The Effect of Aqueous-Alcoholic Extract of Ducrosia anethifolia on Cerebellar Tissue of Diabetic Rat’s Embryo

Background & Aims: Since ancient times, medicinal plants have been used to treat a wide range of diseases. In recent decades, despite the progress of chemical pharmacy, the use of medicinal plants is still considered. Different parts of the plant contain many compounds with different properties, and some plants have antidiabetic properties (1). Although reasons such as cheapness, fewer complications, and easier access are considered for using herbal drugs (1), this class of drugs is not completely safe (2). Moshgak with the scientific name Ducrosia anethifolia has beneficial properties, including antioxidant properties (10) and the ability to reduce the activity of enzymes interfering in the metabolism of carbohydrates, such as alpha glycosidase ( 11). Moshgak is native to Iran and is widespread in the southern regions of the country, including Fars province (12). This plant is used raw and cooked in the diet, especially in the southern regions of the country. In addition, many useful properties have been reported for it, which can be pain-relieving, antimicrobial (13), and antifungal (14). The effect of the leaf extract of this plant on the nervous system and its anti-depressant (15) as well as the enhancement of spatial memory and learning level in mice (16) has also been reported. The presence of flavonoid compounds, monoterpenes, and coumarins such as pangolin, myrcene, and limonene in it have given antioxidant properties to this plant (11). The many benefits reported for this plant, which fortunately has widespread in Iran, become an incentive for further research about its effect on living beings. Maybe by confirming its positive effects as a complementary medicine, it will be suggested to the pharmaceutical industry. Regarding the use of herbal remedies in the treatment of diabetes mellitus, this study aimed to investigate the effect of an aqueous-alcoholic extract of Ducrosia anethifolia on the brain protection of diabetic rat embryos. Methods: In this study, 48 rats after fertilizing were made diabetic with a single dose of 80 mg streptozotocin. To prepare the aqueous-alcoholic extract of mashgak leaves, 40 g of dried powder of the moshgak’s leaf was poured into a flask, 400 ml of ethanol was added to it, and it was mixed with a shaker at 250 rpm for 48 hours. Then it was filtered and concentrated in a rotary (17). To determine the appropriate dose of treatment, the lethal dose was estimated. Then the dose of 500 and 1000 mg/kg/bw of mashgak extract was determined as the minimum and maximum dose, respectively. The rats were diabetic by intraperitoneal injection of a single dose (80 mg) of streptozotocin in citrate buffer and pH = 4.5 kg/bw of rat on the first day of pregnancy. The rats with fasting glucose of more than 130 mg/dL were considered diabetic rats. Rats were divided randomly into 6 groups: control group (healthy rats without any treatment), Sham group1 (healthy rats which receiving meshgak extract), Sham group 2 (diabetic rats without treatment), Sham group 3 (diabetic receiving 5 mg/kg/bw of glibenclamide), treatment groups 1, 2 (diabetic rats receiving 1 ml meshgak extract at doses 500 and 1000 mg/kg/bw respectively). Treatment was done from the 6th to the 20th day of gestation, and blood sampling was done at the end of treatment. 2 cc of blood was taken from the heart of the rats, and centrifuged at 3000 rpm for 10 minutes. The separated serum was analyzed to determine the level of glucose, insulin and malondialdehyde enzyme by enzymatic method and using specific kits in the lab. Then the embryos were dissected, and their brains were removed and fixed in formalin 4%. After tissue passage in the automatic Tissue Processor (model 2000 DS) according to the standard method, sections (5μm) were prepared and their slides were stained with hematoxylin-eosin. About 30 slides of 10 series of longitudinal, sagittal, and cross sections were examined by light microscope. The thickness of the brain layers was measured using the Image analysis software on the micrographs, and counting the cells in each layer using the disector technique and  Grids-stero lite software was determined (18). Obtained data were analyzed by SPSS software (15), and the groups were compared by Duncan test. Results: There was a significant (p < 0.05) increase in maternal blood glucose, malondialdehyde, and reduction of insulin in sham 2 compared to the control group, while it was compensated significantly in the treatment groups. Although these groups had significant differences from the control group (Fig 1-3). The thickness of the cerebral cortex, white matter, and especially gray matter in the diabetic group showed a significant decrease compared to the control group and sham 1, and it was significantly compensated in the treated group with Glibenclamide (sham 3) and Moshgak extract (Fig 4, 5). The observed changes in the different layers of the gray matter also follow the trend of decreasing the number of cells in the control group 2 (diabetic) and a significant increase in the number of cells in the same layers in the rats which received Glibenclamide (sham 3), and Moshgak extract (Fig 6-8). The results indicate a greater effect of the aqueous-alcoholic extract of Moshgak on the brain tissue compared to the antidiabetic drug Glibenclamide, although none of them were able to adjust the changes completely. Conclusion: According to obtained results, the extract of Moshgak leaf can partially compensate for the adverse effects caused by diabetes on the brain tissue in the embryo. The decrease in blood glucose along with the increase in insulin as well as the decrease in malondialdehyde enzyme indicates the decrease in the complications of diabetes on the brain tissue of the fetus, which is obtained as a result of the consumption of Moshgak extract. The compensation of the tissue changes including the changes observed in the thickness of the cortical layers as well as the number of cells in each layer of the cortex also confirms the biochemical results. Therefore, it is possible to mention the Moshgak extract of marigold as a factor in improving the effects of maternal diabetes on the cortical tissue of the fetus. For a definitive statement regarding the anti-diabetic properties of this plant, it is suggested to conduct more studies on the effect of the compounds of this extract, especially on the beta cell line in the pancreas.