It is well known that diabetes mellitus is the commonest endocrine disorder that, according to the World than 176 million people world wide, in Mexico the WHO estimates that the Health Organization, affects more number of diabetic patients will increase from more than 2 million in 2002 to more than 6 million in 2030, which would imply that in a few decades Mexico may have highest rate of diabetes in the world. Because of the complications linked to diabetes like heart disease, retinopathy, kidney disease and neuropathy, it also is a common cause of chronic morbidity and disability among the working population. The term diabetes mellitus describes a metabolic disorder of multiple a etiologies and is characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. The causes of type 2 diabetes are either insulin resistance with relative insulin deficiency or predominantly an insulin secretory defect with or with out insulin resistance (Soleimani et al., 2007).
Recently, there has been increasing interest in use of medicinal plants. The plant kingdom has become a largest for the search by multinational drug and biologically active lead compounds. Ethnobotanical in formation indicates that more than 800 plants are used as traditional remedies for the treatment of diabetes. The hypoglycaemic activity of a large number of these plant has been evaluated and confirmed in different animal models (Eidi et al., 2005). Enhancement of the division of E. arvense protoplasts in culture by activated charcoal and their further development (Akira et al., 1990) and the hepatoprotective effect of E. arvense has been reported by Katikova et al. (2002). Hepatoprotective and free radical scavenging activities of phenolic petrosins and flavonoids isolated from (L) extract reported by Oh et al. (2004). Antidiabetic effect of Equisetum arvense in diabetic rats reported by Soleimani et al. (2007). The aim this study was to investigate the hypoglycemic effects of methanolic extract and histological change in Streptoztocin (STZ) induced rats.
MATERIALS AND METHODS
Equisetum arvense L. subsp. arvense (Equisetaceae) pharmacopeia:
lName Equiseti herba is a traditional plant. Traditional use of the plant was
recorded at market in 2005; by our selves of the plant for the treatment of
kidney problems and diabetes in the Ardabil. Our own ethnopharmacological studies
were performed in the community in Urmia university in 2005. Diabetic people
were identified by the local health services and local healers. All informations
were obtained about the plant and its special usage based on structured and
unstructured interviews with the traditional healers and the diabetic people,
respectively. The data were referred to plant samples (mini-herbarium) collected
at its natural habitats and stored as herbarium vouchers for exact identification.
Plant material: Equisetum arvense is mainly found in northwestern
regions of Iran especially Ardabil. This plant is considered an herbal drug
and is used for renal disorders and diabetic related illness. This powdered
from of this plant was purchased from the Herbal medicine research institute
of Tabriz university, Iran and their identity was confirmed and voucher specimens
were deposited at this Herbarium in Tabriz (No. 313).
Extraction: The dried plant was than milled to fine powder and 500 g of this powder was packed in to Soxhlet apparatus and extracted with methanol. The extract were dried at 45°C in hot air oven till solid to semisolid mass was obtained and were stored in airtight containers in refrigerator below 10°C.
Animals and experimental protocols: A total of 30 male wistar rats were used; eight weeks old weighing 150 to 200 g was obtained from the laboratory animal center of Veterinary faculty of Urmia University. The rats were housed under controlled environmental condition (12 h dark light cycle at 22°C) and had free access to standard rat chow and water. Diabetes was induced by intravenously injecting STZ (50 mg kg-1 body weight) in acetate normal saline.
Experimental groups: The diabetic animals were classified into 5 groups (1-5) each of them with 6 rats.
Group 1: As non-diabetics control received 0.03 mL physiological NaCl-solution (Vehicle).
Group 2: As the diabetic control received also 0.03 mL of physiological NaCl-solution (vehicle).
Group 3: Were given the standard oral hypoglycemic agent glibenclamide
(5 mg kg-1 b.wt.) in the same vehicle Groups (4 and 5) received (50
and 250 mg kg-1 b.wt.) methanolic extracts, respectively.
Collection of blood and determination of blood glucose: Blood samples were taken from the tail vein before oral administration of the extracts or the vehicle. The glucose concentration was measured in plasma serum with Reflotron equipment and confirmed by Accutrend GC and Accu-check compact equipments (Roche).
Histopathology evaluation: Tissue samples from the pancreas were fixed in 10% buffered neutral formalin, embedded in paraffin, sectioned at 5 μm and stained with hematoxylin-eosin and periodic acid-schiff.
Statistical analysis: All the values of body weight, blood sugar and biochemical estimations were expressed as mean+Standard Error of Mean (SEM) and analyzed for ANOVA tukey test.
Activity in diabetic rats: STZ administration at of dosage of 50 mg kg-1 b. wt. to normal rats significantly elevated the blood glucose levels compared with rats injected normal salin alone (Table 1) as in previous reports (Soleimani et al., 2007). In present diabetic rats, the extracts showed significant hypoglycemic effects (Table 1). The methanolic extract at doses of 50 and 250 mg kg-1 bw significant reduction (p<.0001) of plasma glucose level compared with diabetic control from the 1 and 5 weeks of treatment. Gilbenclamide (5 mg kg-1 b. wt.) produced a significant decrease in plasma glucose (Table 1).
These results indicate that there is no significant difference between the tested plant preparations in comparison to glibenclamide (standard hypoglycemic drug).
Animal weights: The mean weight of diabetic animals was significantly lower than that of non-diabetic animals (Table 1) how ever; the glibenclamide regimen was sufficient both to support weight gain in the diabetic animals during the early stages and to maintain their weight at the later stages of the study. Mortality was less than 6% among diabetic animals during the study. The weights of the extracts-treated diabetic animals were statistically identical (Table 1).
The body weight of 5 weeks diabetic rats treated with methanolic extract of E. arvense. At the diabetics rats treated with methanolic extracts (50 and 250 mg kg-1 day) immediately after diagnosis of diabetes had their body weight comparable to no-diabetic control groups (Table 1).
Histopathological examination of pancreas: Figure 1 A
and B represent two islets of langerhans from a normal and
a STZ-induced diabetic rats, respectively. Comparison of Fig. A and B it is
clearly indicates the reduction in the number of β-cells in the diabetic
||Effect of oral administration of methanolic extract of Equisetum
arvense on plasma glucose concentration, body weight in diabetic rats
|1: Non diabetic control; 2: Diabetic control; 3: Diabetic
treated with 5 mg kg-1 bw gilbenclamide; 4: Diabetic treated
with 50 mg kg-1 bw; 5: Diabetic treated with 250 mg kg-1
bw extract; Values are given mean±SEM for groups of six animals each.
a: p<.0001, (tukey-test), diabetic control was compared with
the vehicle control and extract treated groups were compared with the diabetic
As it is evident from Fig. 1B, the islets are irregularly
shaped and relatively small and atrophic. Most cells of the islets are small,
degeranulated and dark with scanty cytoplasm. Severe vaculation and degranulation
are present in the β-cells of a number of islets. A exudates predominantly
of lymphocytes, with a few macrophages and neutrophils is evident within and
around the affected islets. However, compared to untreated diabetic rats, histophatological
examination of the plant extract treated diabetic rats revealed an increase
in the number of pancreatic islets and the number of β-cells along with
a reduction in the number of initiated lymphocytes and macrophages (Fig.
1 C and D). Restoration of normal cellular
population size of islets with hyperplasia by glibenclamide (Fig.1C-E).
||Photomicrographs rat pancreas stained by hematoxylin and eosin
of untreated (A) and STZ-induced diabetic (B) rats, (C) diabetic treated
with 50 mg kg-1 bw extract, (D) diabetic treated with 250 mg
kg-1 b. wt. extract and (E) diabetic treated with 5mg kg-1
b. wt. gilbenclamide (hematoxylin-eosinx200)
In the present investigation, the methanolic extract of Equisetum arvense was investigated for its antidiabetic activity in streptozotocin-induced diabetic rats. Glibenclamide treatment (5 mg kg-1) was not as effective in reducing blood glucose in STZ-diabetic rats as in normoglycaemic rats. It has been reported that glibenclamide was not effective when destruction of ß-cells has occurred and hence more effective in moderate diabetic rats than in severe diabetic animals (Sharma et al., 1997; Andrade-Cetto et al., 2000; Hosseinzadeh et al., 2002). The acute hypoglycaemic effect of glibenclamide results has been shown from the stimulation of insulin release from the residual ß-cells and inhibition of glucagon secretion (Moller, 2001). The extract might possess insulin like effect on peripheral tissues either by promoting glucose uptake and metabolism or inhibiting hepatic gluconeogenesis. The phytochemical studies of C. pentandra revealed the presence of epicatechin isolated from other plants has been found to stimulate insulin secretion or possess an insulin-like effect (Marles and Farnsworth, 1995; Noreen et al., 1998; Kameswara et al., 2001).
In light of the results, present study indicates that E. arvense have good antidiabetic activity. Methanolic extract of E. arvense exhibited significant anti-hyperglycemic activities in streptozotocin-induced hyperglycemic rats with out significant change in body weight. They can also improve the condition of DB as indicated by parameters like body weight. Among them methanolic extract produce a hypoglycemic effect in rats. Similar finding reported by Soleimani et al. (2007 ).
The plant extract treated diabetic samples histopathologically approach the
corresponding healthy pancreatic samples. The regeneration of the β-cells
of the STZ-destructed islets is probably due to the fact that pancreas contain
stable cells which have the capacity of regeneration. There fore, the surviving
cells can proliferate to replace the lost cells (Yazdanparast et al.,
2005). The renewal of β-cells in diabetes has been studied in several animal
models. The total β-cells mass reflects the balance between the renewal
and loss of these cells. It was also suggested that regeneration of islet β-cells
following destruction by alloxan may be the primary cause of the recovery of
alloxan-injected guinea pigs from the effects of the drug (Nagappa et al.,
2003). Similar effects in streptozotocin-treated diabetic animal were reported
by pancreas tonic (Rao et al., 1998), ephedrine (Xiu et al., 2001)
and Gymnema sylvestre leaf extracts (Shanmugasundaram et al.,
1990). In present studies, the damage of pancreas in STZ-treated diabetic control
rats (Fig. 1B) and regeneration of β-cells by glibenclamide
(Fig. 1E) was observed.
In conclusion, our histopathological investigation along with the biochemical evaluations suggests the possibility of the islets regeneration upon plant extract treatment. Further research is required to explore exactly the mechanism of islet regeneration by the plant extract.
I would like to thank the colleagues in the Faculty of Science for their cooperation. Appreciation also goes to Habib Shojaei who helped me in this study.