Trigonella Foenum Graecum Extract Benefits on Hematological, Biochemical and Male Reproductive System of as a Complementary Therapy with Glimepiride in Treating Streptozotocin Induced Diabetic Rats

Diabetes mellitus (DM) is a chronic metabolic disorder. Streptozotocin is a naturally occurring cytotoxic chemical, particularly toxic to the pancreas and insulin producing beta cells in mammals and induces diabetes. Glimepiride is a second generation sulfonylurea, used as second-line or add-on treatment options for type 2 diabetes. Fenugreek (Trigonella foenum graecum) seeds have been documented as a traditional plant treatment for diabetes. Soluble dietary fiber of Fenugreek significantly improved oral glucose tolerance in diabetic rats. It also exerts anti-diabetic effects mediated through the inhibition of carbohydrate digestion and absorption and the enhancement of peripheral insulin action. Most herbal remedies can interact with allopathic drugs resulting in altered activity and toxicity. At the same time, herbal remedies might produce the same kind of effects as the drug produce. Current published research information on herb-drug interactions is scanty. So, the aim of this study was to investigate the possible interaction between conventional drug used for the management of diabetes; (Glimepiride) and a traditional herbal remedy; Fenugreek aqueous extract in Streptozotocin induced diabetic male albino rats. In conclusion, combination therapy induces better hematological, biochemical effects and improves the oxidative stress biomarkers and antioxidant enzymes. Histological studies showed better results on some organ functions. The results emphasize the benefit of using the combination of Fenugreek seeds aqueous extracts as supportive complementary anti-diabetic therapy. DOI: 10.14302/issn.2374-9431.jbd-19-2613 Corresponding author: Eman H. Radwan, Department of Zoology, Faculty of Science, Damanhour University, Egypt, Phone No: 01001089259, Email: dr_eman_hashem@yahoo.com


Introduction
Diabetes mellitus (DM) is a chronic metabolic disorder featured by hyperglycemia and disturbances in protein, carbohydrate and fat metabolism. These metabolic abnormalities result either from a deficiency of the blood sugar-lowering hormone; insulin or from insulin resistance; a defect in the body's capacity to respond to insulin. Streptozotocin (N-Nitro derivative of Glucosamine) is a naturally occurring broad spectrum antibiotic and cytotoxic chemical. It is produced by the bacterium Streptomyces achromogenes and it is particularly toxic to the pancreas and has been used widely to create animal models of diabetes [1]. Amaryl® (Glimepiride) is a sulfonylurea used as the drug of choice in the treatment of type 2 diabetes. The hypoglycemic activity of Glimepiride relied on its ability to enhance insulin release and action [2]. Trigonella foenum graecum (Fenugreek) is a medicinal plant that grows annually and used daily in Africa and Asia.
Fenugreek is known to have several pharmacological effects. One of them is the hypoglycemic effect. The seeds have been documented as a traditional plant treatment for diabetes. Soluble dietary fiber (SDF fraction) of Fenugreek significantly improved oral glucose tolerance in diabetic rats [3]. The aim of this study is to investigate the possible interaction between Amaryl ® tablets (Glimepiride) and Fenugreek seed aqueous extract on Streptozotocin induced diabetes in male albino rats.

Experimental Animals
Male albino rats, weighing between 150-160 g, were housed in a clean and well ventilated animal house, maintained at room temperatures, with a constant 12 hr light and 12 hr dark cycle, with access to tap water and standard diet (SCF; Ewu Feed Mill, Edo State, Nigeria) for a 2-week acclimatization period. The handling of the animals was in accordance with the standard principles of laboratory animal care of the United States National Institutes of Health [4].

Chemicals Streptozotocin (STZ) was purchased from
Sigma -Aldrich chemical, USA). Amaryl® (Glimepiride) was obtained from local pharmacies, Egypt. It was grinded using a mortar. The drugs were administered orally through tube once a day.

Herbal Samples
Trigonella foenum -Graecum (Fenugreek) water extract was made from 100g fresh seed. Seeds were grinded and mixed with 2000 ml distilled water [5]. The water extract was lyophilized and collected as powder.

Experimental Design
Diabetes was induced in the experimental rats by intraperitoneal injection with Streptozotocin at single dose (40 mg/kg body weight). After 72 hours, rats had more than 250 mg/dl random blood glucose concentration. Treatment with Fenugreek and Amaryl ® started 3 days after Streptozotocin injection and continued for eight weeks.

Animal Model
Thirty-five Male Albino rats were randomly divided into five groups (7 rats/group) as follows: Significance of the obtained results was judged at the 5% level. The minimum level of statistical significance was set at P < 0.05.

Body Weight Through Eight Weeks of Treatment
Results showed a reduction in the body weight in STZ injected rats. This reduction may be attributed to insulin depletion provoking a loss of adipose tissues and the changes in carbohydrates and protein metabolism that occur in rats with Streptozotocin-induced diabetes [6]. The increase in the body weight of group (5) exceeds that of group (3) and group (4). It reached the same value of the control group at the eighth week. The body weight of group (3) and group (4) improved during the experiment (Fig. 1).
Fenugreek aqueous extract significantly increase the body weight. It exhibited antioxidant property which protects the functional organs and increase the body weight [7]. Glimepiride increases the body weight significantly [8].

Platelets Count
In diabetes, there is an increased platelet activation and subsequent aggregation favoring thrombosis and also depression of bone marrow stem cells which could be contributory for a reduced platelet count observed. The decrease in circulating platelet number may also means an increase in aggregated platelet typical of atherosclerotic disorders of diabetes mellitus. In Fenugreek group, platelet count increased significantly (p < 0.01). The significant improvement in the levels of circulating platelets in blood could be attributed to inhibitory activity of certain constituents of T. foenum graecum on platelet aggregation [11].
Glimepiride group showed an increase of the platelet count. Glimepiride exhibits inhibitory effects on human platelets aggregation through selective suppression of the cyclooxygenase pathway [12]. Combination of Fenugreek and Glimepiride showed better platelet count with no significant difference compared with normal control group after eight weeks, (Table 1).  (Table 2). Fenugreek seeds play a role in protecting the spleen and increasing the lymphocytes.
So, they might improve the immunity. These observations were in agreement with those obtained by Sindhu et al. [13] and Kandhare et al. [14]. Fenugreek seeds influenced the hemoglobin and lymphocytes count, improving hematopoietic function and survival.
The present study demonstrates that combination of Fenugreek and Glimepiride are more effective than Fenugreek alone or Glimepiride alone in protecting to the altered hematological variables, ( Table 2).

Random Blood Sugar Level
The current study showed some changes in the biochemical measurements due to the injection of Streptozotocin (Fig. 2). Group (5); Fenugreek and Glimepiride combination improved the random blood sugar to reach a value near the normal control values.
The random blood sugar level of group (3) and group (4) showed no significant difference from each other. Injection with STZ induces activation of poly adenosine diphosphate ribosylation and nitric oxide release. As a result, pancreatic cells are destroyed by necrosis [15]. Fenugreek seeds water extract acts as an insulin secretor. Hypoglycemic effect of Fenugreek may be mediated through stimulating insulin synthesis and increasing β pancreatic cells secretion. The primary mechanism of action of Glimepiride in lowering the blood glucose appears to be dependent on stimulating the release of insulin from functioning pancreatic beta-cells [16]. Glimepiride binds to sulfonylurea receptors on β-cells leading to blocking of K + ATP channels, opening of voltage-gated calcium channels and increase in Ca 2+ influx leading to insulin release from pancreatic β-cells.

Insulin Concentration
The present study demonstrated that STZ    results in excess fatty acid accumulation in the liver.

Serum Liver Functions (ALT, AST and ALP Values)
The reported present data ( Table 6)  Fenugreek therapy restores the activity of these enzymes. These seeds may inhibit the liver damage induced by Streptozotocin and improved liver function with return of gluconeogenesis toward its normal level [26]. Treatment of diabetic rats with sulfonylurea drugs was explained by increased insulin secretion, hepatic uptake of glucogenic amino acids and stimulation of amino acids incorporation into protein [27].   Fenugreek seeds to diabetic rats significantly decrease of sperm shape abnormality and improve the sperm count [32]. Glimepiride reduced sperm abnormality and increased testis weights and sperm count by its antioxidant action [30]. The potential protective efficacy of Fenugreek seed extract when added to Glimepiride was observed on reproductive systems.

Pancreas Histopathological Observations
Histological examination of pancreas of the control rats showed normal sized islets of pancreas (Ip) compared to the diabetic rats which showed markedly atrophic islets of pancreas (I.p.) ( Fig. 8 and 9). Pancreas of in groups (3) and (4) rats showed mild atrophic islets of pancreas (I.p.) with inflammatory cellular infiltrate ( Fig. 10 and 11). The pancreas of rats in group (5) showed nearly normal islets of pancreas (I.p.) with few inflammatory cellular infiltrate (I.c.) (Fig. 12).
Histopathological Fenugreek improved blood glucose levels and insulin, lowered pancreatic islet and β-cells damage which may be attributed to its immune modulatory activity and insulin stimulation action along with its antioxidant potential [32].              lined with normal epithelium (Fig. 13). The diabetic rats showed tubular casts, inflammatory cellular infiltration and glomerular atrophy (Fig. 14). Kidney of rats in groups (3 and 4) showed some glomeruli return to be normal (G). Focal tubules casts (T) in Fenugreek group and some normal tubules in the Glimepiride group with no inflammatory cellular infiltrate in both groups ( Fig. 15 and 16). However, kidney of rats in group (5) showed normal glomerular (G), normal tubules (T) with no tubular casts (Fig. 17).

Conclusion
Fenugreek is a natural therapy that has many beneficial health effects while Glimepiride is an efficient