In Vitro and In Vivo Antioxidant and Anti-Hyperglycemic Activities of Moroccan Oat Cultivars

Improvement of oat lines via introgression is an important process for food biochemical functionality. This work aims to evaluate the protective effect of phenolic compounds from hybrid Oat line (F11-5) and its parent (Amlal) on hyperglycemia-induced oxidative stress and to establish the possible mechanisms of antidiabetic activity by digestive enzyme inhibition. Eight phenolic acids were quantified in our samples including ferulic, p-hydroxybenzoic, caffeic, salicylic, syringic, sinapic, p-coumaric and chlorogenic acids. The Oat extract (2000 mg/kg) ameliorated the glucose tolerance, decreased Fasting Blood Glucose (FBG) and oxidative stress markers, including Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), Glutathione (GSH) and Malondialdehyde (MDA) in rat liver and kidney. Furthermore, Metformin and Oat intake prevented anxiety, hypercholesterolemia and atherosclerosis in diabetic rats. In vivo anti-hyperglycemic effect of Oat extracts has been confirmed by their inhibitory activities on α-amylase (723.91 μg/mL and 1027.14 μg/mL) and α-glucosidase (1548.12 μg/mL & 1803.52 μg/mL) enzymes by mean of a mixed inhibition.


Introduction
Oxidative stress (OS) has been implicated as a contributor to both the onset and progression of diabetes [1]. In physiologic concentrations, endogenous reactive oxygen species (ROS) help to maintain homeostasis. However, when ROS accumulate in excess for prolonged periods of time, they cause chronic

Antioxidant Effect in Tetrahymena pyriformis Cell Culture
T. pyriformis is a suitable experimental organism for wide spectrum of functional, pharmacological studies allowing the use of indices common in animal studies, such as growth arrest and metabolic inhibition, kinetics and synthesis of specific molecules or enzymes. In this assay T. pyriformis was used as a cellular model to follow the protective effect of natural products against Hydrogen peroxide (H 2 O 2 )-induced OS. The ciliated protozoa T. pyriformis was grown axenically, without shaking, in the Proteose-peptone yeast Glucose defined medium (PPYG), as described by Mori [21]. The cells were incubated at 28 • C in capped 500 mL Fernbach flasks containing 100 mL of PPYG medium. For the bioassays, the T. pyriformis cultures were always in exponential growth phase, and they were adjusted to a density of 10 4 cells/mL in fresh PPYG medium just before treatment with the tested chemicals. Protective antioxidant effect has been evaluated on T. pyriformis under H 2 O 2 treatments in the presence of Oats extracts (controls were performed without H 2 O 2 ). For this purpose, the 50% inhibitory concentration values (IC 50 ) of tested substances (H 2 O 2 , Am and F11) were determined previously on T. pyriformis by cell counting. Treatments solutions were prepared in deionized water, just before testing; pH was checked, and readjusted to 6.5 if necessary. The IC 10 of Oat extracts has been used for protective effects of Oat extracts simultaneously with H 2 O 2 cytotoxicity under 75 µM doses. To evaluate cell viability, aliquots of 1 mL were taken from untreated and treated T. pyriformis. The samples were diluted in distilled water, and fixed with neutral buffered formalin containing 10% (v/v) formalin in Isoton buffer for 1 h. The cell number was determined with a hemocytometer under an optical microscope. To determine cell viability, a counting without fixation was realized in order to identify immobile and mobile cells (dead and live cells, respectively). The MTT assay [22] using the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to evaluate the effects of treatments on cell proliferation and/or mitochondrial activity. Four hours later the MTT salts were reduced to formazan blue in the metabolic active cells by mitochondrial enzyme succinate dehydrogenase to form NADH (Nicotinamide Adenine Dinucleotide Hydrogen) and NADPH (Nicotinamide Adenine Dinucleotide Phosphate). Absorbance was read at 570 nm. Oat antioxidant protective effect has been evaluated by measuring the protein content and two antioxidant enzymes: total superoxide dismutase (T-SOD) and Catalase (CAT) in the T. pyriformis homogenate [23][24][25].

Digestive Enzymes Inhibition and Kinetics
The α-amylase inhibition assay was conducted according to Kee [26] work, with slight modifications. Briefly, 250 µL of sample was mixed with 250 µL of α-amylase (240 U/mL, in 0.02 M phosphate buffer solution (PBS), pH 6.9, with 0.006 M NaCl). After incubating at 37 • C for 10 min, 250 µL of 1% (w/v) soluble starch (in 0.02 M PBS, pH 6.9) was added and the mixture was further incubated at 37 • C for 30 min, followed by adding 250 µL of dinitrosalicylic acid color reagent (DNS 96 mM, 30% Na-K tartrate, 0.4 M NaOH), and stopped by heating in a boiling water bath for 10 min. After cooling to room temperature, the mixture has been diluted with 2 mL of PBS and the absorbance was measured at 540 nm.
The α-glucosidase enzyme (0.1 U/mL) and substrate p-Nitrophenyl-α-D-glucopyranoside (p-NPG, 1 mM) were dissolved in PBS (0.1 M, pH 6.7), and all samples were dissolved in distilled water. The inhibitor (150 µL) was pre-incubated with the enzyme (100 µL) at 37 • C for 10 min, and then the substrate (200 µL) was added to the reaction mixture. The enzymatic reaction was performed at 37 • C for 30 min. The reaction was then terminated by the addition of Na 2 CO 3 (1 M, 1 mL). All samples were analyzed in triplicate with different concentrations to determine the IC 50 values, and the absorbance was recorded at 405 nm. The inhibition percentage (%) was calculated by the following equation for both assays: Absorbances are abbreviated as follows: AC (control), AC b (control blank), AS (sample) and AS (sample blank).
The mode of inhibition of α-amylase and α-glucosidase was investigated with increasing concentrations of the substrate (Starch and p-NPG) in the presence of different concentrations of extracts. Then, the type of inhibition was determined by Lineweaver-Burk plot analysis of the data, calculated from the results according to Michaelis-Menten kinetics.

Animals
Wistar rats weighing (250-300 g) and Swiss albino mice (20-25 g), are bred in the central animal facility of the Faculty of Medicine and Pharmacy of Rabat, Morocco. Animals were kept in cages under standard laboratory conditions with tap water and standard diet ad libitum, in a 12 h light/12 h dark cycle at a temperature of 21 to 23 • C.
Ethics approval was also obtained from Mohammed V University in Rabat, under the responsibility of the Central Animal Facility and the Laboratory of Pharmacology and Toxicology at the Faculty of Medicine and Pharmacy of Rabat (01DEC2015). The experiments were conducted in accordance with the accepted principles outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health and all efforts were made to minimize animal suffering and the number of animals used.

Acute Toxicity
Acute oral toxicity study was performed as per 425 guidelines (OECD) from the Organization of Economic Co-operation and Development. Four groups of male Swiss albino mice (n = 6), each one selected by random sampling technique, were used for acute toxicity study. The animals were kept fasting overnight providing them only with water. Subsequent to the administration of extracts (2000 mg/kg), the animals were observed closely for the first 3 h in order to detect any toxic manifestations such as increased locomotors activity, salivation, clonic convulsion, coma and death.
Subsequent observations were made at regular intervals for 24 h. The animals were observed for a further two weeks.

Experimental Diabetes
Overnight fasted rats (OFR) were treated with nicotinamide (110 mg/kg, i.p.). Streptozotocin (65 mg/kg, i.p.) was injected 15 min after nicotinamide administration, in all groups except for normal control [27]. Animals were fed with glucose solution (5%) for 12 h to avoid hypoglycemia. Hyperglycemia was confirmed three days later, and the steady state of hyperglycemia was reached after 10 days. Serum glucose was determined by the glucose oxidase peroxidase method using a glucometer (One Touch Ultra, LifeScan, Milpitas, CA, USA). Animals having serum glucose between 200-300 mg/dL were selected for the study.

Experimental Design
After establishment of the diabetic rats model, animals were divided into five treatment groups: Normal control (NC; 1 mL DW/200 g), Diabetic control (DC; 1 mL DW/200 g), Oat treatment (Am and F11; 2000 mg/kg) and Metformin (Met; 300 mg/kg). The dose was selected based on the results of our preliminary oral glucose tolerance tests (OGTTs) assays, in which we found that the effect of the 2000 mg/kg treatment was significantly higher than 500 mg/kg (p < 0.05) (data not shown). The animals received the respective treatment for 42 days (6 weeks). Blood was collected at the end of the experiment for hematological and biochemical analysis. Liver and kidney homogenate were analyzed for oxidative stress markers. Pancreas has been used for the histological characterization. OGTTs were performed on day 1 and 40 from the beginning of the experiment. During the study period of 42 days, the rats were weighed daily using electronic balance, and glycaemia levels were recorded weekly. Food and water intake, and urinary volume were determined for the first and last day before OGTT using metabolic cages.

Oral Glucose Tolerance Test
OFR were administered with glucose (2000 mg/kg) orally by means of gastric intubation. Animals in Am and F11 groups were administered orally with Oat extracts, at a dose of 2000 mg/kg, 30 min before the oral administration of glucose. Whereas animals in group Met were given metformin (300 mg/kg). The animals of control i.e. group NC and DC were given orally equal volume of water only. Blood samples were collected from the tail vein at 0, 30, 60, 90, 120 and 150 min. Total glycemic responses to OGTT were calculated from respective areas under curves (AUC) of glycaemia during the 150 min observation period. The ∆ variation of glycaemia was defined as the difference between glycaemia at t 0 and a following time point.

Behavioral Assays
The elevated plus maze (EPM) is an ethological model of anxiety in rodents. After 34 days of treatments, OFR have received their treatments at the fixed doses. Thirty minutes after administration, each rat was placed in the central square facing an open arm and allowed to freely explore the maze for 5 min. The following measurements were recorded; arm entries, total time spent in open arms and total number of entries in open arms [28]. Two days later (day 36), the OFR have received their treatments at the fixed doses, and thirty minutes later, the animals' spontaneous activity was evaluated in an open field test (OFT). In individual tests, the rats were placed at the same point and allowed to freely explore the apparatus for 10 min. The following measurements were then recorded; total squares entries, central squares entries and time spent in central squares [29].

Key Enzymes and Markers of Oxidative Stress
After sacrifice, liver and kidney were dissected out, and a 10% of organs homogenate was prepared in ice-cold PBS 50 mM using Teflon glass homogenizer. The homogenate was centrifuged at 3500 rpm for 10 min (at 4 • C) using cooling centrifuge (Mikro 220R, Hettick Lab Technology, Tuttlingen, Germany). The pellet was discarded and supernatant obtained was used for the estimation of antioxidant enzymes, oxidative stress markers and protein content in the homogenate.
The protein content was estimated following the method of Lowry [23]. The antioxidant enzymes were expressed as µmol/min/mg protein. Catalase (CAT) activity was determined according to the method of Aebi [30]. T-SOD activity was determined by the method of Beauchamp and Fridovich [22]. The MnSOD activity was measured after addition of 2 mM KCN in the solution. The CuZn-SOD activity was calculated by subtraction as follows: Activities of Glutathione peroxidase (GPx) were estimated according to the method of Mannervik [25]. Glutathione (GSH) was estimated by a previously described method [31], and expressed as µmol GSH/mg protein using GSH standard. Thiobarbituric acid reactive substances were estimated as malondialdehyde (MDA) equivalent (nmol MDA/mg protein), from the calibration curve [32].

Histological Study of Pancreas
The pancreas from each rat was fixed in 10% buffered formalin and processed via classical histology method using the paraffin wax embedding techniques (dehydration, clearing and embedding). The paraffin embedded-sections were cut at 4 µm thickness using microtome (Shandon Hypercut, Runcorn, UK), and then stained by hematoxylin and eosin staining method. Stained sections of pancreas were qualitatively (morphological) analyzed on microscope (Leica Microsystems DM2500, Wetzlar, Germany), and photomicrographs of histological alterations have been taken at ×400 magnification.

Statistical Analysis
Data were expressed as the mean values ± standard deviation (SD) for each measurement. The data were also analyzed by one-way analysis of variance (one-way ANOVA). Post Hoc procedure was used for significance of difference (p < 0.05). Analysis was performed with Graph pad prism 6.0.

Chemical Analysis
Chemical analysis of Oat varieties ( Data are reported to mean (n = 2) ± SD. Values in the same raw not sharing a common letter ( a,b ) differs significantly at p < 0.05.

Antioxidant Effect in T. pyriformis
Different concentrations of Oat extracts and H 2 O 2 were screened to determine their cytotoxicity (IC 50 ) on T. pyriformis. The determined IC 50 of H 2 O 2 (0.75 mM) was used to establish the oxidative stress environment and the IC 10 of Oat varieties (1 µg/mL) were used for antioxidant treatment in comparison with vitamin C at the same dose ( Figure 1).  Data are reported to mean (n = 2) ± SD. Values in the same raw not sharing a common letter ( a,b ) differs significantly at p < 0.05.

Antioxidant Effect in T. pyriformis
Different concentrations of Oat extracts and H2O2 were screened to determine their cytotoxicity (IC50) on T. pyriformis. The determined IC50 of H2O2 (0.75 mM) was used to establish the oxidative stress environment and the IC10 of Oat varieties (1 μg/mL) were used for antioxidant treatment in comparison with vitamin C at the same dose ( Figure 1). H2O2-induced stress reduced T. pyriformis viability by 48.49% compared to the control group. Cell viability in treated groups was improved by 20.15% in Am, 30.60% in F11 and 57.47% in Vc compared to H2O2 group. Antioxidant enzymes, including T-SOD and CAT levels increased significantly by 38.92% and 36.04% in H2O2 compared to the non-treated control (p < 0.05). However Oat treatment reduced the level of the expression of those enzymes. T-SOD release was reduced by  Cell viability in treated groups was improved by 20.15% in Am, 30.60% in F11 and 57.47% in Vc compared to H 2 O 2 group. Antioxidant enzymes, including T-SOD and CAT levels increased significantly by 38.92% and 36.04% in H 2 O 2 compared to the non-treated control (p < 0.05). However Oat treatment reduced the level of the expression of those enzymes. T-SOD release was reduced by −20.16% in Am, −17.80% in F11, and −37.77% in the positive control (Vc). Similarly, the treatments decreased CAT levels by −20.14% in Am, −15.80% in F11 and −23.77% in Vc.

In Vitro Inhibition of Digestive Enzymes
In an array to explore the in vitro antidiabetic activity, Moroccan Oat extracts were screened for the α-amylase and α-glucosidase inhibitory properties. Effects were compared with the commercially available α-glucosidase inhibitor, acarbose (Ac).
Inhibitory activities of the extracts were evaluated at different concentrations and results were given in Figure 2. Acarbose and extracts showed a dose dependent inhibitory effect on enzymes. The IC 50 values for anti-amylase and anti-glucosidase, activity of Am and F11 were lower than that of acarbose, a very well known drug with well-established activity ( Figure 2). IC 50 for amylase inhibition were 723.91 µg/mL and 1027.14 µg/mL for Am and F11 respectively. While, acarbose inhibitory concentration was 396.42 µg/mL. α-glucosidase inhibitory effect of Oat extracts was much more lower than its equivalent of acarbose. In term of IC 50 , significant differences have been registered between Oat varieties and acarbose: Am (1548.12 µg/mL), F11 (1803.52 µg/mL) and Ac (199.53 µg/mL).
A Lineweaver-Burk double reciprocal plot of enzymes activity in the presence of Oat extracts as inhibitor was plotted. Km and Vmax values increased with increasing Oat concentrations. The kinetic study ( Figure 2) suggests that Oat extracts metabolites intersected in the second quadrant indicating a mixed-type of inhibition in both enzymes (α-amylase and α-glucosidase).

In Vitro Inhibition of Digestive Enzymes
In an array to explore the in vitro antidiabetic activity, Moroccan Oat extracts were screened for the α-amylase and α-glucosidase inhibitory properties. Effects were compared with the commercially available α-glucosidase inhibitor, acarbose (Ac).
Inhibitory activities of the extracts were evaluated at different concentrations and results were given in Figure 2. Acarbose and extracts showed a dose dependent inhibitory effect on enzymes. The IC50 values for anti-amylase and anti-glucosidase, activity of Am and F11 were lower than that of acarbose, a very well known drug with well-established activity ( Figure 2). IC50 for amylase inhibition were 723.91 μg/mL and 1027.14 μg/mL for Am and F11 respectively. While, acarbose inhibitory concentration was 396.42 μg/mL. α-glucosidase inhibitory effect of Oat extracts was much more lower than its equivalent of acarbose. In term of IC50, significant differences have been registered between Oat varieties and acarbose: Am (1548.12 μg/mL), F11 (1803.52 μg/mL) and Ac (199.53 μg/mL).
A Lineweaver-Burk double reciprocal plot of enzymes activity in the presence of Oat extracts as inhibitor was plotted. Km and Vmax values increased with increasing Oat concentrations. The kinetic study ( Figure 2) suggests that Oat extracts metabolites intersected in the second quadrant indicating a mixed-type of inhibition in both enzymes (α-amylase and α-glucosidase). Animals treated with Oat extracts did not show any change in their behavioral pattern during the acute toxicity study. There was no significant difference in the body weight and food

Acute Oral Toxicity and Metabolic Parameters
Animals treated with Oat extracts did not show any change in their behavioral pattern during the acute toxicity study. There was no significant difference in the body weight and food consumption when compared to the vehicle treated group. Also, no apparent pathological changes were seen. Thus, it was concluded that Oat extracts were safe at 2000 mg/kg. Table 2 shows the effect of daily administration of Oat extracts on body weight, urinary volume, and food and water intakes in diabetic rats. After six weeks of treatment, DC rats showed a considerably reduction of body weight by 50.91% compared to the initial value. The administration of Oat extracts (Am and F11) resulted in significant weight gain as compared to DC (40.47%, 30.07% and 39.01% for Met, Am and F11 respectively). In the other hand, food intake increased by 29.47% in DC, 11.99% in Met, 19.29% in Am and 26.58% in F11. Also, the elevated water intake and urinary volume (139.43-56.11%) in DC was significantly reduced to (−18.95%, −18.68%) in Met, (−45.96%, −39.33%) in Am and to (−62.57%, −39.20%) in F11. Compared to DC, urinary volume, food and water intake in treated groups has decreased, while body weight has increased significantly (p < 0.05). Data are reported to mean (n = 8) ± SD. Values in the same column and category not sharing a common letter ( a-c ) differs significantly at p < 0.05.

Glucose Tolerance and Anti-Hyperglycemic Effect
In order to evaluate the effectiveness of each treatment, OGTT was performed at the beginning and the end of the study. OGTT revealed that blood glucose reached its peak level at 30 min after the glucose load in all groups before and after treatments (Figure 3). The initial administration of Oat extracts (Am and F11) at 2000 mg/kg did not significantly reduce the AUC of blood glucose levels compared to the vehicle in DC. However, as expected Metformin show decrease (−52.48%) in glucose levels and stabilize glycaemia to normal values in diabetic rats, 90 min after administration. consumption when compared to the vehicle treated group. Also, no apparent pathological changes were seen. Thus, it was concluded that Oat extracts were safe at 2000 mg/kg. Table 2 shows the effect of daily administration of Oat extracts on body weight, urinary volume, and food and water intakes in diabetic rats. After six weeks of treatment, DC rats showed a considerably reduction of body weight by 50.91% compared to the initial value. The administration of Oat extracts (Am and F11) resulted in significant weight gain as compared to DC (40.47%, 30.07% and 39.01% for Met, Am and F11 respectively). In the other hand, food intake increased by 29.47% in DC, 11.99% in Met, 19.29% in Am and 26.58% in F11. Also, the elevated water intake and urinary volume (139.43-56.11%) in DC was significantly reduced to (−18.95%, −18.68%) in Met, (−45.96%, −39.33%) in Am and to (−62.57%, −39.20%) in F11. Compared to DC, urinary volume, food and water intake in treated groups has decreased, while body weight has increased significantly (p < 0.05). Data are reported to mean (n = 8) ± SD. Values in the same column and category not sharing a common letter ( a-c ) differs significantly at p < 0.05.

Glucose Tolerance and Anti-Hyperglycemic Effect
In order to evaluate the effectiveness of each treatment, OGTT was performed at the beginning and the end of the study. OGTT revealed that blood glucose reached its peak level at 30 min after the glucose load in all groups before and after treatments (Figure 3). The initial administration of Oat extracts (Am and F11) at 2000 mg/kg did not significantly reduce the AUC of blood glucose levels compared to the vehicle in DC. However, as expected Metformin show decrease (−52.48%) in glucose levels and stabilize glycaemia to normal values in diabetic rats, 90 min after administration.  Across the six weeks of treatment period, FBG has been shown to be decreased gradually in Am, F11, and Met groups ( Table 2). Glycemic levels in Oat-treated groups decreased significantly (−35.57% and −36.25%) compared to the dramatically increase (64.97%) in DC. Metformin has been shown to possess largely stronger effect (−60.28%), stabilizing FBG to its normal levels (Table 1).
At the end of treatment, the beneficial effects of Oat extracts on glucose metabolism of diabetic rats become evident. In the OGTT, after 30 min of the glucose overload, treated groups (Am, F11 and Met) were able to reduce glycaemia (Δ variation of glycaemia) more efficiently (65.33, 73.14 and 17.14 mg/dL) than DC (118 mg/dL). To the end of the challenge test, Oat extracts reduced blood glucose level significantly compared to DC. Hence, the glucose tolerance was significantly improved after Oat supplementation and metformin treatment.

Hematological and Biochemical Indices
In order to evaluate the protective effect of the above treatments on physiological functions, the hematological and biochemical parameters were analyzed ( Table 3). The total hemoglobin, red blood corpuscles count, neutrophils, lymphocytes, monocytes and eosinophils were not significantly different between the evaluated groups (Table 3). However, the white blood corpuscles count increased significantly in DC (p < 0.05). Moreover, the ALT and AST in Am, F11 and Met treated rats decreased significantly (p < 0.05) as compared to diabetic animals (Table 3). Nevertheless, total serum protein did not show any significant change in DC compared to NC. In the other hand LDH and urea in Oat and Met has shown decreased levels as compared to DC (p < 0.01). Serum creatinine did not show significant variations among groups. In the lipid profile analysis, the diabetic animals showed significant increase (p < 0.01) in the total cholesterol and TG; simultaneously a decrease in HDL compared to normal and treated rats. LDL levels did not show significant variations between DC and NC. Atherosclerosis index demonstrated significant differences in all groups (p < 0.05). Across the six weeks of treatment period, FBG has been shown to be decreased gradually in Am, F11, and Met groups ( Table 2). Glycemic levels in Oat-treated groups decreased significantly (−35.57% and −36.25%) compared to the dramatically increase (64.97%) in DC. Metformin has been shown to possess largely stronger effect (−60.28%), stabilizing FBG to its normal levels (Table 1).
At the end of treatment, the beneficial effects of Oat extracts on glucose metabolism of diabetic rats become evident. In the OGTT, after 30 min of the glucose overload, treated groups (Am, F11 and Met) were able to reduce glycaemia (∆ variation of glycaemia) more efficiently (65.33, 73.14 and 17.14 mg/dL) than DC (118 mg/dL). To the end of the challenge test, Oat extracts reduced blood glucose level significantly compared to DC. Hence, the glucose tolerance was significantly improved after Oat supplementation and metformin treatment.

Hematological and Biochemical Indices
In order to evaluate the protective effect of the above treatments on physiological functions, the hematological and biochemical parameters were analyzed ( Table 3). The total hemoglobin, red blood corpuscles count, neutrophils, lymphocytes, monocytes and eosinophils were not significantly different between the evaluated groups (Table 3). However, the white blood corpuscles count increased significantly in DC (p < 0.05). Moreover, the ALT and AST in Am, F11 and Met treated rats decreased significantly (p < 0.05) as compared to diabetic animals (Table 3). Nevertheless, total serum protein did not show any significant change in DC compared to NC. In the other hand LDH and urea in Oat and Met has shown decreased levels as compared to DC (p < 0.01). Serum creatinine did not show significant variations among groups. In the lipid profile analysis, the diabetic animals showed significant increase (p < 0.01) in the total cholesterol and TG; simultaneously a decrease in HDL compared to normal and treated rats. LDL levels did not show significant variations between DC and NC. Atherosclerosis index demonstrated significant differences in all groups (p < 0.05). Data are reported to mean (n = 8) ± SD. Values in the same row and category not sharing a common letter ( a-d ) differs significantly at p < 0.05.

Oxidative Stress Markers and Antioxidants Enzymes
By the end of the treatment period, oxidative stress markers (MDA and GSH) and antioxidant enzymes (T-SOD, Mn-SOD, CuZn-SOD, CAT and GPx) were analyzed and results are presented in Table 4. Lipid peroxidation products as expressed in MDA equivalents in diabetic non-treated animals were much higher than those found in NC and treated rats. MDA levels at the liver was significantly higher compared to NC (p < 0.001), while treatment registered a lesser increase in Met (30.95%), Am (154.76%) and F11 (407.14%). Likewise, at the kidney level, similar increase in MDA has been registered (405.49%). Treatments of diabetic groups decreased the MDA production in Met (−274.72%), Am (−378.01%) and F11 (−256.58%) compared to DC. The T-SOD, Mn-SOD and CuZn-SOD levels in DC have increased significantly compared to NC and treated groups (p < 0.05). T-SOD increased by 124.51% in DC compared to NC at the liver, while in the kidney the increase was less marked by 52.16%. Am and Met treatments decreased T-SOD and similarly Mn-SOD and CuZn-SOD in both organs (p < 0.05). Also, compared to NC, diabetic animals have shown a significant increase (p < 0.05) of CAT by 218.68% in the liver and 50.05% in the kidney ( Table 4). The treated groups exhibited a significant decrease (p < 0.05) in CAT levels except for the F11 group. Oat extract and metformin induced a reduction of CAT peaks in treated diabetic animals (−194.63%, −170.41% and −63.66% for Met, Am and F11 respectively) in the liver and similarly in the kidney. Moreover, DC rats were also characterized by an increase in GSH by 85.59% in the liver. Administration of the treatments induced a significant (p < 0.05) decrease of GSH in the liver for Met (−37.28%), Am (−55.08%) and F11 (−42.36%), compared to DC. The kidney levels of GSH followed the same tendency. GPx Levels in the other hand increased significantly in liver of the diabetic controls (123.60%). Treated groups (Am and Met) show decreased values compared to diabetic control at the liver level, except for F11 group who did not show significant variations in comparison with DC. GPx levels in the kidney did not show significant differences. Data are reported to mean (n = 3) ± SD. Values in the same row and category not sharing a common letter ( a-e ) differs significantly at p < 0.05. CAT, Mn-SOD, CuZn-SOD and GPx are expressed in in µmol/min/mg protein, GSH in µmol GSH/mg protein, MDA in nmol MDA/mg protein.

Behavioral Analysis
As depicted in Table 5   Data are reported to mean (n = 8) ± SD. Values in the same column and category not sharing a common letter ( a-d ) differs significantly at p < 0.05.

Pancreas Histopathology
The pathologic features of pancreas tissues in each group are shown in Figure 4. Normal control showed normal pancreatic parenchyma cells and islet cells (round or oval-shaped cell mass) whereas DC showed focal necrosis and dilated acini. Metformin and Oat-treated groups showed minimal pathological changes.

In Vitro Antidiabetic Activity
This study reports the inhibitory kinetics of Oat aqueous extracts against key enzymes linked to hyperglycemia. The in vitro assay demonstrated a moderate inhibitory effect compared to the positive control. Regarding the safe profile of Oat extract, it could be of interest to consider Oat formulation for inhibition of digestive enzymes in the small intestine. Oat mixture can display an antidiabetic effect if used as a capsulated complement. The presence of numerous phenolic compounds among the extract mixture e.g., p-Hydroxybenzoic, Syringic and Caffeic acids, resulted in a mixed type of inhibition. Previously, the association of several bioactive compounds revealed to be much more effective and safe in therapeutic strategies [33].
In fact, the heterogeneous character of type 2 diabetes results from the dynamic interaction between defects in insulin secretion and insulin action. Such a deficiency results in increased concentrations of blood glucose, which in turn damages many of the body's systems [34]. Therefore, there is a need to control postprandial blood glucose for diabetes management. One of the strategies in glucose control is the inhibition of digestive enzymes [17], such as α-glucosidase and α-amylase, which results in a significant reduction of the post-prandial blood glucose conferring an important target for diabetes management [35]. Although the wide availability of glucosidase inhibitors, many works sought natural sources in the hope to present nutritional alternatives with minimal side effects and low therapy costs [36]. Actually, a pseudo-tetrasaccharide of microbial origin namely acarbose, inhibits the brush-border enzymes glucoamylase, dextrinase, maltase and sucrase as well as the pancreatic α-amylase [17]. Additionally, other digestive enzymes inhibitors [37] are available (miglitol, emiglitate voglibose). Despite the effectiveness of acarbose as antidiabetic drug with glucosidase inhibitory properties, food alternatives are indeed needed because of acarbose side effects such as diarrhea and flatulence that occurs when colonic bacteria ferment the undigested carbohydrates, resulting in gas formation [38]. Also uncommon hepatotoxicity cases have been registered following long-term intake [39]. Currently, there is renewed interest in nutritional therapies and functional foods with preventive effects on diabetes and obesity [40]. Few papers described the Oat glucosidase and amylase inhibitory activities [16,17]. Nevertheless, none of the previous studies has determined the inhibitory concentrations and the kinetic of inhibition. Also the reported works focused on whole oatmeal and did not especially study the aqueous phenolic rich

In Vitro Antidiabetic Activity
This study reports the inhibitory kinetics of Oat aqueous extracts against key enzymes linked to hyperglycemia. The in vitro assay demonstrated a moderate inhibitory effect compared to the positive control. Regarding the safe profile of Oat extract, it could be of interest to consider Oat formulation for inhibition of digestive enzymes in the small intestine. Oat mixture can display an antidiabetic effect if used as a capsulated complement. The presence of numerous phenolic compounds among the extract mixture e.g., p-Hydroxybenzoic, Syringic and Caffeic acids, resulted in a mixed type of inhibition. Previously, the association of several bioactive compounds revealed to be much more effective and safe in therapeutic strategies [33].
In fact, the heterogeneous character of type 2 diabetes results from the dynamic interaction between defects in insulin secretion and insulin action. Such a deficiency results in increased concentrations of blood glucose, which in turn damages many of the body's systems [34]. Therefore, there is a need to control postprandial blood glucose for diabetes management. One of the strategies in glucose control is the inhibition of digestive enzymes [17], such as α-glucosidase and α-amylase, which results in a significant reduction of the post-prandial blood glucose conferring an important target for diabetes management [35]. Although the wide availability of glucosidase inhibitors, many works sought natural sources in the hope to present nutritional alternatives with minimal side effects and low therapy costs [36]. Actually, a pseudo-tetrasaccharide of microbial origin namely acarbose, inhibits the brush-border enzymes glucoamylase, dextrinase, maltase and sucrase as well as the pancreatic α-amylase [17]. Additionally, other digestive enzymes inhibitors [37] are available (miglitol, emiglitate voglibose). Despite the effectiveness of acarbose as antidiabetic drug with glucosidase inhibitory properties, food alternatives are indeed needed because of acarbose side effects such as diarrhea and flatulence that occurs when colonic bacteria ferment the undigested carbohydrates, resulting in gas formation [38]. Also uncommon hepatotoxicity cases have been registered following long-term intake [39]. Currently, there is renewed interest in nutritional therapies and functional foods with preventive effects on diabetes and obesity [40]. Few papers described the Oat glucosidase and amylase inhibitory activities [16,17]. Nevertheless, none of the previous studies has determined the inhibitory concentrations and the kinetic of inhibition. Also the reported works focused on whole oatmeal and did not especially study the aqueous phenolic rich extract. Oat anti-enzymatic activity is probably linked in part to its phenolic content. As we have adopted a complementary nutritional therapy with multi-target bioactivities (antioxidant and antidiabetic), the safe Oat phenolic extracts have been chosen for in vivo and in vitro activities. Inhibitory effect of hybrid and parent Oat varieties was lower than acarbose, especially for glucosidase inhibition. As a complex mixture, Oat extracts exhibited a mixed inhibition, which is an intermediate mode between the competitive and uncompetitive inhibition [41]. The extracts metabolites were able to bind either to the free enzyme or the enzyme-substrate complex. Phenolic compounds were reported to exhibit a glucosidase inhibitory effect. Especially cereal grains, such as Barley [42], Wheat [43] and Quinoa [44], were reported to inhibit intestinal glucosidase.

Oxidative Damage Prevention and Anti-Hyperglycemic Effect
Recently, we have reported the nutritional characteristics and biochemical composition of Moroccan Oat varieties [10]. This assay is an attempt to investigate the in vivo antidiabetic and antioxidant properties of the Moroccan Oat variety (Amlal) and its derivative new line (F11-5).
Oat plays a role in modulating the metabolic effects observed after fiber-rich meals. As a soluble fiber with viscous characteristics, Oat β-glucans modifies properties of chyme in the upper part of the gastrointestinal tract affecting gastric emptying, gut motility, and nutrient absorption, which are reflected in lower postprandial glycemic and insulin responses [45]. Thus, oat β-glucan intake is beneficial for healthy subjects and patients with type-2 diabetes [46]. Manifestly, many papers described the Oat antidiabetic action linked to its fiber content and viscosity [47]. Moreover, Oat antioxidant effect is well described in literature using in vitro methods [10]. However few papers discussed the preventive effect of Oat extracts on hyperglycemia-induced OS. In our multi-target Oat-therapy approach, we tried to focus on both diabetes and oxidative stress. In this regard we have decided to use the aqueous phenolic extracts instead of other Oat extracts. The in vivo antioxidant and antidiabetic activities were evaluated in male wistar rats, following acute toxicity study in female Swiss mice. The use of female in toxicity studies is mainly due to their higher sensitivity. According to the OECD 425, the preferred rodent species is the rat, although other rodent species may be used. On the other hand, for diabetes models, several animal species, including the mouse and rat are sensitive to the pancreatic β-cell cytotoxic effects of Streptozotocin (STZ). However, some mice strains are less sensitive to this toxin.
In summary, the results from in vivo study of Oat extract intake, revealed an amelioration of glucose tolerance, a decrease in FBG and an antioxidant effect based on oxidative stress markers expression compared to diabetic stressed control. Furthermore, Oat intake prevented anxiety like behavior, hypercholesterolemia and atherosclerosis in diabetic rats.
Animal models of diabetes are an important tool for in vivo screening of antidiabetic compounds. The fungal compound streptozotocin is a hydrophilic nitrosourea analogue with antibiotic and chemotherapeutic activities. Owing to its glucose like similar structure, it enters β-cells via GLUT2 (Glucose transporter 2) transporters in a similar way to glucose [48], resulting in DNA alkylation, nitric oxide release and ROS generation leading to insulin reduced synthesis and diabetogenic state. In the STZ-NA (Streptozotocin-Nicotinamide) model of diabetes the use of NA a poly-ADP-ribose (Poly-Adenosine diphosphate-ribose) synthetase inhibitor, is mainly for its protective effect on β-cells function via the prevention of reduction in the level of nicotinamide adenine dinucleotide; thereby it results in a reverses of the insulin secretion inhibition lowering the degenerescence in the experimental model following the STZ administration, which show similar characteristics to T2DM (Type 2 Diabetes Mellitus) [49].
Actually, we have demonstrated that Oat extracts at 2000 mg/kg reduced significantly the FBG in STZ-NA-induced diabetic rats after 6 weeks of treatment. The OGTT performed at the beginning and the end of the treatment period show significant improvement in impaired glucose tolerance. Body weight loss in diabetic animals has been attributed to the waste in muscle mass [50]. Oat treatment and anti-hyperglycemic effect resulted in a decrease of body weight loss frequency. In the other hand, the increase in urinary volume and water and food intake in diabetic animals is comparable to polyphagia and polydipsia observed in diabetic patients [51]. Oat treatment prevented those manifestations and decreased diabetic symptoms impact. The decreased serum glucose levels after treatment period can probably be linked to Oat ability to inhibit digestive enzymes at the intestinal level. Based upon histopathological results it can be also hypothesized that Oat may probably exhibit protective effect on pancreatic β-cells against STZ toxic effects. Manifestly, β-cells number and size in treated animals support this hypothesis. Furthermore, diabetes complications such as hypercholesteremia and hypertriacylglycerolemia are primary factors involved in the development of atherosclerosis and coronary heart disease [52]. Oat treatment significantly reduced serum TG and total cholesterol in STZ diabetic rats. Consequently, it is suggested that Oat intake and Met treatment could modulate blood lipid abnormalities and reduce atherosclerosis risk. As a vital organ the liver metabolizes nutrients and detoxifies harmful substances. Accordingly, ALT and AST are reliable markers of liver function [53]. In the STZ-NA model of diabetes the increase in ALT and AST activities in plasma indicates the STZ hepatotoxic effect and the liver necrosis [54]. Am and F11 treatments reduced these enzymes levels in plasma compared to the DC and consequently prevented the liver damage; which indicates their hepatoprotective effect. The positive control group has been treated by metformin, which is a guanidine-containing compound currently recommended as first line therapy for all newly diagnosed T2DM patients [51]. Metformin decrease glucose production in the liver via the suppression of gluconeogenesis. Moreover, enhance the insulin suppression of endogenous glucose production and, to a lesser extent, reduce the intestinal glucose absorption and probably improve the glucose uptake and utilization by peripheral tissues [55]. Indeed, there is a growing evidence that hyperglycemia causes OS in a variety of tissues through ROS production [56]. At the same time, free radicals and ROS are implicated in the physiological gluco-regulatory systems. For instance, insulin release that is stimulated by glucose production in β-cells has been linked to H 2 O 2 regulatory action [57]. Recent mathematical modeling described the OS dynamics to be of oscillatory nature [58] and tissue depending [59], which may explain the inconsistency of experimental data related to antioxidant enzymes measurements. Alternatively, focusing on stable OS markers such as MDA may reflect more reliable information on OS damage under long-term dynamics. Correspondingly, antioxidant enzymes and thiols form the first line of defense against ROS in the cells. In fact, SOD activity enhances the spontaneous dismutation of superoxide radicals to H 2 O 2, before it is removed by CAT activity. In contrast to CAT, the GPx act by reducing lipid hydroperoxides to the corresponding alcohols and reduce free H 2 O 2 to H 2 O even at low concentrations. Moreover, the major endogenous antioxidant GSH directly neutralizes the free radicals and ROS, and maintains the exogenous antioxidants such as vitamins C and E in their reduced forms [60]. The increase in antioxidant enzymes activity in various tissues could reflect an adaptation to diabetes-induced OS. Our results showed that the level of MDA and GSH and the activities of T-SOD, Mn-SOD, CuZn-SOD, and CAT in the liver and kidney of diabetic rats significantly increased when compared with the normal control animals. However GPx increased only in the liver. The change in the antioxidant enzymes activities at the kidney and liver, show similarities to those seen in rats subjected to food deprivation-induced weight loss [61]. The reduced lipid peroxidation products and antioxidant enzymes after treatment likely indicates that Oat extracts might be a good source of metabolites with protective effect against diabetic-induced oxidative stress complications. Similarly, our results demonstrated clearly the protective effect of Oat extract against H 2 O 2 oxidative stress in the cellular model of T. pyriformis. Both models indicate the in vivo antioxidant effect of Oat extracts, which can be attributed to its main phenolic compounds. Many phenolics with reported antioxidant effect has been isolated and identified in Oat such as ferulic acid and avenanthramides derivatives [62].
The radical scavenging effect of those Oat metabolites is highly linked to their bioavailability [63] and biotransformation [64].
Diabetes and hyperglycemia-induced oxidative stress can result in neurological complications such as dementia and depression [14]. Also the diabetogenic agent streptozotocin when injected at the intracerebro-ventricular level in rats can produce an Alzheimer disease (AD) model that triggers an insulin resistant brain state [65]. The plausible explanation suggests that low metabolism of brain glucose associated with oxidative stress and neuro-inflammation can result in mitochondrial dysfunction and apoptosis at the hippocampus, leading to accumulation of β-amyloid plaques and neurofibrillary tangles. This oxidative, neuro-inflammatory and degenerative environment induces progressively the memory impairment [66]. Moreover, in previous reports [67], STZ-treated rats displayed increased anxiety-like behavior in different paradigms, such as the open-field test, and the elevated plus maze. In fact, metformin administration significantly attenuated the anxiety-like behavior [68]. In our assays, the Oat and metformin treatment significantly improved cognitive status and prevented anxiety-like behavior in diabetic animals.

Conclusions
The present study suggests that Oat extracts are able to prevent oxidative stress in the diabetic liver and kidney. Also it has been demonstrated that Oat antidiabetic activity is mediated by digestive enzymes inhibition including (α-amylase and α-glucosidase). Oat extracts have been shown to protect antioxidant systems under diabetic stress and to prevent lipid peroxidation. Furthermore, Oat treatment preserved body weight and ameliorated liver functions (ALT and AST), lipid profile (cholesterol, TG) and reduced atherosclerosis index in diabetic animals. Finally, Oat and metformin intake significantly improved the anxiety-like behavior in diabetic animals.