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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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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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