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Background: Type 2 diabetes mellitus is a prevalent systemic disease affecting an important proportion of the population worldwide. It has been suggested that excessive reactive oxygen species generation and therefore development of an oxidative stress status is a key factor leading to diabetic complications. Accordingly, it seems that medicinal plants can offer a wide range of new antidiabetic drugs. Diplotaxis simplex (Viv.) Spreng. (Brassicaceae) is an edible plant largely distributed in the Mediterranean region. D. simplex flowers display important in vitro antioxidant potential and inhibitory activity of the α-glucosidase, a key enzyme linked to type 2 diabetes mellitus. In this paper, the antihyperglycemic potential of D. simplex flowers on diabetic rats were investigated. Methods: Bioactive substances were determined by liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS) analysis. Animals were divided into four groups of six rats each: a normal control group, a diabetic control group, a diabetic group receiving flowers extract (200 mg/kg body mass) and a diabetic group receiving acarbose (10 mg/kg body mass) as standard drug. Results: Many glycosides of rhamnetin, isorhamnetin, quercetin and kaempferol compounds were identified in the ethanolic flowers extract. Alloxan induced hyperglycemia, manifested by a significant (p < 0.05) increase in the blood glucose level as well as in serum α-amylase activity. Furthermore, diabetic rats exhibited oxidative stress, as evidenced by a decrease in antioxidant enzymes activities and an increase in lipid peroxidation level of the pancreas, liver and kidneys. Interestingly, the oral administration of D. simplex flowers extract during 30 days restored the glycemia, α-amylase activity, serum lipid profile and antioxidant enzymes. Moreover, the flowers extract exhibited a renal protective role by decreasing the urea and creatinine levels in diabetic rats serum. Conclusions: D. simplex flowers contained bioactive compounds that possess important antioxidant and hypoglycemic properties and protected pancreas, liver and kidneys against hyperglycemia damage. [ABSTRACT FROM AUTHOR]