Plant materials and authentication
The leaves of Hamelia patens were harvested from a compound bush in Abia State University, Uturu, Abia State, Nigeria. The plant was authenticated at the department of Plant Science and Biotechnology, Abia State University Uturu by a taxonomist and voucher samples deposited in the Departmental herbarium. (Voucher number: ABSU/PSB/00043)
Preparation of plant extract
The extract was prepared using the method described by Daniel et al.,5 with slight modifications. The leaves were air dried for four weeks into a constant weight. The dried leaves were milled into a fine powder and stored in a cellophane bag until use.
Hundred grams (100g) of powdered leaves of Hamelia patens was soaked in 1000ml of water for 24hours with stirring for proper mixing and drained using a muslin cloth. It was filtered using Whatman no. 1 filter paper in other to get a clear filtrate. The concentration of he extract was determined to be 100mg/ml
A total of forty eight (48) apparently assumed healthy male Albino rats (9-10weeks old) weighing between (160 - 180 g) was procured from the animal house, Department of Biochemistry,Abia State University, Uturu. They had access to clean drinking water ad libitum and growers feed. All protocols for animal handling were strictly observed.6
Ethical clearance was obtained from the Ethical Committee of Animal Care Use of the Faculty of Biological Sciences, Abia State University; Uturu, Nigeria
Induction of Diabetes
This was determined by the method described by Etuk E.U.7
Diabetes mellitus was induced by a single intraperitoneal injection of 160mg/kg body weight of Alloxan. Alloxan was dissolved in 0.9% normal saline as vehicle.
Measurement of Body Weight
Body weight was measured on days 0, 7 and 14. Body weight noted was expressed as mean body weight (g).
Randomized complete block experimental design recommended by Ogbeibu8 was used in the study. The experimental animals were divided randomly into six (6) groups (Replicates) of eight (8) animals each:
Group I: Normal control.
Group II: Diabetic group (negative control)
Group III: Diabetic group treated orally with 58mg/ml gliclazide: a standard anti-diabetic drug.
Group IV: Diabetic group treated orally with 400mg/kg body weight of aqueous leaf extract of Hamelia patens.
Group V: Diabetic group treated orally with 800mg/kg body weight of aqueous leaf extract of Hamelia patens.
Group VI: Diabetic group treated orally with 1200 mg/kg body weight of aqueous leaf extract of Hamelia patens.
Liver Function Test
Determination of Aspartate Aminotransferase (AST) and Alanine transaminase (ALT) Activity was determined by the method of Reitman and Frankel9
Determination of alkaline phosphatase (ALP) activity was by the method of Julius et al 1964.10 This method is based on the principle that serum alkaline phosphate hydrolyses a colourless substrate of phenolphthalein monophosphate giving rise to phosphoric acid and phenolphthalein which at alkaline pH value turns into a pink colour that can be photometrically determined.
Determination of Total protein was as described by Weichselbau11
Colorimetric determination of total protein based on the principle reaction (Copper salt in an alkaline medium). Protein in plasma or serum forms a blue coloured complex when treated with cupric ions in alkaline solution. The intensity of the blue colour is proportional to the protein concentration.
Determination of antioxidants activities
Determination of superoxide dismutase activity (SOD)
The method of Sun and Sigma as described by Christine and Joseph,12 was adopted. The reaction mixture (3ml) contained 2.95ml sodium carbonate buffer (0.05M, ph. 10.2), 0.02ml of serum and 0.03ml of epinephrine in 0.005N HCL used to initiate the reaction. The reference cuvette contained 2.95ml buffer, 0.03ml of substrate (epinephrine) and 0.02ml of water. An extinction coefficient for epinephrine at 480nm of 4020 mˉ¹cm̄ ¹ was used in calculating activity.
Determination of catalase activity
The catalase activity was determined according to the method of Christine and Joseph,12
Determination of Glutathione (GSH)
The GSH level was determined using the method described by Christine and Joseph,12 with slight modifications. Serum (0.5ml) was added to 2ml of 5% TCA and centrifuged at 3000 rpm for 10minutes. The supernatant (1ml) was added to 0.5ml of DTNB (10nm) in the presence of 3ml phosphate buffer (0.1M, pH 7.4). Absorbance was read at 420nm.
Determination of Malondialdehyde (MDA) activity
Lipid peroxidation was ascertained by formation of Malondialdehyde (MDA) and measured by thiobarbituric reactive (TBARS) method previously described by Onkawa et al.13 Reaction mixture containing serum (0.5ml), TCA (0.5ml) and TBA (0.5ml) was incubated in boiling water for 15minutes. The pink color of chromogen formed was extracted in butanol solution (2.0ml). The mixture was centrifuged at 3000 rpm for 10minutes and the supernatant was read at 532nm.
Determination of C-peptides:
The C-peptides values were deytermined using the approved guidelines of Clinical and Laboratory Standards Institute.14