesthesia. where, blood
samples were collected in three different tubes, first tube containing sodium fluoride for
blood glucose estimation, second empty tube to obtain serum by centrifugation at 4000 rpm
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for 20 min. Serum and plasma were transferred into Eppendorf tubes and stored frozen at -20
ºC until analysis of different biochemical measurements.
Tissue sample
Different tissues (liver, kidney, and pancreas) were excised from all animal's groups, rinsed in
chilled saline solution, cleared off blood. Liver, kidney and pancreas specimens of the rat
were carefully collected. Small pieces from different tissues were immersed in 10% neutral
buffered formalin for 24 hr. for histopathological examination. The fixed tissue was
processed routinely, embedded in paraffin, sectioned, deparaffinized and rehydrated using the
standard techniques.[17]
Biochemical analysis
Estimation of biochemical parameters
Determination of plasma glucose was performed by glucose oxidase peroxidase activity using
a commercial kit derived from Elitech clinical systems, France.[18]
Liver function tests
Serum Albumin concentration was determined by colorimetric method according to modified
bromcresol green binding assay (BCG).[19]
Serum Alanine aminotransferase ALT activity
was determined by colorimetric method using assay colorimetric kit.[20]
Serum Aspartate
aminotransferase AST activity was determined by colorimetric method using assay
colorimetric kit.[21]
Serum total bilirubin concentration was determined by bilirubin is
converted to colored diazotized sulfanilic acid and Measured photo metrically.[22]
Kidney function tests
Serum Urea concentration was measured by Berthelot enzymatic colorimetric method using a
commercial kit derived from Diamond Diagnostic Company, Germany.[23]
Serum creatinine
was measured by Buffered kinetic jaffe reaction without deproteinization, using a commercial
kit derived from Spin react Company, Spain.[24]
Lipid profile
Serum cholesterol concentration was determined by CHOD-POD colorimetric method using
a commercial kit derived from spin react Company, Spain.[25]
Serum triglycerides
concentration was measured by GPO-PAP enzymatic colorimetric method using a
commercial kit derived from Vitro Scient Company, Germany.[26]
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Histopathological examination
Different tissues were then immersed with molted paraffin wax, then embedded and blocked
out. Paraffin sections (4–5 um) were stained with hematoxylin and eosin then examined
through light electric microscope.[27]
Statistical analysis
All results were analyzed by SPSS software (SPSS, ver.14.00, USA). Data were expressed as
mean ± SEM. Comparison of mean values of studied variables among different groups was
done using ANOVA test. P<0.05 was considered to be significant.[28]
RESULTS
Spectral analyses of the new coumarin derivative
IR spectroscopy results of the new synthesized coumarin derivative „‟cromen‟. It showed a
number of peaks at different positions with different intensities as follow; IR (KBr. cm-1
):
3487 (NH2), 3057 (Ar-H), 1658 (C=O), 1588 (C=N), 1563 (C=C). 1H-NMR (CDCl3) δ: 6.82
(d, 2H, NH2), 7.37-8.27 (m, 1H, Ar-H), 4.74 (s, 1H, CH), 4.08 (q, 2H, CH2), 1.16 (t, 3H,
CH3). m/z: 345.14 (100.0%), 346.14 (23.8%), 347.14 (2.7%). Mass spectrum analysis of the
new synthesized coumarin derivative „‟cromen‟‟ showed that the melting point is 165-170 oC,
its color is brown, it yielded 67%. Its chemical formula is C22H19NO3 and Anal. Calcd. C,
76.50; H, 5.54; N, 4.06; O, 13.90 Found: C, 75.40; H, 6.04; N, 4.56; O, 13.20. Its molecular
weight is 345.40.
Molecular structure
The molecular structures (HOMO & LUMO) of „‟cromen‟‟ are presented in Figure 14. They
indicated „‟cromen‟‟ as a stable compound (Figure 1)
Toxicity study
Studies carried out for determination of the median lethal dose are important to help us to
assess the limit dose recommended, we use procedure described by to calculate the (LD 50),
Doses for all compounds were being safe until 20000 mg/kg.
Effect of cromen on final body weight in all studied groups
Results in Table 1 showed significant decrease in the mean of Final body weight of diabetic
induced group (267±1.0 g) (positive group) which amounted to -20.05 % when compared to
control group (p < 0.001), while group treated with Cromen (Therapeutic) and metformin
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(standard) showed statistically non-significant decrease (307.8±11.5 g), (340.4±1.4 g) which
amounted to – 7.84 % and – 1.91 % respectively when compared to control group (P > 0.05).
Effect of cromen on plasma glucose
Results in Table 2 showed that the mean of final plasma glucose level (405±15.0 mg/dl)
showed significant elevation in positive group which amounted to 292.44% in compared to
control group (P< 0.001), while treated groups with cromen (Therapeutic) and metformin
(standard) groups showed good improvement (237.8±2.1 mg/dl), (232.3±4.8 mg/dl)
respectively which amounted 130.42% in cromen and 125.09% in metformin when compared
in control group.
Effect of cromen on (ALT, AST, ALB and Total bilirubin)
Results in Table 3 showed that the mean value of serum ALT activity (59.8±4.3 U/L) showed
significant elevation in positive group which amounted to 95.42% in compared to control
group (P = 0.001), while treated groups with cromen (Therapeutic) and metformin (standard)
(35.5±3.5 U/L), (45.9±2.7 U/L) respectively showed good improvement which amounted
16.01% in cromen and 50% in metformin when compared in control group.
Also, showed that the mean value of serum AST activity (67.1±2.9 U/L) showed significant
elevation in positive group which amounted to 53.66% in compared to control group (P <
0.05). While treated groups with cromen (Therapeutic) and metformin (standard) (67.1±2.9
U/L), (65.1±4.3 U/L) respectively showed good improvement which amounted 4.68% in
cromen and 1.56% in metformin when compared in control group.
Also, showed that the mean value of serum Total bilirubin (0.51±0.010 mg/dl) showed
significant elevation in positive group this amounted 37.83 % in compared to control group
(p< 0.01). While treated groups with cromen (Therapeutic) and metformin (standard)
(0.46±0.017 mg/dl), (0.41±0.019 mg/dl) respectively showed good improvement which
amounted - 24.32 % in cromen and, -10.81 % in metformin when compared in control group.
In contrast, showed that the mean value of serum ALB activity (3.5±0.55 mg/dl) showed
significant decrease in positive group this amounted -25.53% in compared to control group
(p< 0.01). While Treated groups with cromen (Therapeutic) and metformin (standard)
(4.5±0.55 mg/dl), (4.4±0.14 mg/dl) respectively showed good improvement which amounted
-4.25% in cromen and, -6.38% in metformin when compared in control group.
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Effect of cromen on (Urea, Creatinine, Cholesterol and Triglyceride)
Results in Table 4 showed that the mean level of serum Urea (15.5±5 mg/dl) showed
significant elevation in positive group which amounted to 19.23% in compared to control
group (P ˃ 0.05), while treated group with cromen (Therapeutic) and metformin (standard)
(13.1±0.7 mg/dl), (13.8±0.8 mg/dl) respectively showed good improvement which amounted
16.01% in cromen and 6.15% in metformin when compared in control group.
Also, showed that the mean level of serum Creatinine (0.81±0.015 mg/dl) showed significant
elevation in positive group which amounted to 24.61% in compared to control group (p<
0.001). While treated groups with cromen (Therapeutic) and metformin (standard) (0.72±0.01
mg/dl), (0.74±0.01 mg/dl) respectively showed good improvement which amounted
10.76%in cromen and 13.84% in metformin when compared in control group.
Also, showed that the mean level of serum cholesterol (103.6±3.6mg/dl) showed significant
elevation in positive group this amounted 35.60% in compared to control group (P < 0.05).
While treated groups with cromen (Therapeutic) and metformin (standard) (82.4±4.4mg/dl),
(85.3±3.9mg/dl) respectively showed good improvement which amounted 7.85%in cromen
and, 11.64%in metformin when compared in control group.
Also, showed that the mean level of serum triglyceride (209.2±10.4mg/dl) showed significant
elevation in positive group this amounted 56.82% in compared to control group (P = 0.001).
While treated groups with cromen (Therapeutic) and metformin (standard) (165.4±7.4 mg/dl),
(169.5±5.8 mg/dl) respectively showed good improvement which amounted 23.98%in
cromen and, 27.06% in metformin when compared in control group.
Histopathological examination
Histology of pancreases, liver and kidney was studied.
The normal pancreases section of negative control & DMSO group showed healthy n
parenchyma, normal pancreatic acini and islets. In standard therapeutic (metformin) group
showed pancreatic islets hyperplasia and pancreatic duct dilatation. In treatment group
(cromen) showed group normal pancreatic acini, ducts, and islets. In positive group showed
severely dilated pancreatic duct and sever congestion in the interstitial blood vessel with
thickened muscular wall (Figure 2).
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The normal liver section of negative control & DMSO group showed normal hepatic
parenchyma; note the normal hepatocytes, blood sinusoids, and portal area. In standard
therapeutic (metformin) group showed congestion in the portal vein and dilated hepatic
artery. In treatment group (cromen) group showed normal hepatic parenchyma; note the
normal hepatocytes, blood sinusoids, and portal area. In positive group showed focal areas of
hepatocytes necrosis infiltrated and replaced with mononuclear cells infiltration and Portal
tract inflammatory changes; note the congestion in hepatoportal blood vessel and the
infiltration of mononuclear cells (Figure 3).
The normal kidney section of negative control & DMSO group showed normal renal
parenchyma; note the normal renal glomeruli and renal tubules. In standard therapeutic
(metformin) group showed congestion in the interstitial blood vessel (arrow head) with thick
muscle wall. In treatment group (cromen) group showed normal renal parenchyma; note the
normal renal glomeruli and renal tubules. In positive group showed sever congestion in the
glomerular capillaries, peri renal tubular capillaries, and interstitial blood vessels and
multifocal areas of renal tubular necrosis (Figure 4).
DISCUSSION
Diabetes mellitus (DM) is one of the globally shared enduring metabolic disorders designated
by persistent elevation of plasma sugar level.[29]
It is commonly classified as type 1, type 2,
gestational diabetes, and specific types of DM owing to other bases, of which type-2 diabetes
is the commonest form.[30]
Diabetes or diabetes mellitus is a complex or polygenic disorder,
which is characterized by increased levels of glucose (hyperglycemia) and deficiency in
insulin secretion or resistance to insulin over an elongated period in the liver and peripheral
tissues.[31]
Both the insulin resistance and deficiency lead to high blood glucose levels.
Evidences show that T2DM progression is rapid because of complications such as heart,
renal, nerve injury, retinopathy, developing in early stages of the disease.[2]
Identifying newly
biomarkers for T2D and its complications remains challenging due to the heterogeneous
nature of T2D. The heterogeneity relates not only to glycemic control or treatment
response.[3]
One of the most potent methods to induce experimental diabetes mellitus is chemical
induction by Alloxan.[32]
Alloxan is a urea derivative which causes selective necrosis of the
β-cells of pancreatic islets. In addition, it has been widely used to produce experimental
diabetes in animals such as rabbits, rats, mice and dogs with different grades of disease
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severity by varying the dose of alloxan used.[5,6]
The toxic action of alloxan on pancreatic
beta cells involve oxidation of essential sulphydryl (-SH groups), inhibition of glucokinase
enzyme, generation of free radicals and disturbances in intra cellular calcium homeostasis.[7]
This study was planned to investigate the role of cromen in treatment of type 2 diabetes
induced by alloxan in rats.
The study included 50 rats were divided into 5 groups (10 rats in each group) as following,
Group I (control Group): Rats were administrated orally with 1 ml single saline dose. Group
II (DMSO Group): Rats were administrated by giving 1ml of 0.2% DMSO for 40 days.
Group Ⅲ (Standard therapeutic): Rats were induced for diabetes mellitus (D.M). After 1
week of DM induction, animals were post treated orally with metformin (100 mg/kg daily for
40 day). Group IV (Therapeutic Group): Rats were induced for DM. After 1week of DM
induction, animals were post treated orally with cromen (50mg/kg daily for 40 day). Group V
(Positive control): Rats were injected intraperitoneal (I. p) with alloxan (150 mg/kg b. wt.).
Result in Table 1 showed significant decrease in the mean of Final body weight of diabetic
induced group (positive group) which Amounted to -20.05 % when compared to control
group (p < 0.001), while group treated with Cromen (Therapeutic) and metformin (standard)
showed statistically non-significant decrease which amounted to – 7.84 % and – 1.91 %
respectively when compared to control group (P > 0.05).
The inhibition of insulin secretion caused metabolic disorders of glucose and also lipids and
protein. The decline and inadequacy of insulin changed over anabolism to catabolism of
proteins and lipids.[33]
The good improvement of mean body weight of (standard therapeutic
group) due to using Metformin as type 2 diabetic drug, and helps diabetics to respond
normally to insulin. Like most diabetic drugs, as a result metformin helps on reduce
metabolic disorders of glucose and also lipids and loss protein tissue.[34]
Results in Table 2 showed that the mean of final plasma glucose level showed significant
elevation in positive group which amounted to 292.44% in compared to control group (P<
0.001), while treated groups with cromen (Therapeutic) and metformin (standard) groups
showed good improvement which amounted 130.42% in cromen and 125.09% in metformin
when compared in control group.
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The diabetogenic agent alloxan has two distinct pathological effects interfering with the
physiological function of the pancreatic beta cells. It selectively inhibits glucose-induced
insulin secretion through its ability to specifically inhibit the glucokinase, the glucose sensor
of the beta cell, and it causes a state of insulin-dependent diabetes mellitus through its ability
to induce a selective necrosis of the beta cells. These two effects of alloxan can be assigned to
specific chemical properties of alloxan.[35]
The good improvement of mean glucose level of
(standard therapeutic group) due to using Metformin as type 2 diabetic drug, metformin
improves glycemic control involves reducing hepatic glucose production, enhancing
peripheral insulin sensitivity, and blocking gastrointestinal glucose absorption.[36]
The study
result in agreement with[37]
the injection of alloxan-induced a significant increase (P < 0.001)
in blood glucose level of alloxan group compared to normal control group. Also, the study
result in agreement with[38]
metformin is the First-line, oral, glucose-lowering medication
recommended by the American Diabetes Association for people with type 2 diabetes and
individuals with Prediabetes.
The alloxanization caused significant increase in serum uric acid, urea, and creatinine and
decrease in level of albumin and protein values in diabetic animals when compared with
nondiabetic control. This may be due to the protein glycation in diabetes which may lead to
muscle wasting and increased release of purine. This may be due to the protein glycation in
diabetes which may lead to muscle wasting and increased release of purine, the main source
of uric acid, as well as increased activity of xanthine oxidase.[39]
Results in Table 3 showed that the mean value of serum ALT activity showed significant
elevation in positive group which amounted to 95.42% in compared to control group (P =
0.001), while treated groups with cromen (Therapeutic) and metformin (standard) showed
good improvement which amounted 16.01% in cromen and 50% in metformin when
compared in control group. Also, showed that the mean value of serum AST activity showed
significant elevation in positive group which amounted to 53.66% in compared to control
group (P < 0.05). While treated groups with cromen (Therapeutic) and metformin (standard)
showed good improvement which amounted 4.68% in cromen and 1.56% in metformin when
compared in control group. Also, showed that the mean value of serum Total bilirubin
showed significant elevation in positive group this amounted 37.83 % in compared to control
group (p< 0.01).this treated groups with cromen (Therapeutic) and metformin (standard)
showed good improvement which amounted - 24.32 % in cromen and, -10.81 % in metformin
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when compared in control group. In contrast, showed that the mean value of serum ALB
activity showed significant decrease in positive group this amounted -25.53% in compared to
control group (p< 0.01). While treated groups with cromen (Therapeutic) and metformin
(standard) showed good improvement which amounted -4.25% in cromen and, -6.38% in
metformin when compared in control group.
In diabetic animal‟s alteration in the activity of serum enzymes directly related to changed
metabolism in which enzymes are involved. Increased activity of liver enzymes in diabetic
animals are reported by many researchers. Increased transaminases level in absence of insulin
because of increased amino acid activity in hyperglycemic condition are responsible for
ketogenesis and gluconeogenesis.[40,41]
The study in agreement with[42]
serum AST and ALT levels increased, when compared with
normal rats.
Results in Table 4 showed that the mean level of serum Urea showed significant elevation in
positive group which amounted to 19.23% in compared to control group (P ˃ 0.05), while
treated group with cromen (Therapeutic) and metformin (standard) showed good
improvement which amounted 16.01% in cromen and 6.15% in metformin when compared in
control group.
Also, showed that the mean level of serum Creatinine showed significant elevation in positive
group which amounted to 24.61% in compared to control group (p< 0.001). While treated
groups with cromen (Therapeutic) and metformin (standard) showed good improvement
which amounted 10.76% in cromen and 13.84% in metformin when compared in control
group.
The significant increase observed in the serum urea and creatinine of all diabetic groups
might be due to increased synthesis from the damaged pancreatic cells caused by alloxan
injection.[43]
Renal illness is one of the most widely recognized and extreme difficulties of
diabetes.[44]
The study result is agreement with.[45,46]
Investigations announced that diabetic
rats indicated essentially expanded serum uric acid (SUA), serum creatinine (SCr), and urea
nitrogen (BUN) levels.
The study results in Table 4 showed that the mean level of serum cholesterol showed
significant elevation in positive group this amounted 35.60% in compared to control group (P
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< 0.05). While treated groups with cromen (Therapeutic) and metformin (standard) showed
good improvement which amounted 7.85%in cromen and, 11.64%in metformin when
compared in control group.
Also, showed that the mean level of serum triglyceride showed significant elevation in
positive group this amounted 56.82% in compared to control group (P = 0.001). While treated
groups with cromen (Therapeutic) and metformin (standard) showed good improvement
which amounted 23.98%in cromen and, 27.06% in metformin when compared in control
group.
A deficiency of insulin is associated with increase in cholesterol levels due to the enhanced
mobilization of lipids from adipose tissue to the plasma.[47]
The study results are nearly
similar with the study of[48]
who found that the rise in blood glucose is accompanied with
disturbance of lipid profile including the increase in Total cholesterol, triglycerides, levels,
which make diabetic patient at high risk for several complications including fatty liver
degeneration.
Results of histopathological examination showed severe alterations of liver, kidney and
pancreatic tissues were observed in untreated diabetic rats. Also, cromen attenuated the
histopathological changes in Alloxan diabetic rats. Results are in accordance with who stated
that pancreatic tissues of diabetic control rats showed a decrease of Langerhans islet size and
multiple degeneration and injuries. In addition to, the number of β-cells was reduced, and
some necrosis and destruction were found[49]
Also, in agreement with, ultrastructural findings
of the liver showed significant differences between nondiabetic and diabetic animals.[50]
Also,
in agreement with, diabetes induced in rats by alloxan is associated with the generation of
reactive oxygen species (ROS) which cause oxidative damage, particularly to heart, kidney,
eyes, nerves, liver, small and large blood vessels, and immunological and gastrointestinal
system.[51,52]
Generally, the present obtained findings confirm that the influences of cromen are attributed
to the antidiabetic properties.
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Table 1: The initial and final body weight of rats in all studied groups.
Groups Initial Body weight (g) Final body weight (g)
Control
Mean ± SEM
230.7± 1.3 334±2.6C
DMSO
Mean ± SEM
% change
227± 1.06
-1.60%
330.6±1.11 C
-1.01%
Positive
(Alloxan-induced)
Mean ± SEM
% change
231± 3.00
0.130%
267±1.0***
-20.05%
Metformin
Mean ± SEM
% change
234.6± 1.77
1.69%
340.4±1.4 C
1.91%
Compound
Mean ± SEM
% change
234.5± 0.88
1.64%
307.8±11.5
-7.84%
P value P ˃ 0.05 P˂ 0.001
* P< 0.05 compared to control group, ** P< 0.01, *** P< 0.001 compared to control group.
aP< 0.05,
bP< 0.01,
cP< 0.001 compared to positive control group. The mean difference is
significant at P< 0.05. % change = Percent of change compared to control group
Table 2: Mean level of glucose in different studied groups.
Groups Glucose (Initial) (mg/dl) Glucose (final) (mg/dl)
Control
Mean ± SEM
97±2.1 103.2±4.5c
DMSO
Mean ± SEM
% change
93±0.83
-4.123%
118.3±3.7c
14.63%
Positive
(Alloxan-induced)
Mean ± SEM
% change
330±8.0***
240.2%
405±15.0***
292.44%
Metformin
Mean ± SEM
% change
331.5±5.5***
241.75%
232.3±4.8***c
125.09%
Compound
Mean ± SEM
% change
327.1±4.8***
237.21%
237.8±2.1***c
130.42%
P value P˂ 0.001 P˂ 0.001
* P< 0.05 compared to control group, ** P< 0.01, *** P< 0.001 compared to control group.
aP< 0.05,
bP< 0.01,
cP< 0.001 compared to positive control group. The mean difference is
significant at P< 0.05. % change = Percent of change compared to control group
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Table 3: Mean values of ALT, AST, ALB and total bilirubin in all studied groups.
Groups ALT
(U/L)
AST
(U/L)
ALB
(mg/dl)
T. Bil
(mg/dl)
Control
Mean ± SEM
30.6±1.4C 64.1±4.3
b 4.7±0.11
C 0.37±0.007
DMSO
Mean ± SEM
% change
39.6±1.5 C
29.41%
72.1±8.1 a
12.48%
4.5±0.15 b
-4.25%
0.41±0.010
10.81%
Positive
(Alloxan-
induced)
Mean ± SEM
% change
59.8±4.3***
95.42%
98.5±1.5**
53.66%
3.5±0.55***
-25.53%
0.51±0.010***
37.83%
Metformin
Mean ± SEM
% change
45.9±2.7**a
50%
65.1±4.3 b
1.56%
4.4±0.14 b
-6.38%
0.41±0.019a
10.81%
Compound
Mean ± SEM
% change
35.5±3.5 C
16.01%
67.1±2.9 b
4.68%
4.5±0.55 b
-4.25%
0.46±0.017 b
24.32%
P value 0.001 P< 0.05 P< 0.01 P< 0.01
* P< 0.05 compared to control group, ** P< 0.01, *** P< 0.001 compared to control group.
aP< 0.05,
bP< 0.01,
cP< 0.001 compared to positive control group. The mean difference is
significant at P< 0.05. % change = Percent of change compared to control group
Table 4: Mean level of urea, creatinine, cholesterol, triglyceride in all studied groups.
Groups Urea
(mg/dl)
Creatinine
(mg/dl)
Cholesterol
(mg/dl)
Triglyceride
(mg/dl)
Control
Mean ± SEM
13±0.9 0.65±0.014c 76.4±2.9
c 133.4±7.1
c
DMSO
Mean ± SEM
% change
13.8±0.7
6.15%
0.68±0.02c
4.61%
79±3.4 c
3.40%
148.5±5.9 c
11.31%
Positive
(Alloxan-
induced)
Mean ± SEM
% change
15.5±5
19.23%
0.81±0.015***
24.61%
103.6±3.6**
35.60%
209.2±10.4***
56.82%
Metformin
Mean ± SEM
% change
13.8±0.8
6.15%
0.74±0.01**a
13.84%
85.3±3.9a
11.64%
169.5±5.8**b
27.06%
Compound
Mean ± SEM
% change
13.1±0.7
0.76%
0.72±0.01**b
10.76%
82.4±4.4a
7.85%
165.4±7.4* b
23.98%
P value P ˃ 0.05 P˂ 0.001 P˂ 0.05 P= 0.001
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* P< 0.05 compared to control group, ** P< 0.01, *** P< 0.001 compared to control group.
aP< 0.05,
bP< 0.01,
cP< 0.001 compared to positive control group. The mean difference is
significant at P< 0.05. % change = Percent of change compared to control group.
Figure 1: The Highest Occupied Molecular Orbital (HOMO) and the Lowest
Unoccupied Molecular Orbital (LUMO) of ‘’cromen’’.
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Figure 2: Histopathological examination of pancreas tissue. A (negative control)& B
(DMSO) showed normal pancreatic acini, ducts, and islets, (H&E X 200) (H&E X 400)
respectively, C (Metformin) showed pancreatic islets hyperplasia (arrow) and
pancreatic duct dilatation (arrow head), (H&E X 200), D (cromen) showed normal
pancreatic acini, ducts, and islets, (H&E X 400), E (Positive control) showed severly
dilated pancreatic duct (arrow head),and showed sever congestion in the interstitial
blood vessel (arrow head) with thickened muscular wall (arrows) (H&E X 400).
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Figure 3: Histopathological examination of liver tissue. A (negative control) & B
(DMSO) showed normal hepatic parenchyma; note the normal hepatocytes, blood
sinusoids, and portal area, (H&E X 200), C (Metformin) showed congestion in the
portal vein (Arrow head) and dilated hepatic artery (arrow), (H&E X 200), D (cromen)
normal hepatic parenchyma; note the normal hepatocytes, blood sinusoids, and portal
area, (H&E X 200), E (Positive control) showed focal areas of hepatocytes necrosis
infiltrated and replaced with mononuclear cells infiltration (arrow head),and Portal
tract inflammatory changes; note the congestion in hepatoportal blood vessel (arrow
head) and the infiltration of mononuclear cells (arrow), (H&E X 400).
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Figure 4: Histopathological examination of kidney tissue. A (negative control) & B
(DMSO) showed normal renal parenchyma; note the normal renal glomeruli and renal
tubules, (H&E X 200) (H&E X 400) respectively, C (Metformin) showed congestion in
the interstitial blood vessel (arrow head) with thick muscle wall (arrow), (H&E X 400),
D (cromen) showed normal renal parenchyma; note the normal renal glomeruli and
renal tubules, (H&E X 200), E (Positive control) showed sever congestion in the
glomerular capillaries (thin arrow), peri renal tubular capillaries (thick arrow), and
interstitial blood vessels (arrow head), (H&E X 400) and multifocal areas of renal
tubular necrosis (arrow head), (H&E X 200).
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