Document Type : Research Paper
1 Department of Pharmacology, Isfahan Pharmaceutical Sciences Research Center, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Pharmacognosy , Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
3 Isfahan Pharmaceutical Sciences Research Center, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
Diabetes mellitus is a group of disorders with different etiologies. It is characterized by derangements in carbohydrate, protein and fat metabolism caused by the complete or relative insufficiency of insulin secretion and/or insulin action. It has been estimated that approximately 140 million people worldwide suffer from diabetes mellitus . Life expectancy may fall to half by this disabling disorder, especially in developing countries of the world where its prevalence is increasing steadily, and adequate treatment is often expensive or unavailable . During the last two decades, traditional systems of medicine and medicinal plant research have become topics of global interest and importance.Securigera securidaca (Fabaceae) is one of such medicinal plants that have been reported to be useful as an effective remedy against epilepsy, hypertension, parasitic infections like malaria, and gastrointestinal ailments [3-6]. Both in Persian and Egyptian folk medicine the seeds of S. securidaca have been used as antidiabetic remedy [4, 5]. Phar-macological studies have confirmed such activities like positive chronotropic, diuretic and hypokalemic effects . Antidiabetic activity of the seeds of the plant has been the subject of research in several investigations. Najaragan et al.  reported that an aqueous extract of the seeds reduced blood glucose level in anesthetized cats. In another study, carried out by the same investigators, normal blood glucose levels of mice were significantly higher after oral administration of the seeds and the extract was not effective to reduce the blood glucose level in alloxan-induced diabetes . Hosseinzadeh et al.  reported that S. securidaca seeds extracts (both aqueous and ethanolic) were not effective in reducing blood glucose levels in normoglycemic and glucose-induced hyperglycemic mice; however, oral or intraperitoneal administration of S. securidaca seeds extract resulted in blood glucose reduction in alloxan-induced diabetic mice. The controversy was noted above conducted us to further investigate the antidiabetic or hypoglycemic properties of the plant seeds in rats as another animal species to help the health professionals to decide better about its antidiabetic activity.
2. Materials and methods
2.1. Plant material
S. securidaca was prepared from the market and cultivated in the laboratory of Goldaru Pharmaceutical Co. (Isfahan, Iran). The plant was collected during spring and identified by Mr. Iraj Mehregan, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran. The voucher sample was deposited for reference in the herbarium at the Department of Pharmacognosy, Faculty of Pharmacy Isfahan University of Medical Sciences, Isfahan, Iran. The plant seeds were dried in shade and then finely powdered.
2.2. Preparation of extract
The powdered seeds (250 g) were macerated in 500 ml of 80% ethanol (ethanol/water ratio 4/1) for 48 h. Then the extract was filtered and concentrated in a rotary evaporator under reduced pressure at 40 ± 1 °C . The resulted extract after drying gave 21.32 g (i.e. 8.52% yield) of bright brownish extract. The plant extract was dissolved in 0.2% tween 80 in normal saline (vehicle) for pharmacological experiments.
Male Wistar rats, weighting 200-250 g, were obtained from Razi Institute, Tehran, Iran. All of the animals were kept under the same and normal laboratory conditions of temperature (20-22 °C), humidity (60-70 %), and light cycle (12 h day/ 12 h night) and had access to water ad libitum. Animals were divided randomly into groups of eight. The principles of laboratory animal care and handling were followed throughout the study.
2.4. Experimental procedure
Diabetes was induced by intraperitoneal (i.p.) injection of streptozotocine (STZ, 60 mg/kg in 0.1 M acetate buffer, pH 4.5). Diabetes was identified by polydipsia, polyuria, and measuring fasting plasma glucose level and was allowed to develop in rats over a period of 5-7 days . Before the commencement of each experiment, both the "control" normal and diabetic rats were fasted for 16 h, but still allowed free access to water throughout.
Fasted STZ-treated rats with blood glucose concentrations between 250-400 mg/dl were considered to be diabetic and were used in this study. Blood samples were taken at 0, 1, 2, 3, 4, and 8 h after treatment by heparinized microhematocrit capillaries from orbital sinus plexus and were deposited into NaF containing centrifuge tubes for plasma separation. For blood glucose analysis, method of glucose oxidase was used . Glibenclamide (10 mg/kg, p.o.) was used as standard antidiabetic (hypoglycemic) agent for comparison . The test compounds (i.e. hydroalcoholic extract of S. securidaca, 200, 400, 800 mg/kg, p.o. and 400 mg/kg, i.p.) were administered to four separated groups of normal and diabetic rats.
2.5. Data Analysis
The data are expressed as means ± SD and were analyzed statistically by using ANOVA followed by Post Hoc Scheffe test. Values of psignificance difference from control.
Results indicated that hydroalcoholic extract of S. securidaca was not effective to reduce blood glucose level both in normal and STZ-induced diabetic rats (Figures 1, 2). In normoglycemic rats, oral administration of the extract with different increasing doses caused a negligible increase (~10 %) in blood glucose concentrations; however, the activity was not significant compared to the control levels (p>0.05). Glibenclamide, on the other hand, lowered blood glucose level in diabetic rats and caused hypoglycemia in normal rats (p3 hours after treatment (p2).
Figure 1. Effect of oral administration of Securigera securidaca seed hydroalcoholic extract on fasting blood glucose level of normal rats. Each point represents the mean ± SD, n=8 rats. Scheffe test, NS: not significant,
* p < 0.05; P < 0.01 pvs control. Ctrl: control, Ext: extract with doses of 200, 400, 800 mg/kg, p.o. and 400 mg/kg, i.p., Glib.: glibenclamide, 10 mg/kg, p.o.
Figure 2. Effect of oral administration of Securigera securidaca seed hydroalcoholic extract on fasting blood glucose level of STZ-induced (60 mg/kg) diabetic rats. Each point represents the mean ± SD, n=8 rats. Scheffe test, NS: not significant,*p < 0.05, p < 0.01 vs control. Ctrl: control, Ext.: extract with doses of 200, 400, 800 mg/kg, p.o. and 400 mg/kg, i.p., Glib.: glibenclamide, 10 mg/kg, p.o.
The present study clearly demonstrated that S. securidaca seeds extract had no effect on blood glucose level in STZ-induced diabetic rats, so it has no insulin-like activity or a pharmacological effect similar to biguanids [7, 12]. In addition, the hydroalcoholic extract of plant has no effect on normoglycemia indicating no activity similar to sulfonylureas . Our findings are in contrary with the results obtained by Hosseinzadeh et al.  and Porchezhian et al. . The differences in animal species were used, source of plant which itself determines the content and identity of active constituents, methodological differences in diabetes induction (i.e. STZ vs alloxan or the dose of STZ) and blood glucose analysis may explain some of these discrepancies. On the other hands, our findings in rats with normoglycemia are somewhat similar to those obtained by Nagarajan et al. . In the latter study, blood glucose levels of normal mice were significantly higher after oral administration of the plant seeds. In the present study, the seeds extract by oral intake, increased normal blood glucose concentrations in rats however, the activity was negligible and dose-independent. This effect was not detectable in diabetic rats or those received the treatment intraperitoneal-ly. It is assumed that sugar contents of extract or those active materials are apt to breakdown in the gut to form carbohydrates may be responsible for this property. Preliminary phytochemical analysis of the S. securidaca both aqueous and ethanolic extract indicated the presence of flavonoids, alkaloids, tannins and saponins. Saponins were detected only in the aqueous extract . Whereas the hypoglycemic effect in some plants are substantially attributed to the flavonoids, but this property dose not necessitate the antidiabetic activity [15, 16].
Our results also indicated that glibenclamide, as a hypoglycemic reference drug, was effective in lowering blood glucose levels in STZ-induced diabetic rats. This is in accordance with the findings reported by Courtois et al.  and Andrade-Cetto et al. . They have reported that oral administration of glibenclamide to STZ-induced diabetic rats decreased the blood glucose level and also could be considered as a standard antidiabetic drug to compare the efficacy of hypoglycemic compounds. The major action of glibenclamide is to increase insulin release from the pancreas. Two other additional mechanisms of action, probably more responsible in our results, are a reduction of serum glucagon levels and an extra-hepatic effect to potentiate the action of insulin on its target tissues . On the basis of current investigation it is strongly recommended that the hypoglycemic effect of S. securidaca is controversial and any use of this plant seeds for therapeutic applications needs further confirmations in various animal species and in human.
We are grateful to Mr. Iraj Mehregan for his assistance in plant authentication and to Goldaru Co., for their efforts to supply the plant material. This work was supported by Reaserch Council of the Isfahan University of Medical Sciences, Isfahan, Iran (research project no. 81295).