By

Heena Amreen


Under Supervision of
Dr.Showkat A.Ganie


Introduction


Diabetes mellitus is a complicated metabolic disorder. that has gravely
troubled the human health and quality of life.

Its characterized mainly by hyperglycaemia and glycosuria.

Diabetes mellitus is divided into two main types:

a) Type I (insulin -dependent diabetes mellitus or IDDM).

b) Type II (non -insulin -dependent diabetes mellitus or NIDDM).















In type 1 diabetes mellitus the immune system attacks and destroys
the insulin producing beta cells of the pancreas. There is beta cell deficiency
leading to complete insulin deficiency.

Thus is it termed an autoimmune disease where there are anti insulin or anti-
islet cell antibodies present in blood.

These cause lymphocytic infiltration and destruction of the pancreas islets.

The destruction may take time but the onset of the disease is rapid and may
occur over a few days to weeks.

Type 1 diabetes always requires insulin therapy, and will not respond to insulin-
stimulating oral drugs.







This condition is caused by a relative deficiency of insulin and not an absolute
deficiency.

There is Beta cell deficiency coupled with peripheral resistance. Peripheral
insulin resistance means that although blood levels of insulin are high there is no
hypoglycaemia or low blood sugar.

This may be due to changes in the insulin receptors that bring about the actions
of the insulin.

Obesity is the main cause of insulin resistance.









Plant Material Collection and Extraction

The leaves of Crataegus songarica was collected from Vanaspati medicinal plant nursery
Ganderbal in month of September - October , and were identified by the courtesy of centre
of Plant Taxonomy, Department of Botany.

Preparation of plant extracts:

The authentically identified plant material leaves were dried in shade under room
temperature.

The dried leaf material was grind into powder using mortar and pestle and sieved with a
sieve of 0.3mm aperture size.

The powder obtained was extracted in distilled water, by using soxhlet extractor (60-
80

C).

The powdered plant material was successively extracted in 250ml each of petroleum
ether, ethyl acetate, methanol and distilled water by using Soxhlet extractor.

Experimental animals:

Adult male albino rats of Wister strain weighing 200- 250g were used throughout this
study.
Induction of diabetes:
The animals were fasted for 16 h prior to the induction of diabetes.

Alloxan monohydrate, freshly prepared in normal saline, was immediately injected
intraperitonially (130 mg kg1 BW) to induce diabetes.

Dosage and treatment:

Rats were divided into five groups containing three rats each. The plant extract was
employed at oral doses 100and 300mg/Kg body wt. /day. The aqueous extract was
suspended distilled water in such that the final volume of extract at each dose was 1ml and
was fed to rats with gavage.


Procedure:

Group 1- Served as normal control and received distilled water per day.

Group 2- Served as diabetic control (vehicle) and received 1ml distilled water per
day.
Group 3-Animals were orally administrated with Metformin 50mg/Kg body weight
dissolved in distilled water for fifteen day.

Group 4- Animals received 100mg/Kg body weight of C. songarica extract orally for
all fifteen days.

Group 5.Animals received 300mg/Kg body weight of C .songarica plant extract
orally for all fifteen days.


On the sixteen day the rats were sacrificed and the liver was isolated for enzyme
study.




Evaluation of antidiabetic potential of C. songarica extracts in albino rats
subsequent to short term exposure to Alloxan.

Lipid peroxidation (PMS)
LPO effects of aqueous extract C. songarica were measured by the formation of free MDA in
liver of rat following short term exposure to Alloxan. The extract significantly inhibited the
formation of MDA in the organ tested. The MDA levels significantly decreased to 0.020
0.001 and 0.015 0.001 Table (1) and fig 1with the oral administration of 100mg/kg , and
300mg/kg of aqueous extract of C. songarica in liver tissue compared with the control group
0.021 0.0015.
Tissues
Control
Group 1
Alloxan
treated
Group 11
Metformin
treated
Group 111
100mg/kg
extract
Group IV
300 mg/kg
extract
Group V
Liver
(nm of MDA
formed/g
protein)
0.008 0.001 0.021 0.0015 0.010 0. 001 0.020 0.001 0.015 0.001
The data were presented as means SD for five animals in each
0.000
0.005
0.010
0.015
0.020
0.025
n
m

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f


M
D
A

f
o
r
m
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p
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Lipid Peroxidation
Lipid
Peroxidation

Effect on enzymatic and non enzymatic antioxidant defense system of aqueous
extract of C. songarica

1) Protein levels

A significant decrease in protein levels were noted in group II rat tissue homogenates after a
single dose of Alloxan administration. Pretreatment with Oral doses of C. songarica aqueous
extract increased the protein levels in a dose dependent manner in the tested organ viz liver,
(Table 2 and figure 2).

2) Glutathione- S- transferase levels
The effect of oxidant and antioxidants on Glutathione- S- transferase (GST) activity in liver
tissue homogenates of all the experimental animals have been shown in (Table3). In the organ
tested for the GST activity was decreased in Alloxan treated groups compared to the normal
group. Pretreatment of aqueous extract largely restored the GST activity in a dose dependent
manner in all the three tested organs as depicted in Table 2 and fig 3.

4) Glutathione Peroxidase and Glutathione Reductase levels
Significant decrease was observed in Alloxan administered rats when compared with that of
the normal control animals. Treatment with aqueous extract of C. songarica at the oral dose
of 100 and 300mg/kg significantly restored the level of both enzymes (table 2 and figure 4
both and 5) the tested organ.



Table 2: Shows the effect on level of the antioxidant enzyme in the organ tested (Liver) of liver
of albino rats:
Tissues
Control
Group 1
Alloxan
treated
Group 11
Metformin
treated
Group 111
100mg/kg
extract
Group IV
300 mg/kg
extract
Group V
Protein (mg/100 mg tissue) 720 13.748 430.72 9.23 610 8.56 620 11.72 651 7.368
Reduced sulphydryl group
(nm/g protein)
350.59 3.56 79.393 3.15 290.61 3.13 190.30 2.85 265.42 2.74
Glutathione reductase (g
GSSG utilized/minute/mg
protein)
169.41 3.52 45.55 1.55 70.77 1.71 121.31 1.82 150.75 3.713
Glutathione peroxidase (g
GSH utilized/minute/mg
protein)
269.86 5.13 99.84 2.09 125.87 3.27 176.63 2.81 219.72 4.08
Glutathione- S- transferase
(nmoles of CDNB
conjugated/min/mg protein)
369.41 5.520 109.22 3.31 145.25 3.09 191.00 2.58 220.35 3.54
The data were presented as means SD for five animals in each
Figure 2: Represents the total protein content of liver.

720
430.72
610
620
651
0
100
200
300
400
500
600
700
800
normal alloxan metformine 100 mg 300 mg
P
r
o
t
e
i
n

(
m
g
/
1
0
0

m
g

t
i
s
s
u
e
)

Protein
0
50
100
150
200
250
300
350
400
normal alloxan metformin plant 100mg plant 300mg
Glutathione-s-
transferase
Figure 3: Represents the total GST levels of liver

Figure 4: Represents the total GPx levels of the liver

0
50
100
150
200
250
300
normal alloxan metformin plant 100mg plant 300 mg
Glutathione
peroxidase
0
20
40
60
80
100
120
140
160
180
normal alloxan metformin plant 100mg plant 300 mg
GR
Figure 5: Represents the total GR levels.

CONCLUSION
Based on the oxidative stress hypothesis of alloxan action, it was
considered as an adequate model for investigating the role of free
radicals in the pathology of diabetes mellitus.
The present study demonstrates that aqueous extract of Crataegus
songarica, a potent antioxidant, can exert anti-diabetic effects by
preserving liver cell function.
From this study, it can be concluded that the aqueous extract of
Crataegus songarica possesses the antidiabetic action which is
comparable with that of the standard Metformine drug employed.
The data presented provide additional benefits of aqueous extract of
Crataegus songarica administration and may offer a promising natural
and safe new trend for the prevention or delay of diabetic
complications.
ACKNOWLEDGEMENTS
Prof.Akbar Masood ( Head Department of Biochemistry)
Dr.Shajrul Amin Co-ordinator (Clinical biochemistry),.
Dr.Showkat Ahmad Ganie
Dr.Tanveer Ali Dar
Miss Shagoon Tabin
Mr.Ovais Zargar
Miss Humira Ramzan
Miss Nasreen Rashid