**3. Antidiabetic studies**

#### **3.1. Hypoglycaemic activity test**

The hypoglycaemic effect of the aqueous extract was studied in alloxan-induced diabetic rats. The rats were fasted for 8 hours but allowed free access to water. At the end of the fast‐ ing period, the basal fasting blood glucose (FBG) level of the rats was determined. Subse‐ quently, diabetes was induced by single intraperitoneal injection of alloxan monohydrate (70 mg/kg) (Aruna *et al.,* 1999) and normal feeding maintained thereafter. Five days later, blood was drawn from each rat and the blood glucose level was measured to establish dia‐ betes. Animals with blood glucose level ≥225mg/dl was considered to be diabetic and used for this study. The diabetic animals were randomly divided into four groups (n=5) and re‐ ceived oral administration of aqueous extract (200 and 400 mg/kg), Distilled water (5ml/kg) and Glibenclamide (0.2 mg/kg) respectively. Aqueous extract was dissolved in distil water. Blood glucose was then measured before (i.e. 0 h) and at 0.5, 1, 2 and 4 h after treatment.

ment) as well as on days 14 and 28 after commencement of treatment. The absorbance of each sample containing the reaction mixture with or without serum was read at 540nm in a UV spectrophotometer. Total cholesterol or triglyceride is calculated using the formula: To‐ tal cholesterol (mg/dl) = SAod/STod x 200, where SAod = optical density of test sample and

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The effect of chronic administration of the extract on haemoglobin (Hb) and cell counts [white blood cells (WBC) and red blood cells (RBC)] of diabetic rats was also determined. Blood samples were collected by ocular puncture using haematocrit tubes, transferred into EDTA-containing test tubes and placed in a haematology analyzer (Abacus Junior®, Buda‐ pest-Hungary) for determination of the parameters. Measurements were taken before (bas‐ al) and after the induction of diabetes (Pre-treatment) as well as on days 14 and 28 after the

Cyanohaemoglobin method was used for this purpose (Coles, 1986). Four mls of Drabkin´s solution (diluents) was placed in a tube, 0.02ml of the collected blood sample was put in the tube using pipette and the pipette was rinsed for more than three times. The mixture was stirred well and allowed to stand for 10minutes. This was read in a colorimeter at 540nm wavelength. The equivalent haemoglobin was read from a calibration curve or table. The

Photometer reading of unknown x g% Hb value of standard x DF/Photometer reading of

Erythrocyte diluting pipette marked 101 above the bulb was used to draw the blood sample up to exactly 0.5mark. The tip of the pipette was then inserted into the erythrocyte diluting fluid - Gower´s solution and through a steady suction; the pipette was filled with the fluid to the 101 line above the bulb, rotating it gently while filling. The pipette was brought to a horizontal position and finger tip was placed over the tip before removing the rubber tub‐ ing. The pipette was shaken for more than 30minutes in a mechanical shaker; the haemocy‐ tometer was then filled with the diluted blood and then allowed to stand for a few minutes for the cells to settle. The erythrocytes were then counted under microscope lens (x 40 objec‐ tive) counting all the erythrocytes in the 5 of the 25 small squares in the central area. Each of the 5 small squares to be counted was bordered by double or triple lines and was divided into 16 smaller squares. A total of 80 of these small squares were counted. The cells were

Volume of the whole blood used + volume of diluent/volume of whole blood used.

**3.6. Effects of the extract on haemoglobin and cell counts of diabetic rats**

STod = optical density of standard.

commencement of treatment (Post-treatment).

DF = dilution factor and it was calculated as:

**3.8. Determination of erythrocyte (rbc) count**

It is expressed in gram percent.

haemoglobin value of the blood sample was calculated as:

**3.7. Estimation of haemoglobin**

standard

#### **3.2. Normoglycaemic activity**

Animals fasted overnight were randomly divided into four groups (n=5) and received oral administration of the extract (200 and 400mg/kg), glibenclamide (0.2mg/kg) and vehicle con‐ trol (5ml/kg) respectively. The blood glucose level of each animal was measured prior to (pretreatment) and at 0.5, 1, 2 and 4hour after extract administration (Okoli et al., 2010).

### **3.3. Oral glucose tolerance test**

Animals were fasted for 16h but with free access to water were randomly divided into four groups (n=5) and received oral administration of the aqueous extract (200 and 400mg/kg), glibenclamide (0.2mg/kg) and vehicle control (5ml/kg) respectively. Ninety minutes later, the rats were fed with glucose (4g/kg). The blood glucose level of animals in each group was then measured before (0) and at 30, 60, 90, 120, 150, 180 min after glucose load (Okoli et al., 2010).

#### **3.4. Antidiabetic activity test**

The antidiabetic effect of the plant extract was studied by evaluating the effect of its chronic administration on the blood glucose level of alloxan-induced diabetic rats. The basal fasting blood glucose (FBG) of the rats was determined and diabetes was induced as described be‐ fore. 25 diabetic rats with glucose level ≥225 were selected and used for the study. The rats were fasted for 8h but allowed free access to water (Okoli et al., 2010). They were then divid‐ ed randomly into five groups (n=5) and received oral administration of extract (200 and 400mg/kg), glibenclamide (0.2mg/kg, diabetic control), extract (200mg/kg) and the vehicle (5ml/kg) both of which serve as non diabetic control. The treatment was administered orally to the animals once daily for 28 days. Blood glucose level was then measured as described before (pretreatment) and on days 14 and 28 after commencement of the treatment. The body weight of each animal was also measured on these days.

#### **3.5. Effects of the extract on lipid profile of diabetic rats**

The effect of the extract on the lipid profile of treated diabetic rats was studied by monitor‐ ing the cholesterol and triglyceride levels. Blood samples were collected by ocular puncture, transferred into test tubes and centrifuged at 3000 rpm for 5 mins. The serum was collected and the total cholesterol and triglyceride levels of each sample were separately determined by enzymatic colorimetric method (Muller *et al.,* 1977) using reagent kits. Lipid levels of dia‐ betic animals were measured before (Basal) and after the induction of diabetes (pre-treat‐ ment) as well as on days 14 and 28 after commencement of treatment. The absorbance of each sample containing the reaction mixture with or without serum was read at 540nm in a UV spectrophotometer. Total cholesterol or triglyceride is calculated using the formula: To‐ tal cholesterol (mg/dl) = SAod/STod x 200, where SAod = optical density of test sample and STod = optical density of standard.

#### **3.6. Effects of the extract on haemoglobin and cell counts of diabetic rats**

The effect of chronic administration of the extract on haemoglobin (Hb) and cell counts [white blood cells (WBC) and red blood cells (RBC)] of diabetic rats was also determined. Blood samples were collected by ocular puncture using haematocrit tubes, transferred into EDTA-containing test tubes and placed in a haematology analyzer (Abacus Junior®, Buda‐ pest-Hungary) for determination of the parameters. Measurements were taken before (bas‐ al) and after the induction of diabetes (Pre-treatment) as well as on days 14 and 28 after the commencement of treatment (Post-treatment).

#### **3.7. Estimation of haemoglobin**

blood was drawn from each rat and the blood glucose level was measured to establish dia‐ betes. Animals with blood glucose level ≥225mg/dl was considered to be diabetic and used for this study. The diabetic animals were randomly divided into four groups (n=5) and re‐ ceived oral administration of aqueous extract (200 and 400 mg/kg), Distilled water (5ml/kg) and Glibenclamide (0.2 mg/kg) respectively. Aqueous extract was dissolved in distil water. Blood glucose was then measured before (i.e. 0 h) and at 0.5, 1, 2 and 4 h after treatment.

Animals fasted overnight were randomly divided into four groups (n=5) and received oral administration of the extract (200 and 400mg/kg), glibenclamide (0.2mg/kg) and vehicle con‐ trol (5ml/kg) respectively. The blood glucose level of each animal was measured prior to (pretreatment) and at 0.5, 1, 2 and 4hour after extract administration (Okoli et al., 2010).

Animals were fasted for 16h but with free access to water were randomly divided into four groups (n=5) and received oral administration of the aqueous extract (200 and 400mg/kg), glibenclamide (0.2mg/kg) and vehicle control (5ml/kg) respectively. Ninety minutes later, the rats were fed with glucose (4g/kg). The blood glucose level of animals in each group was then measured before (0) and at 30, 60, 90, 120, 150, 180 min after glucose load (Okoli et al.,

The antidiabetic effect of the plant extract was studied by evaluating the effect of its chronic administration on the blood glucose level of alloxan-induced diabetic rats. The basal fasting blood glucose (FBG) of the rats was determined and diabetes was induced as described be‐ fore. 25 diabetic rats with glucose level ≥225 were selected and used for the study. The rats were fasted for 8h but allowed free access to water (Okoli et al., 2010). They were then divid‐ ed randomly into five groups (n=5) and received oral administration of extract (200 and 400mg/kg), glibenclamide (0.2mg/kg, diabetic control), extract (200mg/kg) and the vehicle (5ml/kg) both of which serve as non diabetic control. The treatment was administered orally to the animals once daily for 28 days. Blood glucose level was then measured as described before (pretreatment) and on days 14 and 28 after commencement of the treatment. The

The effect of the extract on the lipid profile of treated diabetic rats was studied by monitor‐ ing the cholesterol and triglyceride levels. Blood samples were collected by ocular puncture, transferred into test tubes and centrifuged at 3000 rpm for 5 mins. The serum was collected and the total cholesterol and triglyceride levels of each sample were separately determined by enzymatic colorimetric method (Muller *et al.,* 1977) using reagent kits. Lipid levels of dia‐ betic animals were measured before (Basal) and after the induction of diabetes (pre-treat‐

body weight of each animal was also measured on these days.

**3.5. Effects of the extract on lipid profile of diabetic rats**

**3.2. Normoglycaemic activity**

118 Antioxidant-Antidiabetic Agents and Human Health

**3.3. Oral glucose tolerance test**

**3.4. Antidiabetic activity test**

2010).

Cyanohaemoglobin method was used for this purpose (Coles, 1986). Four mls of Drabkin´s solution (diluents) was placed in a tube, 0.02ml of the collected blood sample was put in the tube using pipette and the pipette was rinsed for more than three times. The mixture was stirred well and allowed to stand for 10minutes. This was read in a colorimeter at 540nm wavelength. The equivalent haemoglobin was read from a calibration curve or table. The haemoglobin value of the blood sample was calculated as:

Photometer reading of unknown x g% Hb value of standard x DF/Photometer reading of standard

DF = dilution factor and it was calculated as:

Volume of the whole blood used + volume of diluent/volume of whole blood used.

It is expressed in gram percent.

#### **3.8. Determination of erythrocyte (rbc) count**

Erythrocyte diluting pipette marked 101 above the bulb was used to draw the blood sample up to exactly 0.5mark. The tip of the pipette was then inserted into the erythrocyte diluting fluid - Gower´s solution and through a steady suction; the pipette was filled with the fluid to the 101 line above the bulb, rotating it gently while filling. The pipette was brought to a horizontal position and finger tip was placed over the tip before removing the rubber tub‐ ing. The pipette was shaken for more than 30minutes in a mechanical shaker; the haemocy‐ tometer was then filled with the diluted blood and then allowed to stand for a few minutes for the cells to settle. The erythrocytes were then counted under microscope lens (x 40 objec‐ tive) counting all the erythrocytes in the 5 of the 25 small squares in the central area. Each of the 5 small squares to be counted was bordered by double or triple lines and was divided into 16 smaller squares. A total of 80 of these small squares were counted. The cells were counted beginning at the left of the top row of small squares, then from right to left for the next row and so on.

**4.1. Statistical analysis**

**5. Results and discussion**

means were considered significant at P< 0.05.

Data was analyzed using graph pad prism 5 and the results expressed as mean ± SD. The results were further subjected to one way ANOVA for comparisons and differences between

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Phytochemical screening of the leaves of *P. amarus* showed the presence of alkaloids, tannin, flavonoids, saponin, anthraquinones and cardiac glycosides. Flavonoids and tannins are phenolic compounds and plant phenolics are also a major group of compounds that act as primary antioxidants or free radical scavengers (Adedapo et al., 2008a, 2008b, Ayoola et al., 2008). Tannins and saponins are also found to be effective antioxidants, antimicrobial, and anti-carcinogenic agents (Lai et al., 2010). Polyphenolic compounds are ubiquitous in foods of plant origin, and thus they constitute an integral part of the human diet (Bravo 1998). In‐ terest in polyphenols has greatly increased recently because these phytochemicals are known to suppress rates of degenerative processes such as cardiovascular disorders and cancer (Bravo 1998, Duthie 2000, Huang et al., 2007; Jimoh et al., 2010). Some of these poten‐ tial health benefits of polyphenolic substances have been related to the action of these com‐ pounds as antioxidants, free radical scavengers, quenchers of singlet and triplet oxygen and inhibitors of peroxidation (Li-Chen et al., 2005). As a group, phenolic compounds have been found to be strong antioxidants against free radicals and other reactive oxygen species, the major cause of many chronic human diseases (Kyung-Hee et al., 2005, Chen and Yen 2007).

In the acute toxicity test, no death was recorded in all the groups. All the mice appeared to be normal and none of them showed any visible signs of toxicity. Acute oral administration of *Phyllanthus amarus* to mice indicated that the plant is non toxic even at the dose of 1600mg/kg body weight. It thus showed that this plant is safe for medicinal use at this dose.

The aqueous extract caused a significant (P<0.05) dose related reduction in the fasting blood glucose (FBG) of normoglycaemic rats. Maximum reduction occurred within 2hr post- treat‐ ment with 400mg/kg dose of the extract (Table 1). In this study, experimental evaluation of the antidiabetic potentials of *P*. *amarus* has shown that single oral administration of the ex‐ tract to normal rats reduced fasting blood glucose which suggests an inherent hypoglycae‐ mic effect (Table 2). The extract also suppressed the postprandial rise in blood glucose in normal rats following a heavy glucose meal with maximum suppressive effect coinciding with the time of peak blood glucose level after the meal (Table 3). Chronic hyperglycaemia in DM is a risk factor constantly fuelled by postprandial elevation of blood glucose. Control of postprandial hyperglycaemia in diabetes is of great importance due to its close relation to the risk of micro and macro-vascular complications and death (Balkau, 2000; Ceriello, 2005).

Calculation

Cells counted x 10(0.1mm depth) x 5(1/5 of sqmm) x 200(1:200) dilution = erythrocytes per cu mm.

OR

The sum of the cells in the five small squares multiplied by 10,000 = total erythrocytes per cu mm (Coles, 1986)

### **3.9. Determination of leucocyte (wbc) count**

Leucocyte diluting pipetting was used to draw the blood sample to a point marked 0.5 and filled with leucocyte diluting fluid up to the 11mark above the bulb. The mixture was shak‐ en for 3minutes until well mixed. Two to three drops from the pipette was discarded before filling the counting chamber of haemocytometer. The leucocytes were allowed to settle for 1minute. The leucocytes in the larger squares of haemocytometer chamber were counted and multiplied by 50 to obtain the total number of white blood cells (Coles, 1986).

Calculation:

Cells counted x 20 (1:20 dilution) x 10(0.1mm depth)/4 (no of sq mm counted)

= WBC/cubic mm

OR

The sum of the cell counted in the 4 corner squares multiplied by 50 = total leucocytes per cubic mm.
