Abstract: Urtica dioica has been introduced with hypoglycemic activity in Iranian traditional medicine. There are some reports with different results about the hypoglycemic activity of Urtica dioica. This study was done to determine the chronic effect of the hydroalcholic extract of Urtica dioica leaves on Hyperglycemia and regeneration of β-cells in hyperglycemic rats. 30 Wistar rats were allocated in groups of normal, Diabetic and treatment. Hyperglycemia in Rats induced by 80 mg kg-1 streptozotocin. One week after injection of STZ Animals in treatment group received hydroalcholic extract of Urtica dioica 100 mg kg-1 day-1 for 4 weeks, intraperitoneally. The blood glucose was measured by using a Glucometer in 1st, 3rd and 5th weeks. In the end of 5th weeks the animals in each group were sacrificed by ether anesthesia and whole pancreas in three groups extracted and fixed in bouin and stained by chrome alum hematoxiline-phloxine stain and beta cells percentage were counted in three groups by Olympus microscope. Mean±SE of blood glucose concentrations in end of fifth weeks were 99.4±5.0, 454.7±34.5 and 447.4±42.5 in control, diabetic and treatment groups, respectively (p<0.05).The percentage of β-cells (beta cells) in control, diabetic and treatment groups was 73.6, 1.9 and 1.3, respectively. The percentage of β-cells in diabetic and treatment groups comparing with control group was significant (p<0.05). This study showed that chronic administration of hydroalcholic extract of Urtica dioica has not hypoglycemic effect and regeneration of β-cells of langerhans in hyperglycemic rats.
INTRODUCTION
Plants used in folk medicine to treat diabetes mellitus represent a viable alternative for the control of this disease (Maroo et al., 2002).
Urtica L. Stinging nettle (Urticacea) is annual and perennial herb, distinguished with stinging hairs. Leaves are opposite. Flowers are green with yellow stamens, the male and female flowers on separate plants. Fruits are achene. These are the characters of Urtica genus which belong to the family Urticacea. The main varieties identified under the Urtica dioica L, U. urens L, U. pilulifera L., U. cannabina L., U. Memberanacea Poiret, U. kiovensis Rogoff (Kavalali et al., 2003).
Among these Urtica dioica (U. dioica) has already been known for a long time as medicinal plants in the world.
The blood sugar lowering effect of U. dioica as a medicinal herb has been introduced in old script such as those written by Avicenna (Farzami et al., 2003).
There have been also other reports indicating the benefits of using the herb for the use in different conditions, i.e. diabetes (Roman Ramos et al., 1992), prostatic hyperplasia (Hirono et al., 1994; Kayser et al., 1995). Inflammation (Obertreis et al., 1996), rheumatoid arthritis, hypertension and allergic rhinitis (Miltman, 1990).
For instance U. dioica is amongst several species listed for their use against hypoglycemic and diabetes in folk medicine (Atta-Ur-Rahman and Zaman, 1989; Farzami et al., 2003) in a large pharmacological screen of European species with knows potential anti-diabetic effects. Also, two earlier studies have detected hyperglycemic activity of U. dioica.
With the existence of controversy of reports about the glycemic activity of U. dioica, this study was done to evaluate chronic hypoglycemic effect of the hydroalcholic extract of U. dioica leaves and potential roles in regeneration of β-cells of pancreas.
MATERIALS AND METHODS
Plant material: U. dioica leaves were collected from cultivated plant, from suburb of Gorgan, northern Iran (Golestan, Iran) in October 2004 and taxonomically identified by Department of Pharmacognosy, Mazandaran University of Medical Sciences. A voucher specimen (5-77-1) was deposited in the herbarium of Mazandaran University.
Preparation of plant extract: Powder of U. dioica leaves was perculated by hydroalcoholic (60°) solvent for 48 h. The extract was filtered and concentrated under vacuum at 40°C to made a jellies material. In addition to thin layer chromatography and purity tests (foreign matter, total ash, acid insoluble ash and water insoluble ash) for qualification analysis, monosaccharide-linked another reagent assay (spectrophotometry) have been carried out to determine the concentration of polysaccharides in U. dioica leaves for standardization of the extract. The results of phytochemical analysis showed the existence of high percentage of Tannins, Steroids and low levels of Flavonids, Carotenoids and Saponins in leaves of U. dioica.
Animals: Male adult albino rats (wistar strain) of 125-175 g were fed on pellet diet and tap water for full acclimatization. The animals were kept in air-conditional animal room (22±2°C) under a 12 h light/dark cycle. The rats were divided into three groups of ten animals each.
Experimental design: Diabetes was induced with a single IP injection of Streptozotocin (STZ) (80 mg kg–1) to overnight fast rats. Streptozotocin (STZ) purchased from Sigma was dissolved in saline immediately use and Intrperiteonally injected (80 mg kg–1).
Blood samples for glucose measurements were taken from the tail vein. Diabetes was confirmed by measuring the glucose concentration by using Glucometer method.
In the experiments, ten rats were used in each group.
Group I: Normal control group. Saline daily for 4 weeks
Group II: Diabetic group. Saline daily for 4 weeks after STZ injection.
Group III: Treatment group. diabetic rats administered 100 mg kg–1 daily hydroalcholic extract of U. dioica, for 4 weeks.
Glucose tolerance test: Intraperitoneal glucose tolerance test (GTT) was performed on 16 h fasted rats using 2 g glucose/kg body weight. In all groups, blood was collected from the animals by tail snipping at 0, 30, 90 and 120 min after glucose load. Also glucose test were performed after IP injection STZ in 1, 3 and 5 weeks.
Histopathologic examinations: The animals of three groups are sacrificed by ether anesthesia. Whole pancreas was dissected. The tissue samples were bouin’s fluid fixed and paraffin embedded for microscopic examination in accordance with routine laboratory procedures. Histopathologic examination and grading were carried out on chrome alum hematoxiline-phloxine stain (Gomori, 1941) stained sections. The number of islets and the number of β-cells of each islet were counted by OLYMPUS microscope and we used OLYSIA software. The islet diameters were measured using calibrated micrometer by taking of fixed number of islet from the three experimental groups and one normal group.
Statistical analysis: All the grouped data were statistically evaluated using Student’s t-test, expressed as the Mean±SE from ten rats in each groups and λ2 test.
RESULTS
The mean±SE of blood glucose concentrations before of STZ injection were 85.5±1.7, 102.9±3.7 and 92±4 mg dL–1 in control, diabetic and treatment groups, respectively. One week after injection of STZ, the mean±SE of blood glucose concentration were 214.1±23.8 and 388.2±17.4. mg dL–1 in diabetic and treatment groups, respectively.
The effect of STZ in diabetic group showed a gradually increasing of blood glucose level which it was (214.1±23.8) in 1 week and it reached to (454.7±34.5) mg dL–1 in fifth week after injection of STZ.
The mean±SE of Blood Glucose concentration level in treatment group increased from 388.2±17.4 to 447.4±42.5 mg dL–1 in the fifth week.
In control group the mean±SE. of Blood Glucose concentration had not any changes. Statistically analysis showed that there was a significant difference between treatment and control groups (p<0.05). But this difference between diabetic and treatment group was not significant. Therefore, the treatment with 100 mg kg–1 day–1 for 4 weeks of hydroalcholic extract of Urtica dioica did not show any hypoglycemic activity.
The same finding was appeared in glucose tolerance test such as; we had no change in trend of GTT curve after 4 weeks injection of U.D (Fig. 1).
The histopathologic results depicted in Fig. 2-4.
In this study the number of islets and number of β-cells in pancreas tissue were 14.7±2.2 and 204±15 in control group, respectively.
| Fig. 1: | Glucose tolerance test (GTT) in 5th week in control (I), STZ
(II) and STZ-Urtica (III) groups that showed no effect of U. dioica
on crude of GTT |
| Fig. 2: | Pancreatic section from normal rat (group I). The beta cells
are shown blue and Alpha cells are red. Staining Gomori’s method, original
magnification X400 [Alph cells ( |
| Fig. 3: | Rat pancreatic tissue prepared from the Diabetic group (group
II) The islet is atrophied and beta cells are decreased. Staining Gomori’s
method, Original magnification X400 [( |
| Fig. 4: | Rat pancreatic tissue prepared from the treatment group (group
III). The beta cells are shown blue and alpha cells are red. Staining Gomori’s
method, original magnification X400 alpha cells ( |
These were (5.8±1.1) and 2.7±0.7 in diabetic groups and (9±1.8) and 1.8±0.5 in treatment group, respectively.
The percentage of β-cell in control, diabetic and treatment groups were 73.6, 1.9 and 1.3%, respectively. The percentage of β-cells in diabetic and treatment groups comparing with control group was significant (p<0.05).
According to our finding although the IP injection of STZ caused a significant of decrease in the percent of β-cells but the administration of U. dioica could not change the number of beta cells.
DISCUSSION
The finding of this study indicated that the chronic administration of the extract of U. dioica leaves did not decrease blood glucose concentration and also have not any effect on number of beta cells in pancreatic tissue.
Various studies showed the controversial effects of Urtica species on inducing hyperglycemia (Bnouham et al., 2003; Swanston-Flatt et al., 1989; Roman Ramos et al., 1992).
In recent study extra pancreatic effect of U. dioica was observed (Onal et al., 2005).
Onal et al. (2005) established that antidiabetic effect of U. dioica can be related to be inhibition of alpha-glycosidase enzyme in rat intestine (Onal et al., 2005).
The results of present study show that the exact mechanism of effect of U. dioica. is not dependent on beta cells of pancreas. This finding is accordance to study of Onal et al. (2005), showed extra pancreatic effect of U. dioica. Earlier study about STZ showed that in high dose we have irreversible damage of beta cell of pancreas (Saini et al., 1996). Therefore we can conclude that U. dioica couldn’t regenerate necrotic beta cell of pancreas. The other species of this genus, U. pilulifera has been reported to has hyperglycemic effects (Kavalali et al., 2003). Kavalali showed that the chronic effect of seed of U. pilulifera can induce the hypoglycemia and prevent cellular damage of pancreas on diabetic rats (Kavalali et al., 2003).
The major differences between the kavalali study and our study are: 1. in present study we used leaves of U. dioica while in kavalali study seed of U. dioica was used. 2. Difference between two species may be responsible for different results.
Acute hyperglycemic activity of U. dioica was demonstrated in STZ diabetic rats by Farzami et al. (2003) and Fathi-Azad et al. (2005). Farzami demonstrated that an hydroalcholic extract of U. dioica was a potent stimulator of insulin release in short time (Farzami et al., 2003) on the basis of present observation, we can not rule out potential short time hyperglycemic activity of U. dioica, but this effect dose not observed in long time and dose not solely due to regeneration of beta cells of pancreas.
Taken together we have shown that chronic administrations of the leaves extract of U. dioica in Hyperglycemic rats have not hypoglycemic activity. Moreover, we have not any effect on regeneration of beta cells of pancreas.
These results put under question on usefulness of U.dioica leaves in the treatment of diabetes mellitus in long time. Further pharmacological studies are necessary to confirm potential usefulness of this plant as a prophylactic agent in preventing development of diabetes.
ACKNOWLEDGMENTS
We thanks research department of Gorgan University of Medical Sciences, Mr. Behnampour and Dr. Soleimani for helping.