MATERIALS AND METHODS
Collection and Preparation of sample
Clerodendrum volubile leaves were obtained locally within Ikale land in Okitipupa Local Government Area of Ondo State, Nigeria and identified in the Department of Microbiology and Botany, University of Ibadan, Nigeria. Herbarium specimen, with voucher number UIH22481, was deposited at the Herbarium of the University of Ibadan, Nigeria.
The leaves were removed from their stalks, thoroughly washed with tap water and rinsed with distilled water to remove sand and other impurities. They were air-dried in the laboratory for twenty (20) days. The dried leaves were subsequently ground into fine powder by using a commercial blender. The powdered samples were stored in polythene bag until used. All analyses were carried out in triplicates.
Analysis of the Sample
Proximate composition and Mineral Analysis
Crude fibre, moisture content, crude fat and total ash were carried out according to the methods of the Association of official Analytical Chemists [9]. The nitrogen was determined using the micro-Kjedahl method as described by Pearson [10] and the nitrogen content obtained was converted to protein by multiplying by a factor of 6.25.
Mineral element composition was determined using Atomic Absorption Spectrophotometer after acid digestion and measurements taken were reported as mg/100g.
Amino Acid Analysis
4g of the ground sample was defatted with chloroform/methanol mixture for 15 hours in soxhlet extraction apparatus. Between 30 – 35mg of the defatted sample was weighed into a glass test tube. 7ml of 6M HCl was added and oxygen was expelled by flushing with nitrogen gas. The glass test tube, well sealed, was put in an oven for 22hours at 110oC. This was allowed to cool before the content was filtered. The filtrate was evaporated to dryness at 40oC under vacuum using a rotator evaporator. The residue obtained was dissolved with 5ml acetate buffer (pH 2.0).
The method of amino acid was by ion-exchange Chromatography using the Technicum Sequential Multi Sample Amino Acid Analyzer (TSM) (Technicum Instruments Corporation, New York).