**2.2. Animals**

Several plants have been used in folkloric medicine for the treatment and prevention of in‐ fectious and non-infectious diseases in man and his animals and this has led to renewed sci‐ entific interest in the use of plants for these purposes (Oridupa et al. 2011). There is global resurgence in the use of herbal preparations and in some developing countries like Nigeria; it is being gradually integrated into the primary and secondary health care systems. Nearly all societies have used herbal materials as sources of medicines and the development of

*Phyllanthus amarus* belongs to the family Euphorbiaceae (the spurge family) of which the largest genus is the genus Euphorbia. The plant is known to originate from Malaysia. The species *Phyllanthus amarus* is a small tropical herb, which occurs widely as a rainy-season weed throughout the hotter parts of India (Bagchi et al., 1992). The widespread usage of this herb has prompted several investigations (Calixto et al., 1998; Odetola and Akojenu, 2000; Adeneye et al., 2006). The plant has a history of use in Ayurverdic medicine for over 2000 years as well as a wide variety of traditional applications. The plant is commonly found in Southern Nigeria, Sierra Leone and Equatorial Guinea. It also occurs in Ghana and other parts of tropical Africa (Irvine, 1930; Adedapo et al., 2005). It is a weed of cultivated land and in waste spaces, it is common to find it growing and spreading freely along the road sides, under flower beds and in many other places (Burkill, 1994). For this reason, grazing animals are prone to consuming this plant along with their feed particularly in drier tropical

Many studies have thus been carried out on the plant in various parts of the world but there is a resurgence of interest in this plant as antidiabetic agent. The present study was therefore undertaken to investigate the phytochemical constituents, anti-diabetic and safety potentials of the aqueous leaf extract of *Phyllanthus amarus* Schum in experimental animals especially

Fresh leaves of *Phyllanthus amarus* Schum were collected from the campus of the University of Ibadan, Nigeria in March 2012. The leaves were identified by botanists and a voucher specimen (UIH ADE/003/2012) deposited at the herbarium of the Department of Botany, University of Ibadan. The ground plant material (200 g) was shaken in 1 L distilled water for 48 h on an orbital shaker at room temperature of 24°C. The extract was filtered using a Buck‐ ner funnel and Whatman No 1 filter paper. The filtrate was concentrated to dryness under reduced pressure at 40ºC using a rotary evaporator. The thick solution was lyophilized us‐

ing freeze drying system for biological investigations. The extract yield was 13.85%.

these herbal medicines depended on local botanical flora (Adedapo et al., 2009).

climates where lush green grass is not often available (Adedapo, 2002).

**2. Material and methods**

116 Antioxidant-Antidiabetic Agents and Human Health

**2.1. Plant material and preparation of extracts**

that diabetes has assumed a global dimension as a non-communicable disease.

The animals used in this study were male Wistar rats weighing between 100 and 200g as well as mice weighing between 15 and 30g. They were maintained at the Experimental Ani‐ mal House of the Faculty of Veterinary Medicine, University of Ibadan in rat cages and fed on commercial rabbit cubes (Ladokun and Son Livestock Feeds, Nigeria Ltd). The animals were allowed free access to clean fresh water in bottles *ad libitum*. All experimental protocols were in compliance with University of Ibadan Ethics Committee on Research in Animals as well as internationally accepted principles for laboratory animal use and care.

## **2.3. Chemicals**

Alloxan used in this study was obtained from Sigma-Aldrich (Chemie Gmbh, Steinheim, Denmark). The standard drug used in the various experiments was glibenclamide. The chemical and drug used were of analytical grade. Normal saline and distilled water were al‐ so used in this study.

#### **2.4. Phytochemical screening**

The phytochemical analysis was performed on the ground (powered) leaf of *P. amarus* for identification of the constituents. The constituents tested for were alkaloids, tannins, sapo‐ nins, anthraquinones, cardiac glycosides and flavonoids as described by Trease and Evans (1983); Abate (1989); Shale et al., (1999); Evans, (2002); Moody et al., (2006) and Sawadogo et al., (2006).

#### **2.5. Acute toxicity test**

The acute toxicity of *P. amarus* aqueous was determined in mice according to the method of Hilaly *et al.* (2004) with slight modifications. Mice fasted for 16 h were randomly divided in‐ to groups of six mice per group. Graded doses of the plant's extract (100, 200, 400, 800 and 1600 mg/kg p.o.) were separately administered to the mice in each of the groups by means of bulbed steel needle. All the mice in the groups were then allowed free access to food and water and observed over a period of 48 h for signs of acute toxicity. The number of deaths within this period of time was recorded.
