O A

749
Advances in Environmental Biology, 7(4): 749-754, 2013
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Stereological and Histopathological Study of Ovarian Tissue after Hydro-alcoholic
Extract of Artemisia Plants Compared with Metformin in Diabetic Rats
1
Kouhpeyma Farhad, 2Zahiri Shahla, 1Kargar Jahromi Hossein, 3Azhdari Sara, 3Farzam
Mohammad, 4Mahmoudi Teimourabad Saeid
1
MSc, Animal Physiology, Young Researchers Club, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
Shiraz University of Medical Sciences, Department of Anatomy, Shiraz, Iran.
3
Phd, The Department of Anatomy and Embryology, International branch, Shiraz University, Shiraz, Iran
4
The Department of Histology, International branch, Shiraz University, Shiraz, Iran.
2
Kouhpeyma Farhad, Zahiri Shahla, Kargar Jahromi Hossein, Azhdari Sara, Farzam Mohammad,
Mahmoudi Teimourabad Saeid; Stereological and Histopathological Study of Ovarian Tissue after
Hydro-alcoholic Extract of Artemisia Plants Compared with Metformin in Diabetic Rats
ABSTRACT
Introduction: Diabetes is an endocrine and metabolic disorder, a variety of effects on the structure and
function of many body systems leaves. Reproductive system, the rule is different. The aim of this study was to
investigate the effects of the herb Artemisia extracts of ovarian tissue is improved due to diabetes complications.
Materials and Methods: 50 rats, adult female aged approximately 10 weeks of Wistar rats randomly divided into
5 groups: control, diabetic (streptozotocin 50 mg/kg), diabetic + extract of Artemisia (200 & 300 mg/kg) and a
positive control (metformin 250mg/kg) were included. The streptozotocin was administered intraperitoneally,
and other materials as gavage. Treated mice continued for 8 weeks. At the beginning and end of the experiment,
mice were weighed and blood glucose was measured, so the mice were dissected and the right ovary weighed
after fixation in 3% formaldehyde and cutting and preparing for special studies were conducted respectively.
Mason with three more chrome slides, were stained with hematoxylin eosin and periodic acid Schiff. Few
studies include estimates of mean ovarian volume and follicle determines the quality and histopathological
studies were performed. The results indicate an increase in blood sugar levels, ovarian volume, the percentage of
atretic follicles quantitatively and cause congestion, hyperemia, and increased thickness of the germinal
epithelium and fibrosis in diabetic rats was qualitatively, in the other groups, these parameters showed a
decreasing trend (p ≤ 0.05). Conclusion: This study suggests that as plant extract could significantly reduce the
complications of diabetes in female reproductive system changes. Key words: diabetes, ovarian, moose,
mountain sagebrush, stereology, histopathology
Key words:
Introduction
Infertility related problems as one of the major
problems in the couple's life is known [3]. According
to the statistics 35% of men and 25% of infertile
couples, infertility is related to the second pair
[16,11]. Because diabetes is a disease associated with
sexual activity in men and women is about 90% of
patients deficient in sexual activity in reduced libido
and reduced fertility are included [6]. This causes
ovarian dysfunction, including changes in estrous
behavior, follicular growth, oocyte maturation and
ovulation is reduced or not [5,21], this shifts the
focus from disrupting the hypothalamic - pituitary gonadal axis is [5] FSH and LH hormone levels,
which in effect reduced then the ability of the ovaries
to produce the hormones estrogen and progesterone
are decreased [5]. In addition, histopathological
changes in the ovary, including diabetes, polycystic
ovary syndrome, ovarian enlargement [2,9] and
atretic follicles have also been reported [2,21],
although the exact mechanism is well known to have
diabetes mellitus but construction has not increased
free radical damage is the major mechanism [23] of
antioxidants such as vitamins, flavonoids in the diet
can have protective effects in diabetic patients [17].
Free radicals are reduced flavonoid [20] recently for
the treatment of diabetes blood glucose lowering
effects of herbs that are used and expected to have
similar effects, but drugs have less side effects [18,4]
Such as Artemisia plants are [1] that recent research
has demonstrated its ability to reduce blood sugar
[10,1]. Metformin is a combination - Govanidi
lowering of blood glucose that is used in the
Corresponding Author
Farhad Kouhpeyma, 1MSc, Animal Physiology, Young Researchers Club, Jahrom Branch, Islamic
Azad University, Jahrom, Iran.
Tel: 09171335717 E-mail: [email protected]
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Adv. Environ. Biol., 7(4): 749-754, 2013
treatment of diabetes. The drug works by a series of
reactions in various organs and tissues can be
obtained as follows: Reduction of hepatic
gluconeogenesis in hepatocytes and Gelicozhenz and
Govanidi increase, decrease glucose uptake by
muscle and fat cells. Oral administration of
intravenous administration of metformin has better
performance [8,24]. Since stereological and
histopathological study on the effect of
hydroalcoholic extract of Artemisia compared with
metformin on ovarian structures on diabetic rats has
been reported, this is exactly the outcome sought to
do.
Materials and Methods
In this study, 50 adult female Wistar rats,
approximately 10 weeks of age, education and care
of laboratory animals were purchased from Shiraz
Medical School. Initially, rats were randomly divided
into 5 groups: control, diabetic, diabetic + extract
(200 & 300 mg/kg) and diabetic + metformin (250
mg / kg) into the chamber Animals Branch arranged
at a temperature of 2 24 ° C and 12 h light and 12 h
dark photoperiod and free access to enough food and
water, were kept. To induce diabetes in rats injected
intraperitoneally dissolved streptozotocin as a single
dose or dose 50mg / kg on 9/0% Nacl, 100 mM
sodium citrate buffer PH = 4/5 solution were used
[5]. After 4 days of STZ passed blood sugar with
glucometer in All Departments Czech made (prior to
glucose for 12 hours feeding of rats was used) after
diabetic They (300mg / dl) were treated for 12 weeks
starting at the end of the experiment, blood glucose
Czech animals were anesthetized by ether and
autopsied. Right ovaries of all animals completely
separated and washed with normal Saylin the
formalin 3% for 3 days were performed after fixation
adequate tissue passage was cut by 5 mm with three
painted a chrome Mason (for diagnosis of fibrosis),
periodic acid Schiff (glycogen deposition in tissue
diagnosis) and H & E preparations. Stereological
studies: Cavalieri 's method was used to study the
ovarian volume. Cavalieri showed [14] that uses a
uniform random sections and parallel and the
distance, the sample size can be cut to size and the
distance between them can be calculated. Different
stages of the procedure are as follows: The right to
be selected. Samples were cut parallel to the interval
(t) so in cases where the preparation can be difficult
to cut with the same average thickness (t) can be
used for cutting. Should be identical to the first
accident cut subsequent random and uniform will be
provided. Cut random number m of area A1, A2...
Am, and the distance (t) are prepared and the formula
for calculating volume is estimated non Bias [14].
estV=t.( A1+A2+…+Am )
To calculate the area (A) from a network point
that is randomly placed on the cut surface is used.
First, should the range or scope of any point (a / p)
can be computed in the network. Section area (A)
multiplied by the total points of the network which
has the cutting surface, is obtained [14].
The area of the section thickness (t) and thus
multiplying the volume (V) is calculated by the
following equation:
After preparing the slides and put them on the
micro projector and after dropping the image on the
display of the cross-shaped probe is quite clear that
the scope of each point (Fig. 1). (Point lattice probe)
On transparent paper copy is accidentally unleashed
on the image and then counting the number of points
that have to deal with image size of the following
formula is easily obtained.
In the distance, v the volume, ΣP is the total
number of points counted in the area or anywhere in
the frame and t is the thickness of the initial slides.
Meanwhile, a / p or scope of any point can be
obtained by the formula where Δy and ΔX linear
dimensions of each point and M is the magnification
Linear Micro Devices Projectors [14].
Data analysis:
Results using SPSS version 15 software and
ANOVA tests (one way ANOVA) and Duncan's test,
were analyzed (p ≤ 0.05). Mean and standard
deviation were calculated for the data and the graphs
were plotted by Excell software.
Blood Sugar:
Secondary blood sugar in diabetic rats showed a
significant increase compared to the other groups (p
<0/0001) that the amount of glucose in the treated
group showed a significant decrease. It should be
noted that the extract significantly increased mean
ovarian weight in the diabetic group than the control
group, metformin, diabetes + Artemisia is 200mg/kg.
It should be noted that the ovarian weight in diabetic
rats compared to diabetic + extract of Artemisia
300mg/kg soon was reduced but this reduction was
not significant (p ≤ 0.05) (Table 1). Ovarian volume
in diabetic rats increased significantly compared to
the control group from other groups of diabetic +
metformin, diabetes + Artemisia 200 and 300 (mg /
kg) significantly decreased in size compared to the
diabetic group show (p ≤ 0.05) (table 1).
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Adv. Environ. Biol., 7(4): 749-754, 2013
Image 1: Probe or cross network used to determine ovarian volume. Histopathological studies: The
histopathological studies, in terms of congestion and ovarian vasculature, fibrosis, vacuolization of
cells of ovarian tissue, follicular atresia, the thickness of the germinal epithelium were studied.
Pre-antral follicles:
Average pre-antral follicles was significantly
lower in the diabetic group compared to other groups
show (p ≤ 0.05) (Table 2).
Antral follicles:
Average antral follicles in the diabetic and
diabetic + metformin with other groups shows a
significant decrease (p ≤ 0.05) (Table 2).
The number of luteinizing
The results of counting the number of CL among
different groups suggests that increased significantly
between diabetic + metformin and diabetes +
Artemisia 200mg / kg and 300 to the control group
but are different in other groups together Seen is not
(p ≤ 0.05) (table 2).
Atretic follicles:
Results related to oocyte diameter, antral
follicles, corpus luteum and the germinal epithelium
Average oocyte diameter decreased significantly in
diabetic rats as compared to diabetic + extract of
Artemisia 300mg/kg show. But other groups have no
significant difference in the diameter of antral
follicles in groups of no exist, the mean diameter of
the corpus luteum in the diabetic + metformin group
decreased significantly as compared to the diabetic
group show (p ≤ 0.05). The mean diameter of the
germinal epithelium significantly increased in the
diabetic group compared to the control group. Side
diameter of germinal epithelium in diabetic rats
compared to other groups declined, but this decline
was not significant.
Histopathological changes:
Histopathological changes in the ovaries of rats
in different groups, there is congestion and
vasculature and fibrosis were observed in the diabetic
group.
Average atretic follicles significantly increased
in diabetic rats compared to other groups show
(05/0> p) (Table 2).
Table 1: Comparison of primary and secondary blood glucose testing at the beginning and end of the comparison groups examined ovary
weight and volume.
Groups
Ovarian volume (mm3)
Ovarian weight (g)
Secondary blood
Initial blood glucose (mg
glucose (mg / dl)
/ dl)
Control
ab
a
1/44 92
0/40 3/67
0/0029
/032
a1/77
96/6
b
Diabetic

59 0/  5/40
c
a
0/34
 2/88
 3/79
0/79  3/82

0/0091

25/12  469/6
 0/053
a
 0/031
0/014  0/031
0/022  /038
b*
0/0052
31/17
 207/6
 195/4
18/45  247/2

20/70
 467/4
13/18
 472/2
 465
13/73  496/2
Diabetic + metformin
Diabetes + Artemisia
200mg/kg
Diabetes + Artemisia
ab
ab
b*
300mg/kg
Marked * showed no significant difference before and after treatment is. Letters indicate significant differences at the end.
0/ 51
ab
a
b
45/49*
19/14
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Adv. Environ. Biol., 7(4): 749-754, 2013
Table 2: Comparison of mean number of follicles in groups.
Atretic follicles
CL
Antral follicles
1/17  4/38
a
 38/18
0/46  1/85
0/26  4/65
0/21  2/16
9/99
b
a
a
a
4/05  18/85
ab
a
c
c
c
 10/08
6/26  34/45
4/9  26/52
4/99  34/98
0/49
b
 19/47
1/39  6/77
1/44  7/78
1/94  14/82
1/35  17/25
3/23
Pre-antral follicles
a
a
a
b
b
b
 57/13
3/03  24/45
5/05  56/67
6/49  48/34
4/49  54
4/36
b
b
b
Table 3: Comparison of mean CL diameter, germinal epithelium, antral follicles and oocytes in groups.
Oocyte diameter
Antral follicle diameter (mμ)
Aptiliom diameter
CL diameter (mμ)
(mμ)
(mμ)
 73/04
2/07  65/5
ab
a
ab
2/13
b
a
4/28  72/96
ab
 74/8
4/09  78/4
 388/96
32/29  370/88
22/72  391/04
16/25  408
22/15  390/56
a
a
3/18
a
a
0/38  5/20
ab
39/57
 12/12
0/62  6/64
0/56  8/03
0/44  8/16
cd
bc
c
/92
c
Discussion:
In this study, a significant decrease in blood
glucose levels in the treated group compared to the
diabetic group was soon, the results Tastekin [10]
and colleagues in 2006, Jouad [13] and colleagues in
2001, Eddouks [19] And colleagues in 2002 and Eidi
[29] and colleagues in 1386 is consistent with most
species of the genus Artemisia contains flavonoids
seskoee terpene lactones, especially derivatives 11
and Persian date 13 Di Hydroseskoee terpenes, are
[1] the current use of medicinal plants containing
flavonoids, due to their pharmacological properties
and therapeutic for the treatment and prevention of
diabetic complications, researchers have noted [29].
Flavonoid, polyphenols are a diverse biological
activities such as nitric oxide and reduced
performance of low-density lipoprotein and an
increase in plasma antioxidant body [27]. So the
above can be said that the mechanism of the plant
Artemisia extract on blood glucose levels of insulin
in the diabetic group receiving the extract is another
mechanism that can be considered for these extracts
are the reabsorption of glucose from the intestine,
which may reduce or inhibit the uptake of glucose by
the kidney tubules or by decreasing glucagon lowers
blood glucose [10]. Another possible mechanism of
increased sensitivity to insulin receptors and can also
be considered such a mechanism similar to that of
second-generation sulfonylureas such a mechanism
has been shown from the plant Artemisia pallens
[29]. Although the mechanism of action of
metformin reduces glucose production in the liver by
inhibiting gluconeogenesis No direct effect on
insulin resistance or reduced insulin effectively
reduce serum glucose levels [8,24]. In the present
study the mean ovarian weight in diabetic rats
compared to diabetic + plant extract (200 and 300 mg
ab
b
64/144  902
 992/56
32/180  758
75/016  872/8
60/56  870/8
58/55
a
ab
ab
Groups
Control
Diabetic
Diabetic + metformin
Diabetes + Artemisia
200mg/kg
Diabetes + Artemisia
300mg/kg
Groups
Control
Diabetic
Diabetic + metformin
Diabetes + Artemisia
200mg/kg
Diabetes + Artemisia
300mg/kg
kg body weight), and diabetic + metformin
significant increase that could possibly be due to the
increased number of stromal cells , and the remaining
follicles in the polycystic ovary syndrome or be
diabetic [28]. Koudner studies and colleagues (2006)
Histopathological changes including polycystic
ovary syndrome, and enlargement of the ovaries are
ovaries in women with type 1 diabetes have been
reported [9]. In a study in 1388 by Soleimani et
Mehranjani diabetic mice was performed on a total
volume of ovarian cortex and medulla increased
significantly in the diabetic group than in the other
groups [28], which results We are in agreement. In
the present study a significant increase in the
thickness of the germinal epithelium was observed
that likely cause of persistent follicles and
subsequent ovulation and ovarian volume is another
reason is the increase in size of the ovary and
Sekoulareh and creates congestion and cortical
vasculature [28]. Artemisia plants with antioxidant
compounds such as flavonoids types can reduce the
adverse effects of free radicals. So that a significant
reduction in ovarian volume in the metformin group
receiving juice 200 and 300 mg / kg can be observed
as compared to the diabetic group. The study
Moghetti and colleagues in 2000 increased
significantly after metformin ovulation has been
reported [22]. Thus, metformin and Artemisia plant
extracts can prevent the accumulation of follicles and
ovulation increases ovarian volume is reduced. Grace
and colleagues (1982) in a study to reduce the
number of secondary follicles and increases the
number of follicle atresia were reported in diabetic,
Significant differences between the control group
and the diabetic soon saw the number of corpus
luteum in the ovary [12]. The results of the present
study is similar in this study, a significant increase in
the mean CL in the group treated diabetic group as
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Adv. Environ. Biol., 7(4): 749-754, 2013
compared to controls was observed indicates that the
process of ovulation in these groups is increasing. In
this study, the significant reduction in the average
primary follicles was observed in diabetic rats
compared to other groups that Grace & Associates,
which is consistent with the research of the Balstr
Research (2007) can be said that serum LH and FSH
levels in the diabetic animal models [5] and thus
reduce estrogen and eventually slow the proliferation
of granulose cells and unravel the layers. The change
in ovarian function and pathological changes such as
increasing and becoming Sekoulareh ovarian
granulose cell necrosis, fibrosis and vacuolization so
they are one of the causes of atretic follicles and
pathological changes of diabetes based on the
hypothalamus - pituitary - gonadal that low levels of
gonadotropins and ovarian cell sensitivity to these
hormones is [5]. The plant extract and metformin
have a lower blood sugar and decrease lipid
peroxidation and oxidation of DNA and proteins to
prevent the primary follicle atresia [12]. In a study
conducted in 2005 by Chanj and colleagues smaller
diameter and size of the oocyte and ovarian follicle
development in rats has been reported in diabetic rats
[7]. The present study is similar, in this study, a
significant difference in diameter antral follicles
were observed in the groups tested with other studies,
Grace and colleagues (1982) show the follicles which
are more evolved or at a higher level of growth is
more likely to stay healthy and continue their growth
[12]. In this study, a reduction in the diameter of the
corpus luteum in the diabetic + metformin was
compared with the other groups Luteal cells is
probably due to decreased lipid reserves because
metformin addition to the above-mentioned effects
and plasma leptin levels, reduce fat stores [25,26].
2.
3.
4.
5.
6.
7.
8.
9.
Conclusions:
10.
Because of the complications of diabetes appear
on the ovarian tissue is a cause of reduced fertility in
females so the disease can be controlled with the
reproductive capacity of women said. As the survey
results indicate Hydroalcoholic extract of Artemisia
could be somewhat similar to metformin
significantly reduced the blood glucose and other
complications of diabetes on female reproductive
system created and can be used as a traditional
medicine with fewer complications useful in
improving diabetes.
11.
12.
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