**2. Materials and methods**

#### **2.1. Experimental animals and reagents**

36 Albino rats (*Rattus norvegicus*) with an average weight of 132.5 g were obtained from the Animal Holding Unit of the Department of Biochemistry, University of Ilorin, Ilorin, Kwara State, Nigeria. Animal husbandry and experimentation were consistent with Guiding Princi‐ ples in the use of Animals in Toxicology [20]. All the reagents used for this study were of analytical grade and were prepared in all glass-distilled water.

#### **2.2. Plant extract preparation**

The bark of *Acacia ataxacantha* was obtained from Akure, Ondo State, Nigeria. Identification was carried out at the University Ilorin Herbarium, Department of Plant Biology, Ilorin, Nigeria where voucher number 872 was deposited. The bark was air-dried under shade and pulverized into fine powder. 200 g of the fine powder was percolated in 500 ml of absolute ethanol (60-80 o C) for 24h. The percolated mixture was filtered and evaporated at room temperature according to the method of Majekodunmi *et al*. [21]. A homogenous aqueous suspension of the extract was made before administration to the experimental animals [21].

#### **2.3. Induction of experimental diabetes**

The animals were fasted overnight and diabetes was induced by a single intra-peritoneal injection of freshly-prepared STZ (55 mg/kg body weight of rats) in 0.1 M citrate buffer (pH 4.5) [22]. The animals were allowed to drink 5% glucose solution overnight to overcome the drug-induced hypoglycaemia. Control rats were injected with citrate buffer alone. The animals were considered as diabetic, if their blood glucose values were above 250 mg/dL on the third day after the STZ injection. The treatment was started on the fourth day after the STZ injection and this was considered the first day of treatment.

#### **2.4. Animals grouping**

All rats were maintained under standard laboratory conditions (12 h light/dark cycle, 25 ± 2 o C). The rats were acclimatized for a week in the laboratory. They were fed with standard rodent diet and tap water *ad libitum.* The animals were then randomly divided into 6 groups-

Group 1: Received distilled water (Control)

Group 2: Diabetic untreated rats

Group 3: Diabetic and treated with125 mg/kg b.w of ethanolic extract of *Acacia ataxacantha* bark

Group 4: Diabetic and treated with 250 mg/kg b.w of ethanolic extract of *Acacia ataxacantha* bark.

Group 5: Diabetic and treated with 500 mg/kg b.w of ethanolic extract of *Acacia ataxacantha* bark

Group 6: Diabetic and treated with standard drug (Metformin)

#### **2.5. Sample preparation**

Hence, the aim of this study is to investigate the anti-diabetic potential and safety evaluation of ethanolic extract of *Acacia ataxacantha* bark in streptozotocin-induced diabetic rats. The

36 Albino rats (*Rattus norvegicus*) with an average weight of 132.5 g were obtained from the Animal Holding Unit of the Department of Biochemistry, University of Ilorin, Ilorin, Kwara State, Nigeria. Animal husbandry and experimentation were consistent with Guiding Princi‐ ples in the use of Animals in Toxicology [20]. All the reagents used for this study were of

The bark of *Acacia ataxacantha* was obtained from Akure, Ondo State, Nigeria. Identification was carried out at the University Ilorin Herbarium, Department of Plant Biology, Ilorin, Nigeria where voucher number 872 was deposited. The bark was air-dried under shade and pulverized into fine powder. 200 g of the fine powder was percolated in 500 ml of absolute

temperature according to the method of Majekodunmi *et al*. [21]. A homogenous aqueous suspension of the extract was made before administration to the experimental animals [21].

The animals were fasted overnight and diabetes was induced by a single intra-peritoneal injection of freshly-prepared STZ (55 mg/kg body weight of rats) in 0.1 M citrate buffer (pH 4.5) [22]. The animals were allowed to drink 5% glucose solution overnight to overcome the drug-induced hypoglycaemia. Control rats were injected with citrate buffer alone. The animals were considered as diabetic, if their blood glucose values were above 250 mg/dL on the third day after the STZ injection. The treatment was started on the fourth day after the STZ injection

All rats were maintained under standard laboratory conditions (12 h light/dark cycle, 25 ± 2

C). The rats were acclimatized for a week in the laboratory. They were fed with standard rodent diet and tap water *ad libitum.* The animals were then randomly divided into 6 groups-

Group 3: Diabetic and treated with125 mg/kg b.w of ethanolic extract of *Acacia ataxacantha* bark

C) for 24h. The percolated mixture was filtered and evaporated at room

results were compared with standard anti-diabetic drug, metformin.

analytical grade and were prepared in all glass-distilled water.

**2. Materials and methods**

**2.2. Plant extract preparation**

ethanol (60-80 o

**2.4. Animals grouping**

o

**2.1. Experimental animals and reagents**

6 Antioxidant-Antidiabetic Agents and Human Health

**2.3. Induction of experimental diabetes**

and this was considered the first day of treatment.

Group 1: Received distilled water (Control)

Group 2: Diabetic untreated rats

At the end of the experimental period, venous blood was collected from the experimental animals and serum was prepared by centrifuging the blood samples at 3000 rpm for 5 minutes [23] and serum collected by pippeting. The animals were thereafter quickly dissected and the liver and pancreas removed. The pancreas and liver were suspended in ice-cold 0.25 M sucrose solution (1:5 w*/*v) and homogenized. The homogenates were kept frozen overnight to ensure maximum release of enzymes [24].

#### **2.6. Estimation of hepatic glucose and glycogen**

Hepatic glucose and glycogen were estimated using the method of Barham and Trinder [25] and Passoneau and Lauderdale [26] respectively.

#### **2.7. Determination of liver enzyme activities**

The method described by Reitman and Frankel [27] was used for assaying the activity of alanine aminotransferase and aspartate aminotransferase.
