Pathological, Immunohistochemical and Biochemical Studies on The Therapeutic Effect of Raphanus...

Egypt. J. Comp. Path &Clinic Path. Vol. 28 No.1, 2015 ; 1- 17 ISSN 1110-7537

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Pathological, Immunohistochemical and Biochemical

Studies on The Therapeutic Effect of Raphanus

Sativus Oil on Streptozotocin Induced Diabetic Rats

Safaa Abbas Abed , M.O. El-Shazely, Kawkab A. Ahmed, Essam M.

Abdel-mawla and Adel K. Ibrahim

Ministry of Science and Technology, Iraq.

Department of Pathology, Faculty of Veterinary Medicine, Cairo University

Arab Academy for Science , Technology and maritime Transport, Alexandria.

Department of clinical Pathology, Faculty of Veterinary Medicine, Cairo University

ABSTRACT— This study was conducted to investigate the Pathological,

Immunohistochemical and Biochemical effects of Radish Oil on Streptozotocin induced diabetic

male rats. Eighty Albino rats (weighing about 250-300 g) were used in this study, they were

randomly divided into 4 equal groups, each containing 20 rats as follows, group 1; control

negative rats, group 2; rats administrated radish oil (300 mg/kg.b.w. orally); group 3: STZ

diabetic rats (single intra-peritoneal injection of streptozotocin (STZ, 60 mg/kg) and group 4:

STZ diabetic rats treated with radish oil (300 mg/kg.b.w) . Blood samples and tissue specimens

were collected from pancreas, liver and kidneys from all rats of all groups at 15, 30 and 45 days

from the start of the experiment for biochemical and histopathological examinations respectively.

The biochemical analysis revealed significant increase in lipase, α-amylase, AST, ALT, ALP,

urea and creatinine levels in the serum of diabetic rats, while the diabetic rat treated with radish

oil showed a significant amelioration in those previously mentioned parameters. The

histopathological examination revealed alterations in the pancreas of diabetic rats characterized

by necrosis and atrophy of β-cells of islets of Langerhans. Activation of kupffer cells with

cytoplasmic vacuolation of hepatocytes, vacuolation and necrosis of renal tubular epithelium

were also noticed. However, regeneration in the pancreas, liver and kidneys was observed in

diabetic rats treated with radish oil. In Pancreas of diabetic rats, the immunoreactivity for anti-

insulin antibodies was markedly decreased (decrease in the number of insulin positive cells).

However, after treatment with radish oil the positive immunoreactions of B-cells for antiinsulin

antibodies were obviously increased. From this study we could conclude that Raphanus sativus

oil contains many antioxidant compounds that stimulate regeneration and reactivation of B- cells

to produce more insulin and this improvement was confirmed by biochemical parameters,

histopathological findings in the pancreas, liver and kidneys as well as the

immunohistochemistry of anti-insulin antibodies in the pancreatic tissue.

Key words: Raphanus Sativus Oil, Streptozotocin, diabetes, histopathology,

immunohistochemistry, rat

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Egypt. J. Comp. Path &Clinic Path. Vol. 28 No.1, 2015 ; 1- 17 ISSN 1110-7537

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INTRODUCTION

Diabetes mellitus is a group of

metabolic alterations characterized by

hyperglycemia resulting from defects in insulin

secretion, action or both. It is made up of two

types: Type I and Type II. Type I diabetes often

referred to as juvenile diabetes, is insulin

dependent and known to affect only 5% of the

diabetic population. Type II, which is non-

insulin dependent, usually develops in adults

over the age of 40. It has already been

established that chronic hyperglycemia of

diabetes is associated with long term damage,

dysfunction and eventually the failure of

organs, especially the eyes, kidneys, nerves,

heart and blood vessels. Diabetes mellitus is

most common metabolic disorder considered

among five leading causes of death in the

world. It is now recognized as one of the killer

diseases, leading causes of morbidity and

mortality in the world Rahimi et al., (2005)

and Abo et al., (2008).

In animals diabetes mellitus is

considered as a common metabolic disease

diagnosed frequently in canine and feline. On

the other hand, clinical syndrome of diabetes

is described rarely in other domestic species

(cattle, small ruminants, swine and horses)

Nelson, (2010).

Over the years, several animal models

have been developed for studying diabetes

mellitus or testing anti-diabetic agents. These

models include chemical, surgical

(pancreatectomy) and genetic manipulations

in several animal species to induce diabetes

mellitus. The chemical model such as

streptozotocin prevents DNA synthesis in

mammalian and bacterial cells. The

biochemical mechanism results in

mammalian cell death is that streptozotocin

prevents cellular reproduction with a much

smaller dose than the dose needed for

inhibiting the substrate connection to the

DNA or inhibiting many of the enzymes

involved in DNA synthesis (Holemans and

van Assche, 2003). Streptozotocin enters the

pancreatic B-cell via a glucose transporter-

GLUT2 and causes alkylation of

deoxyribonucleic acid (DNA). Furthermore,

STZ induces activation of poly adenosine

diphosphate ribosylation and nitric oxide

release. As a result of STZ action, pancreatic

B-cells are destroyed by necrosis ( Mythili et

al., 2004).

Raphanus sativus had anti-diabetic

potential and the active principles responsible

may be flavonoids, terpenes and phenolic

compounds. Flavonoids like anti-oxidants,

may prevent the progressive impairment of

pancreatic beta-cell function due to oxidative

stress and thus reduce the occurrence of type

2 diabetes (Rizvi, 2005).

In accordance with this, the present

study was focused on investigating the

therapeutic effect of Raphanus sativus oil in

diabetic male rats. Biochemical parameters,

histopathology and immunohistochemistry

were done to investigate the effects of this

natural material on pancreas, liver and

kidneys of diabetic rats.

MATERIALS AND METHODS

Materials:

Chemicals:

1- Streptozotocin, Zansor (STZ; product

number S45688), was obtained from Sigma-

Aldrich (St. Louis, MO) Chemical Co.

2- Raphanus sativus oil was obtained from

Gaara Quality Seeds Company in Cairo.

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 28 NO.1, 2015 ; 1- 17

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Method:

Induction of diabetes:

Diabetes was induced by

Streptozotocin a single intra-peritoneal

injection of 60 mg/kg body weight (b.w.)

STZ (Sigma Company, Aldrich, USA),that

was dissolved in citrate buffer (0.1 M, PH:

4.5) (Rakieten. 1963; Cennet and

Ebubekir ,2010; Abeer. 2013;Cheng and

Li. 2013). STZ was dissolved in cold citrate

buffer (0.1 M, PH: 4.5) according to Yan

Lab Protocol (Xinhe Yin and Ben Zhang

.2009). After 72 hours of STZ administration,

the blood was collected to determine fasting

blood glucose level. Only rats with fasting

blood glucose over (250) mg/dL were

considered diabetic and were included in the

experiment (Cheng and Li, 2013) .

Preparation and administration of radish

oil:

Before extraction of oil, R. sativus

seeds (5kg) were cleaned manually, washed

with distilled water and oven dried at a

temperature of 40°C. The oil extracted by an

electrically driven machine by cold press

(Robert et al. 2012) without the use of any

solvent. The R. sativus oil obtained was

1250ml dispensed in 30ml washed and

sterilized brown glass bottles having a

dropper. Raphanus sativus oil was given to

rats in the dose of 300mg/kg body weight by

gavage ( Surekha et al. 2010).

Experimental Design:

A total number of 80 Albino rats

(weighing about 180±10 g) were obtained

from the animal house, Faculty of Veterinary

Medicine, Cairo University, Egypt. Rats were

left for acclimatization on lab. conditions for

7days, prior to the onset of the experiment.

The rats were fed with standard laboratory

diet and allowed to drink water ad libitum.

Rats were randomly divided into 4 groups,

each containing 20 rats as follow:

Group 1: Non-diabetic control negative rats.

Group 2: Control Raphanus sativus oil (300

mg/kg.b.w) .

Group 3: Diabetic control rats (single intra-

peritoneal injection of STZ 60 mg/kg body

weight).

Group 4: Diabetic rats treated with

Raphanus sativus oil (300 mg/kg.b.w) .

Serum biochemical analysis:

Blood samples were collected from

rats of all groups from the retro-orbital

venous plexus in a clean centrifuge tube and

allowed to clot, then centrifuged at 3000 rpm

for 10 minutes for serum separation.

Different biochemical parameters were

estimated included: The activities of amylase

and lipase enzymes (Henry and Chiamori,

1960). The values of (ALT and AST)

transaminases (Reitman and Frankel,

1957). Alkaline phosphatase enzyme

(Belfield and Goldberg, 1971). Urea and

creatinine (Fawcett and Scott, 1960). All

the previous parameters were analyzed by

auto analyzer (STATLAB SZSL60-

SPECTRUM) using commercial kits of

"SPECTRUM Diagnostic, GERMANY”.

Histopathology:

Tissue specimens were collected from

pancreas, liver and kidneys of rats from all

groups at 15, 30 and 45 days post

experiment. Tissues were fixed in buffered

neutral formalin solution 10%. Formalin

fixed specimens were routinely processed,

embedded in paraffin, sectioned at 4-6 um

thickness and stained with Hematoxyline and

Eosin (Bancroft and stevens ,2010).


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Immunohistochemistry:

To determine the insulin content in

the pancreatic islets, insulin

immunohistochemistry was performed on

paraffin-embedded pancreatic tissue using

anti-insulin antibody (Weidenheim et al.,

1983).

Statistical analysis:

Data were analyzed statistically by

analysis of variance, for statistical

significance using L.S.D. test, two way

ANOVA, post hoc multiple comparisons

according to Snedecor and Cochron (1989).

RESULTS Biochemical analysis:

The results presented in table (1) and

figure(1) summarized as the diabetic rats

(group 3) showed a significant increase in

lipase , α-amylase , AST , ALT , ALP , urea

and creatinine levels when compared with

non-diabetic control negative rats and control

Raphanus sativus treated rats. However,

diabetic rats treated with radish oil showed

highly significant decrease in lipase , α-

amylase , AST , ALT , ALP , urea and

creatinine levels when compared with

diabetic rats.

Histopathology:

Pancreas:

Microscopically, sections of pancreas

from control, untreated rat sacrificed at 15,

30, and 45 days revealed normal histological

structure of pancreatic parenchyma; lobules

of pancreatic acini and islets of Langerhans

(Fig.2). Moreover, Pancreas of rats treated

with radish oil showed no histopathological

changes.

However, microscopical examination

of pancreas of diabetic rats sacrificed at 15,

30 and 45 days showed more or less similar

histopathological alterations which consisted

mainly of cytoplasmic vacuolation of acinar

epithelium( Fig. 3) and B-cell in the center of

islets of Langerhans (Fig.4). Perivascular

mononuclear inflammatory cell infiltration

was observed in some rats 15 days post

treatment. At day 45, diabetic rats showed

necrosis of islets of Langerhans and

dilatation of pancreatic duct ( Fig.5 ).

Histopathological examination of

pancreatic tissue of diabetic rats treated with

radish oil at a dose of 300 mg / kg. B.W did

not show improvement in islet of Langerhans

during the first fifteen days. Most of

pancreatic sections revealed necrosis in the

center of the islets of langerhans . Whereas,

atrophy in the islet of Langerhans and

exocrine acinar damage represented by

cytoplasmic vacuolation of acinar epithelium

with pyknotic nuclei were observed in some

cases (Fig.6). Interacinar mononuclear cells

infiltration associated with dilatation in

pancreatic duct were also observed in some

examined sections.

At 30 days post treatment with radish

oil the histological alterations were more or

less similar to those observed in diabetic rats

but were less severe and less extensive. The

pancreas of some diabetic rats revealed

vacuolation of cells of islet langerhans and

mononuclear cells infiltration around

pancreatic duct (Fig.7).

However, at 45days, most sections of


regeneration in pancreatic tissue represented

by hypertrophy and hyperplasia of islets of

Langerhans (Fig .8).


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Liver:

Microscopical examination of liver of

rats from groups 1 and 2 revealed normal

histological structure of hepatic lobules along

the entire experimental period (Fig. 9). At 15

day, liver of diabetic rats showed cytoplasmic

vacuolation of hepatocytes with large

vesicular nuclei as well as congestion of

hepatic sinusoids were commonly observed

in all cases (Fig.10).While, at 30 day, liver of

diabetic rats showed fibroplasia in the portal

triads around bile duct with dilatation and

congestion of hepatoportal blood vessel and

hyperplasia of epithelial lining bile ducts .

Some examined sections showed focal

hepatic haemorrhage dispersing the

hepatocytes far away from each other.

Kupffer cells activation and large vesicular

hepatocytic nuclei were commonly observed.

At 45 day, liver of diabetic rats

showed focal hepatic necrosis associated with

mononuclear infiltration, sinusoidal dilatation

and kupffer cell activation (Figs. 11).

Concerning group 4, at 15 day of

experiment, liver of diabetic rats treated with

radish oil showed vacuolation of

centrolobular hepatocytes and kupffer cell

activation together with congestion of

central vein and hepatic sinusoids.

At 30 day of experiment, liver of


strands of fibroblast proliferation in portal

tract around bile duct and kupffer cell

activation. At 45 day of experiment, some

examined sections showed necrosis of

epithelial lining of bile duct and portal

infiltration with mononuclear cells (Fig.12)

and activation of kupffer cells.

Kidneys:

Microscopical examination of

Kidneys of control rats (group1) and radish

oil control (group 2) revealed normal

histological structure of renal parenchyma

along the experimental period (Fig.13).

Meanwhile, kidneys of diabetic rats (15 and

30d) showed vacuolation of the glomerular

tuft, dilatation of renal tubules, perivascular

edema associated with inflammatory cells

infiltration (Fig.14). At 45 day, kidneys of

diabetic rats showed hypertrophy of

glomerular tuft, thickening of glomerular

basement membrane, periglomerular strands

of fibroblasts proliferation as well as marked

vacuolar degeneration of renal tubular

epithelium (Fig. 15). Necrosis of the

epithelial lining of many renal tubules

associated with focal inflammatory cells

infiltration were also observed .

Regarding group 4, at 15 and 30 days,

the examined sections of kidneys of diabetic

rats treated with radish oil showed congestion

of intertubular blood vessels and necrobiotic

changes of renal tubular epithelium

associated with congestion and vacuolation

of glomerular tuft (Figs. 16). However, at 45

days, most examined sections of kidneys of

diabetic rat treated with radish oil showed

mild histopathological changes.

Immunohistochemistry:

Immunohistochemical staining of

control pancreas as well as pancreas of rat

treated with radish oil showed B-cells stained

strong positive for anti-insulin antibodies

which appeared as brown granules occupying

the cytoplasm of great numbers of the B-cells

(Fig.17 ). However, in the pancreas of

diabetic rats, the immunoreactivity for anti-

insulin antibodies was markedly decreased

(decreased number of insulin positive cells)

(Fig. 18). On other hand, after treatment of

diabetic rats with radish oil, the positive

immunoreaction of B-cells for anti-insulin

antibodies were obviously increased (Fig.

19).

EGYPT. J. COMP. PATH &CLINIC PATH. VOL. 28 NO.1, 2015 ; 1-

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Table (1): showing the levels of serum lipase, α-amylase, AST, ALT, ALP, Urea and

creatinine in different groups

parameter

Group 1 (Control negative)

Group 2 (Control Radish oil)

Group 3 (Diabetic group)

Group 4 (Diabetic Radish oil)

Lipase

28.87 ± 4.00 29.49± 3.24 46.69±13.35Ea*** 39.64± 8.70b***

α-amylase

76.89 ± 2.26

85.71± 14.54 120.66±30.81a*** 94.55± 17.4b***

AST

34.84 ± 1.91

38.31± 1.33

65.80±3.06 a***

44.75± 2.73b***

ALT 40.38 ± 2.49 43.07± 2.74 56.88±4.22 a *** 50.14± 1.95b***

ALP 28.87 ± 2.80 29.49± 3.25 46.69±3.76 a*** 39.64± 3.35b***

Urea 22.30 ± 1.72 19.69± 1.21 33.56±2.34 a*** 32.23± 2.81 b*

Creatinine 0.76 ± .09 0.90 ± 0 .21 1.66 ±0 .56 a*** 1.01 ±0 .34 b**

Each value is expressed Mean ± where n=20. -a Diabetic control positive group. Vs. Normal control negative

group -b Diabetic control positive group vs. All treated group p < 0.05*, p < 0.01** ,p < 0.001.***

0

20

40

60

80

100

120

140

Lipase α-amylase AST ALT ALP Urea creatinine

CONSTERATION G1

G2

G3

G4

Figure ( 1): chart showing the mean values ± SD of lipase, α-amylase, AST, ALT, ALP , urea

and creatinine) in different experimental groups.


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Fig. ( 2 ): Pancreas of control rat (45 days

post treatment) showing lobules of

pancreatic acini and islets of Langerhans

were embedded within the exocrine portions

(H & E X 400).

Fig.(3):Pancreas of diabetic rat

(15 days post treatment)showing cytoplasmic

vacuolation of acinar epithelium

(H & E X 400).

Fig.( 4):Pancreas of diabetic rat (15 days

post treatment ) showing vacuolation of B-

cell in the center of islet of Langerhans

(H&E X 400).

Fig. (5): Pancreas of diabetic rat (45 days post

treatment ) showing necrosis of islets of

Langerhans with pyknosis of their nuclei and

dilatation of pancreatic duct.

(H&E X 400).


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Fig.( 6 ): Pancreas of diabetic rat treated with

radish oil group (15 days post treatment)

cytoplasmic vacuolation of acinar epithelium

(H & E X 400).

Fig.(7): Pancreas of diabetic rat treated with

radish oil (30 days post treatment) showing

vacuolation of cells of islet Langerhans and

mononuclear cells infiltration around

pancreatic duct.

(H & E X 400).

Fig. (8): Pancreas in diabetic rat treated with radish oil (45days post treatment) showing

hypertrophy and hyperplasia of islets of Langerhans. (H & E X 400).


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Fig. (9): liver of rat treated with radish oil

(45days post treatment) showing the normal

histological structure of hepatic lobule

(H & E X 400).

Fig.(10): Liver of diabetic rat (15days post

treatment) showing cytoplasmic vacuolation of

hepatocytes with large vesicular nuclei as well

as congestion of hepatic sinusoids.

(H & E X 400).

Fig.(11): Liver of diabetic rat (45 days post

treatment ) showing focal hepatic necrosis

associated with mononuclear cells

infiltration, sinusoidal leukocytosis and

kupffer cell activation (H & E X 400).

Fig. (12): Liver of diabetic rat treated with

radish oil (45days post treatment) showing

necrosis of epithelial lining bile duct and portal

infiltration with mononuclear cells (H & E X

400).


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Fig.(13): Kidney of rat treated with radish oil

(45days post treatment) showing no

histopathological changes (H & E X 400).

Fig. (14): Kidney of diabetic rat (30days post

treatment) showing vacuolation of the

glomerular tuft, dilatation of renal tubules,

perivascular edema associated with

inflammatory cells infiltration. (H & E X

400).

Fig. (15): Kidney of diabetic rat (45days post

treatment) showing hypertrophy of

glomerular tuft, thickening of glomerular

basement membrane, periglomerular strands

of fibroblasts proliferation as well as marked

vacuolar degeneration of renal tubular

epithelium. (H & E X 400).

Fig. (16): Kidney of diabetic rat treated with

radish oil (30days post treatment) showing

slight congestion and vacuolation of

glomerular tufts (H & E X 400).


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Fig. (17): pancreas of control rat (45days post

treatment) showing strong positive reaction of

B cells for anti-insulin antibodies which

appeared as brown granules occupying the

cytoplasm of great numbers of the β-cells

(X 400).

Fig.(18): Pancreas of diabetic rat (45 days

post treatment) showing decrease

immunoreactivity for anti-insulin

antibodies. Noticed decrease in the number

of insulin positive cells (X 400).

Fig. (19): Pancreas of diabetic rat treated with radish oil (45days post treatment) showing

positive immunoreactions of β-cells for anti-insulin antibodies. Notice increase in the number

of insulin positive cells (X 400).


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DISCUSSION

Diabetes mellitus is the most common

metabolic disorder considered among five

causes leading to death in the world. Type 2

diabetes mellitus is the world’s largest

endocrine disorder which is characterized by

decrease in insulin secretion, defect in

glucose uptake in skeletal muscle and fat and

increased glucose production in the liver.

(Taniguchi et al., 2006).

Many synthetic oral anti-diabetic

drugs are associated with drawbacks such as

resistance and side effects ranging from liver

toxicity, increased cardiovascular risk,

abdominal discomfort, flatulence and

diarrhea Cheng and Fantus ,

(2005).This has led to the usage of medicinal

plants for the treatment of diabetes, but most

of them lack scientific evidence to validate

their usage and efficacy Morelli and

Zoorob (2000).

Streptozotocin is used as a well-

known chemical agent to induce their

experimental diabetes mellitus by specific

cytotoxicity effect on pancreatic β-cells.

Streptozotocin was employed to chemically

induce diabetes in experimental animal

models and affects the endogenous insulin

release/action or both and as a result

increases fasting blood glucose level

(Nastaran, 2011).

In the present study, the diabetic rats

revealed significant increase in pancreatic

lipase and α-amylase which are in agreement

with Yadavucs, et al., (2005) and Quiros et

al.,(2008) who found that the highly

significant elevation of serum amylase in the

included patients might be attributed to the

hyperglycemia, where amylase activity

increases according to the degree of

hyperglycemia . Moreover, these results

confirmed with the histopathological changes

observed in the pancreas of diabetic rats

which characterized by vacuolation of

epithelial lining pancreatic acini, necrosis and

atrophy of β-cells of islets of Langerhans, as

well as focal hemorrhages and cystic

dilatation of pancreatic duct . These results

are in accordance with the findings of Cook

et al.,(2005); Cristina et al.,(2008); Gandhi

and Sasikumar (2012) and Kulkarni et

al.,(2012) .

In the current study, the diabetic rats

treated with radish oil showed reduction in

lipase and α-Amylase when compared with

control diabetic rats. These results are in

agreement with He Q and Yao, (2006) and

Ramachandran., et al (2012) who

explained the antidiabetic effect of radish oil

by inhibitory effect on α-amylase when

demonstrated that α-Amylase is catalyses the

hydrolysis of a-1, 4-glucosidic linkages of

starch, glycogen and various oligosaccharides

and α–glucosidase further breaks down the

disaccharides into simpler sugars, readily

available for the intestinal absorption.

In the present work, the

histopathological changes in the pancreas

from diabetic rats treated with radish oil were

partial regeneration in pancreatic tissue,

represented by hypertrophy and hyperplasia

of islets of Langerhans in some rats. These

results were in agreement with Tahany et

al.,(2015) who stated that Egyptian radish

(Raphanus sativus) may lead to the

regeneration of Beta-cells of the pancreas and

potentiating of insulin secretion from

surviving cells. The increase in insulin

secretion and consequent decrease in blood

glucose level may lead to inhibition of lipid

peroxidation and control of lipolytic

hormones. The authors concluded that the

histopathological examination of pancreas

tissues revealed significant changes

attributable to the STZ- induced diabetic rats,

while radish oil showed partial recovery of

STZ effects on pancreas tissues.


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The results of this study showed

increase in liver function enzyme AST, ALT

and ALP when compared with control

negative rats. These results are harmonious

with Hsueh CJ, et al (2011) who confirmed

that the release of these enzymes into the

serum is a result of tissue injury or changes in

the permeability of liver membranes; hence

the concentration may increase with acute

damage to liver cells with acute damage to

liver cells. Rai et al., (2010) who found that

the STZ induces hepatocellular damage

association high serum levels of AST, ALT

and ALP in untreated diabetic rats.

In the current study, the results of

histopathological changes in liver of diabetic

rats characterized by activation of kupffer

cells, apoptosis of hepatocytes, dilatation and

congestion of central vein with necrosis of

sporadic hepatocytes, focal hepatic necrosis

associated with mononuclear cell infiltration.

fibroplasia in the portal tracts were also

observed . Similar results reported by Hala et

al.,(2013) reported that the Liver tissues in

diabetic rats after STZ injection showed

activation of kupffer cells, sinusoidal

leukocytosis, apoptosis of hepatocytes ,

marked dilatation and congestion of central

vein with necrosis of sporadic hepatocytes ,

as well as congestion of central vein and

focal hepatic necrosis replaced by

mononuclear infiltration.

Concerning the treatment with radish

oil, our results were in accordance with those

of Eman A.Sadeek ( 2011 ) who stated that

oral administration of radish juice leads to

decrease in the levels of serum AST, ALT ,

ALP which indicated the effectiveness of the

Radish against hepatotoxicity. The hepato-

treated effect of radish may be due to its

antioxidant contents. Also, Kalantari et al.

(2009) showed that crude radish seed extract

significantly decreased AST and ALT

activities indicates protection of liver from

injury.

Many previous studies were

conformity about the radish as a better repair

for liver tissue. Kalantari H et al.,(2009)

evaluated the liver protective activity of

radish seed crude extract as it decreased AST

and ALT activities and may be good enough

to protect liver damage.

In view of the current results, we

observed that radish oil treatment may have

therapeutic benefits in diabetes due to its

ability to reduce oxidative stress and to

improve lipids profile and liver enzyme. The

chemical nature of radish have a high rate of

antioxidant enzymes used to stop the

complication and development of diabetes

mellitus and working to repair tissue injury.

In the current study, there was an

increase in the level of plasma urea and

creatinine in the diabetic rats which are in

agreement with Arulselvan et al.,2006;

Almadal and Vilstrup, 1988 who reported

increases in the levels of kidney functional

markers such as urea and creatinine which

may attributed to that STZ-induced metabolic

disturbances and renal dysfunction. The later

were assisted with our histopathological

observation in the kidneys of diabetic rats

which summarized as marked tubular

damage, haemorrhage and congestion of

intertubular blood capillaries and focal

necrosis of renal tubules associated with

inflammatory cells infiltration. These results

are in agreement with Leegwates et al.,

(1984) who demonstrated two types of

effects of STZ on the kidneys of rats. The

primary effect, the diabetes factor was

associated with hyperglycaemia and was

responsible for dilatation of proximal and

distal tubules in the cortex. The secondary

effect, named the individual response factor,

was associated with inflammatory processes.

In the present study, the diabetic rats

treated with radish oil showed decrease in

creatinine and urea nitrogen concentrations


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,these results are in agreement with

Shirwaikar et al. (2005) and Kishor

Kumar et al.,(2013) who demonstrated that

the Raphanus sativus induce significant

reduction in serum creatinine and urea .

These results came in harmony with our

histopathological findings in kidney tissue.

Moreover, these results were in accordance

with those of Beevi et al., (2010 )and Beevi

et al.,( 2012) who explain that the reduction

of kidney function (urea and creatinine) of

diabetic rats treated with radish oil was due

to the improvement in the kidney

dysfunctions and kidneys pathological

changes and reduction in the inflammatory

reaction when compared with the diabetic

untreated rats ,this improvement may be due

to the presence of poly phenolic compounds

in the radish oil.

Other nephroprotective effect of radish

have been reported as it inhibit xenobiotic-

induced nephrotoxicity in experimental

animal models due to their potent anti-

oxidant or free radicals scavenging effects (

Shirwaikar et al. 2005). In addition

alkaloids in radish have been reported to be

strongly inhibit lipid peroxidation induced in

isolated tissues via its antioxidant activity

(Shirwaikar et al., 2005; Kumaran and

Karunnakaran, 2007).

Concerning the immunohistochemical

examination, our results were in agreement

with Ozlem et al., (2003) who reported that

the pancreatic β-cells of untreated control

animals were stained strongly for insulin in

addition that β-Cells were mainly located in

the center of the islets. While , the untreated

diabetic rats β-cells were particularly

degenerated in central parts of the islets and

the insulin staining was poor because of

degeneration of islet cells , weak

immunohistochemical staining of insulin was

observed in diabetic rats. Moreover, Siham

et al.,(2014)and M. Abdollahi et al (2011)

stated that the positive insulin expression was

seen in the form of dark brown granules

present in the cytoplasm of beta-cells and the

diabetic group showed a marked reduction in

beta-cells number.

Conclusion: Raphanus sativus oil contains

many antioxidant compounds stimulate

regeneration and reactivation of B- cells to

produce more insulin and this improvement

is confirmed by biochemical parameters and

histopathological and immunohistochemical

findings on pancreas, liver and kidneys

tissue.

REFERENCES Abeer A. Abd El-Baky.,(2013): Clinicopathological Effect of Camellia

sinensis Extract on Streptozotocin-Induced

Diabetes in Rats. World Journal of Medical

Sciences 8 (3): 205-211, 2013.

Abo, K. A., Fred-Jaiyesimi, A. A. and

Jaiyesimi, A. E. A. (2008): Ethnobotanical

studies of medicinal plants used in the

management of diabetes mellitus in South

Western Nigeria, J. Ethnopharmacol., vol.

115: 67-71.

Almadal TP and Vilstrup H (1988): Strict

insulin treatment normalizes the organic

nitrogen contents and the capacity of urea-

nitrogen synthesis in experimental diabetes in

rats. Diabetol, 31: 114-118.

Aly, Tahany A.A., S.A. Fayed, Amal M.

Ahmed and E.A. El-Rahim, (2015):

Antidiabetic properties of Egyptian radish

and clover sprout in experimental rats. J.

Biol. Chem. Environ. Sci., 10(1): 11-22.

Arulselvan P, Senthilkumar GP, Sathish

Kumar D and Subramanian S (2006): Anti-diabetic effect of Murraya koenigii

leaves on streptozotocin induced diabetic

rats. Pharmazie., 61: 874-877.


15

Bancroft,J.D.and stevens,A.(2010): Theory

and practice of Histolology Technique

chrichil livingstonE,Edinburgh,London and

New York.

Beevi SS, Mangamoori LN and Gowda

BB.(2012): Polyphenolics profile and

antioxidant properties of Raphanus sativus L.

Nat Prod Res. 2012; 26(6): 557-563.

Beevi SS, Mangamoori LN, Subathra M

and Jyotheeswara Reddy E.(2010): Hexane extract of Raphanus sativus L. roots

inhibits cell proliferation and induces

apoptosis in human cancer cells by

modulating genes related to apoptotic

pathway. Plant Foods Hum Nutr. 65(3): 200-

209.

Belfield A, Goldberg D M. (1971): Revised

assay for serum phenyl phosphatase activity

using 4- amino- antipyrine. Enzyme, 12, 561

– 573.

Cennet Ragbetli and Ebubekir Ceylan

,(2010): Effect of Streptozotocin on

Biochemical Parameters in Rats. Asian

Journal of Chemistry Vol. 22, No. 3, 2375-

2378

Cheng AYY, Fantus IG (2005): Oral

antihyperglycemic therapy for type 2 diabetes

Mellitus, Can Med Assoc J;172(2): 213-226

Cheng, D., B. Liang and Y. Li, (2013):

Antihyperglycemic effect of Ginkgo biloba

extract in streptozotocin-induced diabetes in

rats. Bio. Med. Res. Intern. J., 1: 1-7.

Cook MN, Girman CJ, Stein PP(2005):

Alexander CM, Holman RR: Glycaemic

control continues to deteriorate after

sulfonylureas are added to metformin among

patients with type 2 diabetes. Diabetes Care

.,28:995–1000.

Cristina L, Roberto L, Stefano DP(2008): β-cell failure in type 2 diabetes mellitus. Curr

Diab Rep ., 8:179–184.

Eman A.Sadeek ( 2011 ): Protective effect

of fresh Juice from red beetroot (Beta

vulgaris L.) and radish (Raphanus sativus L.)

against carbon tetrachloride - induced

hepatotoxicity in rat models. African J. Biol.

Sci., 7(1): 69-84

Fawcett, J.K., & Scott, J.E. (1960): Determination of urea. Journal of Clinical

Pathology ,13, 156

Gandhi, G.R. and Sasikumar, P. (2012):

Antidiabetic effect of Merremia emarginata

Burm. F. in streptozotocin induced diabetic

rats, Asian Pacific J. of Ropical Biomed., vol.

63: 281-286.

Hala A. H. Khattab, Nadia S. Al-Amoudi

and Al-Anood A. Al-Faleh( 2013 ): Effect

of Ginger, Curcumin and Their Mixture on

Blood Glucose and Lipids in Diabetic Rats.

Life Science Journal;10(4).

He, Q., Y. Lv and K. Yao., (2006): Effects

of tea polyphenols on the activities of

α‐amylase, pepsin, trypsin and lipase. Food

Chemistry 101:1178–1182.

Henry RJ, Chiamori N.(1960):

Determination of -amylase activity in plasma

and urine of pancreatic diseases. Clin Chem;

6:434–41

Holemans K.I, Van Assche F.A (2003). Fetal growth restriction and consequences for

the offspring in animal models. Journal of the

Society for Gynaecologic Intervention

10:392- 399.

Hsueh CJ, Wang JH, Dai L, Liu

CC.(2011): Determination of alanine

aminotransferase with an electrochemical

nano Ir-C biosensor for the screening of liver

diseases. Biosens; 1(3): 107-117.


16

Kalantari H1, Kooshapur1 H, Rezaii1 F,

Ranjbari N, Moosavi1 M.,( 2009 ): Study of

protective effect of Raphanus sativus

(Radish) seed in liver toxicity induced by

carbon tetrachloride in mice . Jundishapur

Journal of Natural Pharmaceutical Products .

4(1): 24-31

Kishor Kumar S, Sridher Rao K, Sridhar

Y, Shankaraiah P(2013): Effect of

Raphanus sativus Linn. Aginst gentamicin

induced nephrotoxicity in rats..

JAPS/Vol.3/Issue.1

Kulkarni, C. P., Bodhankar, S. L., Ghule,

A. E., Mohan, V. and Thakurdesai, P.A.

(2012): Antidiabetic activity of Trigonella

Foenumgraecum L. seeds extract(IND01) in

neonatal streptozotocin-induced (N-STZ)

rats, Diabetologia.Croatica., vol. 18: 41-42.

Kumaran A and Karunnakaran

RJ.(2007): In vitro antioxidant activities of

methanol extracts of Phyllanthus amarus

species from India. LWTSwiss Society of

Food Science and Technol. 2007; 40: 344-

352.

Leegwates D.C and Kuper C.F. (1984):

Evaluation of histological changes in the

kidneys of the alloxan diabetic rat by means

of factor analysis. Food Chem. Toxicol.,

22(7), 551 - 557.

M. Abdollahi1, A.B.Z. Zuki1, Y.M. Goh1,

A. Rezaeizadeh2 and M.M. Noordin

(2011): Effects of Momordica charantia on

pancreatic histopathological changes

associated with streptozotocin-induced

diabetes in neonatal rats . Histol Histopathol

26: 13-21

Morelli V, Zoorob RJ(2000): Alternative

Therapies: Part 1.Depression, Diabetes,

Obesity, Amer Fam Physician, 62(5): 1051-

1060.Mythili, M.D., Vyas, R., Akila, G.,

Gunasekaran, S., (2004): Effect of

streptozotocin on the ultrastructure of rat

pancreatic islets. Microscopy Research and

Technique 63, 274–281.ynaecologic

Intervention 10:392- 399.

Nastaran JS (2011): Antihyperglycaemia

and antilipidaemic effect of Ziziphus

vulgaris L on streptozotocin induced diabetic

adult male Wistar rats. Physiol. Pharmacol.,

47: 219-223.

Nelson, R.W., Canine Diabetes

Mellitus,(2010): Textbook of Veterinary

Internal Medicine, E.C.F. Univ. Press, Ames,

Lowa., USA.

Ozlem Yavuz1, Meryem Cam, Neslihan

Bukan, Aysel Guven, and Fatma Silan (

2003): Protective effect of melatonin on -cell

damage in streptozot ocin-induced diabetes

in rats. acta histochem. 105(3) 261–266

Quiros JA, Marcin JP, Kupper M, Ann N,

Nasrollzadeh F, Rewers A, et al.(2008): Elevated serum amylase and lipase in

pediatric diabetic ketoacidosis. Pediatr Crit

Care Med ;9:418-22. Back to cited text no.

18

Rahimi R, Nikfar S, Larijani B, Abdollahi

M. (2005): A review on the role of

antioxidants in the management of diabetes

and its complications. Biomed Pharmacother,

59, 365–373.

Rai PK, Jaiswal D, Mehta S, Rai DK,

Sharma B, Watal G(2010): Effect of

Curcuma longa freeze dried rhizome powder

with milk in STZ- induced diabetic rats. Ind J

Clin Biochem 2, 175-181.

Rakieten N, Rakieten ML, Nadkarni

MV.(1963): Studies on the diabetogenic

action of streptozotocin (NSC-37917).

Cancer Chemo. Ther. Rep 29:91–102.


17

Ramachandran Vadivelan, Sanagai

Palaniswami Dhanabal , Ashish

Wadhawani and Kannan Elango.,(2012): α-Glucosidase and α-amylase inhibitory

activaties of RAPHANUS SATIVUS.

IJPSR, Vol. 3(9): 3186-3188

Reitman, S., Frankel, S.A. (1957). Colorimetric method for the determination of

serum oxaloacetic and glutamic pyruvate

transaminase. Am. J. clin. Pathol. 28:56-63.

Rizvi SI, Anis, MA, Zaid R & Mishra

N.(2005): Protective role of tea catechins

against oxidation-induced damage of type 2

diabetic erythrocytes. Clinical and

Experimental Pharmacology and Physiology.

32(1–2), 70–75.

Robert Rusinek , Rafał Rybczyński , Jerzy

Tys , Marzena Gawrysiak-Witulska,

Małgorzata Nogala-Kałucka and

Aleksander Siger .(2012): The Process

Parameters for Non-Typical Seeds during

simulated Cold Deep Oil Expression. Vol.

30, No. 2: 126–134 Czech J. Food Sci.

Shirwaikar A, Rajagopal PL and Malini

S(2005): Effect of Cassia auriculata Linn.

root extract on cisplatin and gentamicin-

induced renal injury. Phytomed. , 12(3): 555-

560.

Siham K. Abunasef , Hanan A. Amin1,

Ghada A. Abdel-Hamid,(2014): A

histological and immunohistochemical study

of beta cells in streptozotocin diabetic rats

treated with caffeine. Folia Histochmica ET

Cytobiologica . Vol. 52, No. 1. pp. 42–50.

Snedecor, G.W. and Cochron, W.G.

(1989): Statistical methods. 8th ed., Lowa

State

Surekha Shukla, Sanjukta Chatterji,

Shikha Mehta, Prashant Kumar Rai,

Rakesh Kumar Singh,Deepak Kumar

Yadav, and Geeta Watal.(2010):

Antidiabetic effect of Raphanus sativus root

juice. Pharmaceutical Biology, 5(3). 1–6.

Taniguchi H, Kobayashi-Hattori K,

Tenmyo C, Kamei T, Uda Y, Sugita-

Konishi Y, Oishi Y, Takita T.(2006): Effect

of Japanese radish (Raphanus sativus) sprout

(Kaiware-daikon) on carbohydrate and lipid

metabolisms in normal and streptozotocin-

induced diabetic rats. Phytother Res; 20:274-

8.

Weidenheim KM, Hinchey WW, Campell

WG Jr.(1983): Hyperinsulinemic

hypoglycemia in adults with islet-cell

hyperplasia and degranulation of exocrine

cells of the pancreas. Amer Clin Pathol.

79:14–24.

Xinhe Yin and Ben Zhang (2009):

Streptozocin Injection, Yan Lab Protocol

Edited by: The sigma Aldrich. Germany.

Yadyvucs ,moorthy K, Baquer NZ (2005): Combined treatment of sodium

orthovanadate and Momordica charantia fruit

extract prevents alterations in lipid profile

and lipogenic enzymes in alloxan diabetic

rats, Mol Cell Biochem, 268(1–2):111–120.

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