J. MOHAMMADI, P. R. NAIK

211

Turk J Biol

36 (2012) 211-216

© TÜBİTAK

doi:10.3906/biy-1008-51

Th e histopathologic eff ects of Morus alba leaf extract on the

pancreas of diabetic rats

Jamshid MOHAMMADI1, Prakash R. NAIK

2

1Department of Physiology, Herbal Medicine Research Center, College of Medicine,

Yasouj University of Medical Sciences, Yasouj - IRAN2Endocrinology 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. Th e purpose of this study was to examine the histopathologic eff ects of Morus alba

leaf extract on the pancreas of diabetic rats. Th e animals were treated with mulberry leaf extract at dosages of 400 and

600 mg/kg body weight for 35 days. Th e 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

Diabetes is a chronic disease characterized by high blood glucose levels and abnormal metabolism of carbohydrates, proteins, and fat associated with a relative or absolute insuffi ciency of insulin secretion and with various degrees of insulin resistance (1). Such alterations result in increased blood glucose, which causes long-term complications in many organs. Despite important progress in the management of diabetes using synthetic drugs, many traditional plant treatments are still being used throughout the world. Plants are valued in indigenous systems of medicine for the treatment of various diseases (2). Medicinal plants provide a useful source of oral hypoglycemic compounds for the development of new pharmaceutical leads as well as dietary supplements to existing therapies (3). Some

of the plants that are being used for the treatment of diabetes have received scientifi c or medicinal scrutiny and even the World Health Organization’s expert committee on diabetes recommends that this area warrants further attention (4).

Recent evidence shows that leaves and shoots from the mulberry tree possess several medicinal properties, including hypoglycemic, hypotensive, and diuretic eff ects (5). Mulberry root bark or leaf extracts were shown to possess hypoglycemic eff ects in animal models of type 1 diabetes mellitus (6). We recently reported that the extract of Morus alba leaves promoted signifi cant hypolipidemic activity in experimental animals (7). Th e aim of the present study was to clarify the eff ect of M. alba leaf extracts on blood glucose and to determine any possible eff ect on pancreatic tissue.

Th e histopathologic eff ects of Morus alba leaf extract on the pancreas of diabetic rats

212

Materials and methods

Animals

Adult Wistar rats were procured from the animal house of the Department of Zoology. Irrespective of sex, the animals had body weights ranging between 150 and 200 g. Protocols of the experiment were approved by the Institutional Animal Ethics Committee. Th e animals were maintained under standard conditions, with a temperature of 20 ± 5 °C, a regular 12-h light and 12-h dark cycle, and a humidity level of 50%-68%. Th e rats were allowed free access to standard laboratory food and water ad libitum throughout the experiment.

Procurement and preparation of the plant material

Th e mulberry leaves used in this experiment were collected from the garden of the Sericulture Department of the University of Mysore. Th e fourth and fi ft h leaves were plucked from the apex of healthy plants, washed thoroughly under running tap water, shade-dried for 5 days, and eventually ground to a fi ne powder in an electric grinder. Th e powdered plant material (1250 g) was extracted twice, 24 h each time, with 90% ethanol at room temperature. Th e extract was fi ltered with Whatman fi lter paper No 1. Th e fi ltrate was evaporated using a Soxhlet evaporator until dry, and 135.5 g of extract was obtained. Th e dried powder was diluted in distilled water at concentrations of 400 and 600 mg/kg body weight.

Experimental protocols

Th e animals were classifi ed into 5 groups, each containing 8 rats. Th ese groups were identifi ed as follows: control group (I), control group with mulberry leaf extract treatment (II), diabetic control group (III), diabetic group treated with 400 mg/kg per day mulberry leaf extract (IV), and diabetic group treated with 600 mg/kg per day mulberry leaf extract (V). Th e animals in groups III, IV, and V were rendered diabetic by a single intraperitoneal injection of streptozotocin (STZ; 60 mg/kg) freshly prepared in 0.1 mol citrate buff er (pH 4.5). Th e animals in groups I and II were injected with the buff er alone. Next, 72 h aft er the injection, blood was drawn from the tail of conscious rats in order to estimate their glucose levels using a glucometer. Blood glucose was similarly estimated every week until autopsy. Ten days aft er the STZ injection, animals in groups II and

IV began to receive 400 mg/kg of the mulberry leaf

extract daily; those in group V received 600 mg/kg of

the extract daily. Doses were administered orally for

5 weeks. Body weight was recorded at the beginning

and end of the experiment in every group. At the

end of the experimental period, animals were fasted

overnight and autopsied under light ether anesthesia.

Morphometric analysis

Pancreatic tissue was taken from all groups. Th e

weight of the pancreas was also recorded in every

group. Th e tissue samples were washed, fi xed in

Bouin-Hollande, and dehydrated with alcohol in

Bouin-Hollande for 18-20 h. Serial sections of 4-5

μm in thickness were cut using a microtome, and

every fi ft h slide was stained using the chrome alum

hematoxylin and phloxine (CHP) method. Th e slides

were mounted using DPX mountant, and 100 islets

were measured from 100 randomly selected cross-

sections of the pancreas from each rat. Th e β cells

were also counted (8). Th e relative weight of the

pancreas was calculated using the following formula:

relative weight of pancreas = weight of pancreas/

weight of animal × 100.

Statistical analysis

Results were analyzed statistically using analysis

of variance (ANOVA) and represented as mean ±

standard error (SE). Wherever the variance value

was found to be signifi cant at 5%, Duncan’s multiple

range test was applied.

Results and discussion

Th e eff ect of M. alba leaf extract on the blood glucose

levels of experimental rats is shown in Figure 1.

Th roughout the experimental period, the control rats

(I) did not show any signifi cant variation in blood

glucose level with group II. Th e administration of

STZ (60 mg/kg) led to a more than 3.5-fold elevation

of blood glucose levels, which was maintained over

a period of 5 weeks. At a daily dosage of 400 mg/

kg, the mulberry leaf extract signifi cantly reduced

hyperglycemia compared to the diabetic group,

although this amount failed to restore the level to that

of the control group. Daily treatment with 600 mg/

kg, however, almost reached the levels demonstrated

by the control group (I) (P < 0.05).

J. MOHAMMADI, P. R. NAIK

213

Figure 2 shows the changes of the relative weight

of the pancreas in all groups. Th e relative weight of the

pancreas with type 1 diabetes did not diff er signifi cantly

across groups I, II, IV, and V, but there was a signifi cant

increase in the relative weight of the pancreas in diabetic

group III (P < 0.5).

Changes in the diameter of islets in all groups are

given in Figure 3. Th e diameter of islets did not diff er

signifi cantly in groups I and II of the experimental

animals. Since the β cells were damaged due to the

induction of diabetes, the islet size decreased signifi cantly

in group III of the experimental animals (P < 0.5). Th e

administration of the mulberry leaf extract to groups

IV and V of the experimental animals resulted in the

recovery of damaged β cells and the restoration of the

diameter of the islets to that of the control group (I).

A decrease in the number of β cells of the islets of Langerhans was observed in the diabetic group in comparison to the control group (Figure 4). Th e number of β cells in animals from groups I and II did not show signifi cant variation. When compared to group III, the number of β cells seen in specimens from groups IV and V increased signifi cantly (P < 0.5) aft er daily treatment with 400 and 600 mg/kg mulberry leaf extract, respectively.

Figure 5A shows an islet of Langerhans from an

animal in the control group (I). Th e islet shows a large

number of β cells (hematoxylin-stained blue cells)

distributed throughout the islet. A few α cells (phloxine-

stained pink cells) can also be observed. Th e islet of

Langerhans shows that the β cells did not diff er in group

II, neither in number nor in diameter, from those of the

control group (I).

0

50

100

150

200

250

300

350

400

450

0 7 14 21 28 35

mg/

dL

Treatment time (day)

I II III IV V

Figure 1. Eff ects of M. alba extract on blood levels (mg/

mL), showing mean blood glucose levels during the

experiment.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Groups

Rel

ativ

e b

od

y w

eigh

t

I II III IV V

Figure 2. Eff ects of M. alba leaf extract on the relative weight of

the pancreas in type 1 diabetes. Th e vertical bars show

mean values of the relative weight of the pancreas.

Lines above the bars indicate SE.

0

20

40

60

80

100

120

140

160

180

Groups

Dia

met

er

I II III IV V

Figure 3. Eff ects of M. alba leaf extract on the diameter of islets

(μm) in type 1 diabetes. Th e vertical bars show mean

values of the diameter of the islets of Langerhans.

Lines above the bars indicate SE.

0

40

80

120

160

200

Groups

Num

ber

of β

cel

ls

I II III IV V

Figure 4. Eff ects of M. alba leaf extract on the number of β cells

in the islets of Langerhans in type 1 diabetes. Th e

vertical bars show the mean number of β cells in the

islets of Langerhans. Lines above the bars indicate SE.

Th e histopathologic eff ects of Morus alba leaf extract on the pancreas of diabetic rats

214

In the diabetic group, a decrease in the number of β cells of the islets of Langerhans was observed in comparison to the control group (Figures 5A and 5B). Th e damage or necrosis of β cells was caused by the streptozotocin used to induce diabetes. Few functional β cells were observed and α cells were more prominent.

Figures 5C and 5D show the islets of Langerhans from the diabetic groups treated with M. alba leaf extract at concentrations of 400 and 600 mg/kg body weight, respectively. Th e damaged β cells seen aft er the initial induction of diabetes were no longer observed aft er treatment with M. alba leaf extract. Th e recovery of necrotic β cells was especially more pronounced aft er treatment with 600 mg/kg of M. alba leaf extract than in the group treated with 400 mg/kg of the extract.

Th is study was carried out histologically by light microscopy and biochemically in order to detect whether this plant decreased blood glucose and whether it had an eff ect on the pancreatic tissue. Th e M. alba leaf extract was found to be eff ective in controlling hyperglycemia in diabetic rats. Although many suggestions have been made about plants’ roles, the precise mechanism is unclear. Plants may act on blood glucose through diff erent mechanisms,

some of them may have insulin-like substances, some

may inhibit insulin activity, and others may increase

β cells in the pancreas by activating the regeneration

of these cells. Th e fi ber of plants may also interfere

with carbohydrate absorption and thereby aff ect

blood glucose (9-12).

Diff erent studies have demonstrated that certain

plant extracts (Scoparia dulcis, Teucrium polium, and

Salvadora oleoides) eff ectively increased the number

of β cells in streptozotocin- and alloxan-induced

diabetes mellitus (13-15). Th e diminished islet size

and β cell number resulted in the histopathology of

the diabetic pancreas. A recent report showing that

adult β cell numbers are formed by self-duplication/

self-proliferation supports our fi nding of an increase

in the β cell number in diabetic islets aft er mulberry

treatment (16).

Taniguchi et al. (17) showed that the compounds

of mulberry leaf, particularly fagomine, are capable

of inducing insulin secretion in isolated rat islet cells.

Kim et al. (18) observed that aqueous extracts of C.

coronarium and M. alba eff ectively decreased blood

glucose in alloxan-induced diabetic rats.

Shirwaikar et al. (19) reported on histopathology

studies in which STZ was suspected of partially

Figure 5A. Photomicrograph of a pancreatic section from the

control group showing the exocrine region and islets

of Langerhans, with scattered β cells and red blood

cells visible in the vicinity. EX: Exocrine pancreas, β

cell: beta cells, RBCs: red blood cells; CHP staining,

400×.

Figure 5B. Photomicrograph of STZ-induced type 1 diabetic

pancreatic section showing the exocrine region

and islets of Langerhans with damaged β cells due

to necrosis and a decreased number of β cells. EX:

Exocrine pancreas, β cell: beta cells, RBCs: red blood

cells; CHP staining, 400×.

J. MOHAMMADI, P. R. NAIK

215

destroying the pancreas. Diabetic rats showed reduced islet cells, which were restored to near normal upon treatment with the extract. Al-Eryani and Naik (20) observed the eff ects of T. cordifolia extract on high fat-fed type 2 diabetic rats; in that study, the extract signifi cantly restored the diameter of islets and the number of β cells in diabetic rats.

Similarly, Yin et al. (21) observed that high-dose STZ in diabetes indicates β cell destruction. Aft er 120 days of normoglycemia, a statistically signifi cant 3.7-fold increase in β cell mass was observed.

Th is study supports the possibility of regeneration as a means for generating new β cells, even in severely diabetic animals or individuals with signifi cantly reduced β cell mass. As other possible mechanisms, the extract from mulberry leaves may increase β cells and sensitize the insulin receptor to insulin, or stimulate the stem cells of the islets of Langerhans in the pancreas of STZ-induced diabetic rats.

In conclusion, the histopathologic studies undertaken on the islets demonstrated the recovery of damaged islets and an improvement in the number of β cells aft er treatment with the plant extract. It can thus be assumed that M. alba leaf extract has a therapeutic eff ect that alleviates diabetes mellitus. Th e actual chemical compound that is responsible for this eff ect requires further investigation.

Acknowledgments

Th e fi rst author gratefully acknowledges Yasouj University of Medical Sciences, Iran, and the Chairman of the Department of Zoology, University of Mysore, Mysore, India, for support and access to facilities.

Corresponding author:

Jamshid MOHAMMADI

Department of Physiology,

Herbal Medicine Research Center,

College of Medicine,

Yasouj University of Medical Sciences,

Yasouj - IRAN

Email: jamshidm2005@yahoo.com

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

400 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×.

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×.

Th e histopathologic eff ects of Morus alba leaf extract on the pancreas of diabetic rats

216

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