Turkish Journal of Biology

Volume 36 Number 2 Article 9

1-1-2012

The histopathologic effects of Morus alba leaf extract on the

pancreas of diabetic rats
JAMSHID MOHAMMADI

PRAKASH R. NAIK

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Recommended Citation
MOHAMMADI, JAMSHID and NAIK, PRAKASH R. (2012) "The histopathologic effects of Morus alba leaf
extract on the pancreas of diabetic rats," Turkish Journal of Biology: Vol. 36: No. 2, Article 9.
https://doi.org/10.3906/biy-1008-51
Available at: https://journals.tubitak.gov.tr/biology/vol36/iss2/9

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 J. MOHAMMADI, P. R. NAIK

Turk J Biol
36 (2012) 211-216
© TÜBİTAK
doi:10.3906/biy-1008-51

The histopathologic effects of Morus alba leaf extract on the

pancreas of diabetic rats

Jamshid MOHAMMADI1, Prakash R. NAIK2

1
Department of Physiology, Herbal Medicine Research Center, College of Medicine,
Yasouj University of Medical Sciences, Yasouj - IRAN
2
Endocrinology Laboratory, Department of Zoology, University of Mysore, Mysore, 570006 - INDIA

Received: 11.08.2010

Abstract: Many traditional treatments have been recommended in the alternative system of medicine for the treatment
of diabetes mellitus. Mulberry (Morus alba) is a nontoxic natural therapeutic agent shown to possess hypoglycemic,
hypotensive, and diuretic properties. The purpose of this study was to examine the histopathologic effects of Morus alba
leaf extract on the pancreas of diabetic rats. The animals were treated with mulberry leaf extract at dosages of 400 and
600 mg/kg body weight for 35 days. The various parameters studied included blood glucose, the relative body weight
of the pancreas, the diameter of islets, and the number of β cells in all groups. Blood glucose level, the diameter of the
islets, and the number of β cells were increased in treatment groups as compared to the diabetic group. According to the
histological and biochemical results obtained, it was concluded that the extract of this plant may reduce blood glucose
levels by regeneration of β cells.

Key words: Morus alba, type 1 diabetes, islets, β cell, glucose

Introduction of the plants that are being used for the treatment
Diabetes is a chronic disease characterized by high of diabetes have received scientific or medicinal
blood glucose levels and abnormal metabolism of scrutiny and even the World Health Organization’s
carbohydrates, proteins, and fat associated with a expert committee on diabetes recommends that this
relative or absolute insufficiency of insulin secretion area warrants further attention (4).
and with various degrees of insulin resistance Recent evidence shows that leaves and shoots
(1). Such alterations result in increased blood from the mulberry tree possess several medicinal
glucose, which causes long-term complications in properties, including hypoglycemic, hypotensive,
many organs. Despite important progress in the and diuretic effects (5). Mulberry root bark or leaf
management of diabetes using synthetic drugs, extracts were shown to possess hypoglycemic effects
many traditional plant treatments are still being in animal models of type 1 diabetes mellitus (6).
used throughout the world. Plants are valued in We recently reported that the extract of Morus alba
indigenous systems of medicine for the treatment leaves promoted significant hypolipidemic activity
of various diseases (2). Medicinal plants provide a in experimental animals (7). The aim of the present
useful source of oral hypoglycemic compounds for study was to clarify the effect of M. alba leaf extracts
the development of new pharmaceutical leads as well on blood glucose and to determine any possible effect
as dietary supplements to existing therapies (3). Some on pancreatic tissue.

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The histopathologic effects of Morus alba leaf extract on the pancreas of diabetic rats

Materials and methods IV began to receive 400 mg/kg of the mulberry leaf
Animals extract daily; those in group V received 600 mg/kg of
the extract daily. Doses were administered orally for
Adult Wistar rats were procured from the animal 5 weeks. Body weight was recorded at the beginning
house of the Department of Zoology. Irrespective and end of the experiment in every group. At the
of sex, the animals had body weights ranging
end of the experimental period, animals were fasted
between 150 and 200 g. Protocols of the experiment
overnight and autopsied under light ether anesthesia.
were approved by the Institutional Animal Ethics
Committee. The animals were maintained under Morphometric analysis
standard conditions, with a temperature of 20 ± 5 Pancreatic tissue was taken from all groups. The
°C, a regular 12-h light and 12-h dark cycle, and a weight of the pancreas was also recorded in every
humidity level of 50%-68%. The rats were allowed group. The tissue samples were washed, fixed in
free access to standard laboratory food and water ad Bouin-Hollande, and dehydrated with alcohol in
libitum throughout the experiment. Bouin-Hollande for 18-20 h. Serial sections of 4-5
Procurement and preparation of the plant material μm in thickness were cut using a microtome, and
every fifth slide was stained using the chrome alum
The mulberry leaves used in this experiment
were collected from the garden of the Sericulture hematoxylin and phloxine (CHP) method. The slides
Department of the University of Mysore. The fourth were mounted using DPX mountant, and 100 islets
and fifth leaves were plucked from the apex of healthy were measured from 100 randomly selected cross-
plants, washed thoroughly under running tap water, sections of the pancreas from each rat. The β cells
shade-dried for 5 days, and eventually ground to a were also counted (8). The relative weight of the
fine powder in an electric grinder. The powdered plant pancreas was calculated using the following formula:
material (1250 g) was extracted twice, 24 h each time, relative weight of pancreas = weight of pancreas/
with 90% ethanol at room temperature. The extract was weight of animal × 100.
filtered with Whatman filter paper No 1. The filtrate Statistical analysis
was evaporated using a Soxhlet evaporator until dry, Results were analyzed statistically using analysis
and 135.5 g of extract was obtained. The dried powder of variance (ANOVA) and represented as mean ±
was diluted in distilled water at concentrations of 400 standard error (SE). Wherever the variance value
and 600 mg/kg body weight. was found to be significant at 5%, Duncan’s multiple
Experimental protocols range test was applied.
The animals were classified into 5 groups, each
containing 8 rats. These groups were identified
Results and discussion
as follows: control group (I), control group with
mulberry leaf extract treatment (II), diabetic control The effect of M. alba leaf extract on the blood glucose
group (III), diabetic group treated with 400 mg/ levels of experimental rats is shown in Figure 1.
kg per day mulberry leaf extract (IV), and diabetic Throughout the experimental period, the control rats
group treated with 600 mg/kg per day mulberry leaf (I) did not show any significant variation in blood
extract (V). The animals in groups III, IV, and V were glucose level with group II. The administration of
rendered diabetic by a single intraperitoneal injection STZ (60 mg/kg) led to a more than 3.5-fold elevation
of streptozotocin (STZ; 60 mg/kg) freshly prepared of blood glucose levels, which was maintained over
in 0.1 mol citrate buffer (pH 4.5). The animals in a period of 5 weeks. At a daily dosage of 400 mg/
groups I and II were injected with the buffer alone. kg, the mulberry leaf extract significantly reduced
Next, 72 h after the injection, blood was drawn from hyperglycemia compared to the diabetic group,
the tail of conscious rats in order to estimate their although this amount failed to restore the level to that
glucose levels using a glucometer. Blood glucose was of the control group. Daily treatment with 600 mg/
similarly estimated every week until autopsy. Ten kg, however, almost reached the levels demonstrated
days after the STZ injection, animals in groups II and by the control group (I) (P < 0.05).

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 J. MOHAMMADI, P. R. NAIK

Figure 2 shows the changes of the relative weight A decrease in the number of β cells of the islets of
of the pancreas in all groups. The relative weight of the Langerhans was observed in the diabetic group in
pancreas with type 1 diabetes did not differ significantly comparison to the control group (Figure 4). The number
across groups I, II, IV, and V, but there was a significant of β cells in animals from groups I and II did not show
increase in the relative weight of the pancreas in diabetic significant variation. When compared to group III, the
group III (P < 0.5). number of β cells seen in specimens from groups IV and V
increased significantly (P < 0.5) after daily treatment with
Changes in the diameter of islets in all groups are
400 and 600 mg/kg mulberry leaf extract, respectively.
given in Figure 3. The diameter of islets did not differ
significantly in groups I and II of the experimental Figure 5A shows an islet of Langerhans from an
animals. Since the β cells were damaged due to the animal in the control group (I). The islet shows a large
induction of diabetes, the islet size decreased significantly number of β cells (hematoxylin-stained blue cells)
in group III of the experimental animals (P < 0.5). The distributed throughout the islet. A few α cells (phloxine-
administration of the mulberry leaf extract to groups stained pink cells) can also be observed. The islet of
IV and V of the experimental animals resulted in the Langerhans shows that the β cells did not differ in group
recovery of damaged β cells and the restoration of the II, neither in number nor in diameter, from those of the
diameter of the islets to that of the control group (I). control group (I).
180
450 I II III IV V 160
400 140
350
Diameter

120
300 100
mg/dL

250 80
200 60
150
40
100
20
50
0
0 Groups
0 7 14 21 28 35
Treatment time (day) I II III IV V
Figure 1. Effects of M. alba extract on blood levels (mg/ Figure 3. Effects of M. alba leaf extract on the diameter of islets
mL), showing mean blood glucose levels during the (μm) in type 1 diabetes. The vertical bars show mean
experiment. values of the diameter of the islets of Langerhans.
Lines above the bars indicate SE.

200
Number of β cells

0.4 160
Relative body weight

0.35
0.3 120
0.25
0.2 80
0.15
0.1 40
0.05
0 0
Groups Groups
I II III IV V I II III IV V
Figure 2. Effects of M. alba leaf extract on the relative weight of Figure 4. Effects of M. alba leaf extract on the number of β cells
the pancreas in type 1 diabetes. The vertical bars show in the islets of Langerhans in type 1 diabetes. The
mean values of the relative weight of the pancreas. vertical bars show the mean number of β cells in the
Lines above the bars indicate SE. islets of Langerhans. Lines above the bars indicate SE.

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The histopathologic effects of Morus alba leaf extract on the pancreas of diabetic rats

In the diabetic group, a decrease in the number some of them may have insulin-like substances, some
of β cells of the islets of Langerhans was observed may inhibit insulin activity, and others may increase
in comparison to the control group (Figures 5A and β cells in the pancreas by activating the regeneration
5B). The damage or necrosis of β cells was caused of these cells. The fiber of plants may also interfere
by the streptozotocin used to induce diabetes. Few with carbohydrate absorption and thereby affect
functional β cells were observed and α cells were blood glucose (9-12).
more prominent.
Different studies have demonstrated that certain
Figures 5C and 5D show the islets of Langerhans plant extracts (Scoparia dulcis, Teucrium polium, and
from the diabetic groups treated with M. alba leaf Salvadora oleoides) effectively increased the number
extract at concentrations of 400 and 600 mg/kg of β cells in streptozotocin- and alloxan-induced
body weight, respectively. The damaged β cells seen diabetes mellitus (13-15). The diminished islet size
after the initial induction of diabetes were no longer and β cell number resulted in the histopathology of
observed after treatment with M. alba leaf extract. the diabetic pancreas. A recent report showing that
The recovery of necrotic β cells was especially more adult β cell numbers are formed by self-duplication/
pronounced after treatment with 600 mg/kg of M. self-proliferation supports our finding of an increase
alba leaf extract than in the group treated with 400 in the β cell number in diabetic islets after mulberry
mg/kg of the extract.
treatment (16).
This study was carried out histologically by light
Taniguchi et al. (17) showed that the compounds
microscopy and biochemically in order to detect
of mulberry leaf, particularly fagomine, are capable
whether this plant decreased blood glucose and
of inducing insulin secretion in isolated rat islet cells.
whether it had an effect on the pancreatic tissue.
Kim et al. (18) observed that aqueous extracts of C.
The M. alba leaf extract was found to be effective in
controlling hyperglycemia in diabetic rats. Although coronarium and M. alba effectively decreased blood
many suggestions have been made about plants’ glucose in alloxan-induced diabetic rats.
roles, the precise mechanism is unclear. Plants may Shirwaikar et al. (19) reported on histopathology
act on blood glucose through different mechanisms, studies in which STZ was suspected of partially

Figure 5A. Photomicrograph of a pancreatic section from the Figure 5B. Photomicrograph of STZ-induced type 1 diabetic
control group showing the exocrine region and islets pancreatic section showing the exocrine region
of Langerhans, with scattered β cells and red blood and islets of Langerhans with damaged β cells due
cells visible in the vicinity. EX: Exocrine pancreas, β to necrosis and a decreased number of β cells. EX:
cell: beta cells, RBCs: red blood cells; CHP staining, Exocrine pancreas, β cell: beta cells, RBCs: red blood
400×. cells; CHP staining, 400×.

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 J. MOHAMMADI, P. R. NAIK

Figure 5D. Photomicrograph of type 1 diabetic islet treated with

600 mg/kg of M. alba leaf extract showing evenly
distributed β cells and an increased number of β cells.
EX: Exocrine pancreas, β cell: beta cells, RBCs: red
blood cells; CHP staining, 400×.

In conclusion, the histopathologic studies

undertaken on the islets demonstrated the recovery
Figure 5C. Photomicrograph of type 1 diabetic islet treated with of damaged islets and an improvement in the number
400 mg/kg of M. alba leaf extract showing evenly of β cells after treatment with the plant extract. It
distributed β cells and an increased number of β
can thus be assumed that M. alba leaf extract has a
cells. EX: Exocrine pancreas, β cell: beta cells, RBCs:
red blood cells; CHP staining, 400×. therapeutic effect that alleviates diabetes mellitus.
The actual chemical compound that is responsible for
this effect requires further investigation.
destroying the pancreas. Diabetic rats showed reduced
islet cells, which were restored to near normal upon Acknowledgments
treatment with the extract. Al-Eryani and Naik (20)
observed the effects of T. cordifolia extract on high The first author gratefully acknowledges Yasouj
fat-fed type 2 diabetic rats; in that study, the extract University of Medical Sciences, Iran, and the
significantly restored the diameter of islets and the Chairman of the Department of Zoology, University
number of β cells in diabetic rats. of Mysore, Mysore, India, for support and access to
facilities.
Similarly, Yin et al. (21) observed that high-dose
STZ in diabetes indicates β cell destruction. After 120
days of normoglycemia, a statistically significant 3.7- Corresponding author:
fold increase in β cell mass was observed. Jamshid MOHAMMADI
This study supports the possibility of regeneration Department of Physiology,
as a means for generating new β cells, even in severely
diabetic animals or individuals with significantly Herbal Medicine Research Center,
reduced β cell mass. As other possible mechanisms, College of Medicine,
the extract from mulberry leaves may increase β Yasouj University of Medical Sciences,
cells and sensitize the insulin receptor to insulin, or
stimulate the stem cells of the islets of Langerhans in Yasouj - IRAN
the pancreas of STZ-induced diabetic rats. Email: jamshidm2005@yahoo.com

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The histopathologic effects of Morus alba leaf extract on the pancreas of diabetic rats

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