World Journal of Advanced Research and Reviews, 2020, 06(03), 012–029 World Journal of Advanced Research and Reviews e-ISSN: 2581-9615, Cross Ref DOI: 10.30574/wjarr Journal homepage: https://www.wjarr.com (RESEARCH ARTICLE) Antidiabetic and anti-inflammatory effects of two Fabaceae extracts against streptozotocin induced diabetic impairment in male rats Abdou Heba Mohamed 1, *, Mohammed Nema Abd El-Hameed 1, Hussein Hussein Khamis 1, Ghazaly Kamal Sharkawy 2 and Abd El-latif Heba Samir 2 1 Zoology Department, Faculty of Science, Alexandria University, Egypt. 2 Zoology, Biological and Geological Sciences Department, Faculty of Education, Alexandria University, Egypt. Publication history: Received on 20 May 2020; revised on 02 June 2020; accepted on 08 June 2020 Article DOI: https://doi.org/10.30574/wjarr.2020.6.3.0162 Abstract The present study aimed to investigate the antidiabetic and anti-inflammatory effects of Trifolium alexandrinum (TA) and Trifolium Pretense (TP) against streptozotocin induced diabetic impairment in male rats. Forty adult Wistar albino male rats were divided into four groups: Group 1, Control; Group 2, streptozotocin (STZ); Group 3, STZ + TA –treated group and Group 4, STZ + TP–treated group. TA and TP showed a significant reduction in the blood glucose, glycosylated hemoglobin (Hb A1C) levels and elevation in insulin and C-peptide. They restored the glycogen content of liver and skeletal muscle, also showed improvements in the activities of α-amylase, carbohydrate metabolic enzymes, lipase and lipid profile. Treatment with TA and TP extracts resulted in an improvement of the oxidative parameters and enhancement in the antioxidant enzymes in the pancreas and liver. While, pancreatic TNF-α, IL-1β, IL-6, NO, iNos and caspase-3 levels were significantly reduced with the treatment of TA and TP extracts. Administration of TA and TP extracts to diabetic rats revealed elevation in the GLUT4 levels of the skeletal muscle and pancreas. They caused up regulation in pancreatic GLUT2 gene expression. TA and TP extracts showed signs of regeneration of β-cells and improvement in the pancreatic tissue. It could be concluded that TA and TP extracts possess antioxidant and hypoglycemic potential resulting in reduction of the elevated oxidative stress and pro-inflammatory cytokines. Keywords: Trifolium alexandrinum; Trifolium pratense; Diabetes; Carbohydrate metabolic enzymes; Pro- inflammatory cytokines 1. Introduction Diabetes mellitus (DM) is a chronic metabolic disease that is related to a variety of genetic and environmental factors [1]. DM is a heterogeneous metabolic disorder resulting from defective insulin secretion, resistance to insulin action or both leading to prolonged hyperglycemia with disarrangement carbohydrate, lipid and protein metabolism [2]. Diabetes mellitus type 2 (T2DM; non-insulin-dependent diabetes or adult-onset diabetes), is an endocrine disorder characterized by high blood sugar causing insulin resistance (IR) and relative insulin deficiency [3]. Hyperglycemia is responsible for the formation of free radicals, autooxidation of glucose and lipid peroxidation as well as disturbance of the antioxidant defense system [2]. According to the ADA, the chronic hyperglycemia is associated with long-term damage, dysfunction and failure of different organs, especially the eyes, kidneys, nerves, heart and blood vessels [4]. In spite of the availability of various antidiabetic agents and its secondary complications continue to become a major problem in the world population, medicinal plants and their bioactive compound are used as an alternative method to treat the diabetic patient throughout the world and popular as nutraceutical [5]. Corresponding author: Abdou Heba Mohamed Copyright © 2020 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Abdou et al. / World Journal of Advanced Research and Reviews, 2020, 06(03), 012–029 The health properties of Trifolium sp., Shows antiseptic, analgesic, antioxidant and anti-inflammatory properties [6]. The biological activity of Trifolium sp., due to the presence of interesting natural bioactive compounds, such as flavonoid glycosides, clovamides and polyphenols [7]. TA and TP possess great amounts of phytoestrogens [8]. These plants are used to treat and relieve symptoms in postmenopausal women [9]. Estrogen regulates several metabolic processes, including glucose and lipid metabolism, body weight, adipose tissue distribution, caloric intake and energy expenditure in both men and women [10]. The present study was carried out to investigate the efficiency of the two Fabaceae extracts; TA and TP in reducing the hyperglycemia, diabetic impairment and oxidative stress in the STZ induced diabetic rats and to assess their therapeutic potential for treatment of diabetes mellitus. 2. Material and methods 2.1. Chemicals Streptozotocin (STZ) (2-Deoxy-2-(3-methyl-3-nitrosourea) -1-D-glucopyranose) is a synthetic nitrosoureido glucopyranose, pale yellow, sterilized powder. It was purchased from Sigma-Aldrich Chemical Co (www.sigmaldrich.com) St. Louis, MO, USA. Trifolium pratense (TP) Linn. (Family: Fabaceae) (common name Red clover) was purchased from Nature’s Way, Inc. Springville, Utah 84663 (USA) and then the powdered material in the capsules were soaked in methanol then filtered and lyophilized. Kits were purchased from Biodiagnostic and research reagents Co. (Cairo, Egypt). All reagents and chemicals used were of analytical grade, other chemicals were of the highest purity commercially available. 2.2. Preparation of methanolic extracts of Trifolium alexandrinum Aerial parts (leaves, stem and flowers) of the plant, Trifolium alexandrinum (TA) Linn. (Family: Fabaceae) (common name: Egyptian clover, Berseem clover) were collected during the vegetative and flowering growth stage from different localities of Alexandria, Egypt by Safaa Abd Elsalam Mohamed, lecturer of plant taxonomy and palynology, Biological and Geological Sciences Department, Faculty of Education, Alexandria University. The plant was collected from fields in the morning and immediately taken to the laboratory and it was taxonomically identified, according to its morphological characters [11]. The plant extract was done according to Khan et al [12]. The aerial parts of it were collected, washed with tap water, then with distilled water, dried under shade. The chopped plant was grinded into fine powder using an electric grinder to obtain a fine powder. The powdered material was soaked in 80% methanol for a period of 1 week at room temperature and then filtered using a Buchner funnel and Whatman No.1 filter paper. The filtrate was lyophilized for dryness using a Freeze-dry system/Lyph lock 4.5 (Labconco) at the Environmental Sciences Department, Faculty of Science, Alexandria University. After lyophilization, the resulting the dried extracts of TA plant. The TA crude extracts were kept in a sterile labeled container and refrigerated at 4 °C for further assessment. 2.3. Induction of diabetes Thirty adult Wistar albino male rats were injected intraperitoneally (i.p) by a single dose of streptozotocin (STZ) at a dose level of 40 mg/kg b.w. that freshly dissolved in cold sodium citrate buffer (0.1 M/L; pH 4.5) [13]. Three days after the STZ injection, the blood was withdrawn from the tail vein and the glucose level was determined. Rats were diabetic when their fasting blood glucose levels were more than 200mg/dL. 2.4. Experimental animals and groups In the present study, forty adult male albino rats weighing 180–200 g, 11–12 weeks old were used. Rats were obtained from the animal Care Unit, Department of Home Economics, Faculty of Agriculture, Alexandria University, Egypt. Rats were housed in stainless steel wire cages placed in a well-ventilated animal house kept on basal diet and tap water ad libitum and maintained under constant laboratory conditions of (temperature: 22±3 °C, photoperiod: 12/12-h light/dark cycle). The local committee approved the design of the experiments and protocols were carried out according to the guidelines of the National Institutes of Health (NIH). After induction of diabetes, the diabetic rats were randomly allocated to 3 groups (10 rats each): 1. (Diabetic group); STZ-treated group; 2. (Diabetic+TA); Diabetic+Trifolium alexandrinum -treated group): diabetic rats treated orally by 13 Abdou et al. / World Journal of Advanced Research and Reviews, 2020, 06(03), 012–029 gavage with TA methanolic at a dose of 200 mg/kg B.W. [12] once a day for 15 consecutive days; 3. (Diabetic+TP); Diabetic+ Trifolium pratense -treated group): diabetic rats treated orally by gavage with TP methanolic extract at a dose of 250 mg/kg B.W. [14] once a day for 15 consecutive days. Ten rats received distilled water and were used as a control group. 2.5. Blood and tissue sampling 2.5.1. Blood collection At the end of the experiment, rats were fasted overnight and blood samples were collected from anesthetized rats of all groups by cardiac puncture using a sterile syringe for determination of biochemical analysis. Part of the blood samples were put immediately into silicon disposable glass tubes with ethylene diamine tetra-acetic acid (EDTA) (El-Gomhorya Co., Egypt) as an anticoagulant and placed immediately on ice for the determination of glycosylated hemoglobin (Hb A1c) concentration. The other part of collected blood sample from all experimental groups was allowed to coagulate in a sterilized centrifuge tube in frozen