Streptozotocin (STZ), Thiobarbituric acid (TBA), butylated hydroxytoluene (BNT), reduced glutathione (GSH), 2,2,- dipyridyl, xynol orange, aspartic acid, alanine, α-ketoglutarate, 2,4 – dinitro phenyl hydrazine (DNPH), 5,5 – dithiobis-2- nitrobenzoic acid (DTNB) were obtained from Sigma Chemical Co (St Louis, MO-USA). The rest of the chemical utilized were of analytical grade and were obtained from local firm (Nigeria).

Mistletoe (Viscum album) leaves used for this study were obtained from kola nut tree (host plant) from Anara in Isiala Mbano Local government Area of Imo State. Dr S.E Okeke, Department of Plant Biology and Biotechnology, Imo State University, Owerri confirmed the botanical identification of the plant leaf. The voucher samples are kept in the university herbarium for reference.

The method for leaf process was carried out according to (₈). Fresh leaves of Viscum album collected from the host plant were first washed free of sand and debris. Grinding was done using Thomas Contact Mill (Pye Unicam, Cambridge, England). A quantity o the ground sample was soxhlet extracted using 500ml distilled water for 12 hour at 100°C. The extract was evaporated to dryness using Grant instrument, Cambridge, England) at 45°C -50°C to a yield of 20g plant material which was dissolved with appropriate volume of water.

The chemical classes of constituent in the freshly prepared extract were detected using standard photochemical reagents and procedures as described by Tease and Evens (₂₀).

In general, test for the presence or absence of phytochemical compound using the above methods involve the addition of an appropriate chemical agent to the crude material in a test tube. The mixture is then shaken vigorously or gently as the case may be. The presence or absence of saponins, flavonoids, tannins, alkaloids etc. was observed.

Male Wistar rats (200-300g) bred in the Animal House of College of Medicine and Health Sciences, Imo State University, Owerri were used in this study. They were housed in stainless steel cages and kept in a room where a 12-hour light/dark cycle was maintained. They were allowed free access to water and feed diet (product of Pfizer Nigeria Ltd) throughout the period of the experiment.

After one week of acclimatization, the rats were subjected to a 16 hour fast. Diabetes was induces with a single intraperitoneal injection of streptozotocin (STZ) at a dose of 65mg/kg body weight. The STZ was freshly dissolved in citrate buffer (0.01m, pH 4.5) (Ozsoy, Sacan, 2000). The injection volume was prepared to contain 1.0 ml/kg (Murali et al, 2002). After 5 days, blood glucose levels were measure and the animals with a concentration of more than 230 mg/dl were classified as diabetic (Cetto et al, 2000).

Twenty four male Wistar rats were used in this study. The rats were randomized and divided into four groups of six animals each.

Group 1: Normal, received normal saline solution (0.9% NaCl w/v, 5 ml/kg).

Group 2: Diabetic, received STZ (65 mg/kg body weight) once

Group 3. Diabetic, receive STZ (65 mg/kg body weight) once before receiving aqueous extract of Viscum album (100 mg/kg body weight)

Group IV: Diabetic received STZ (65 mg/kg body weight) once before receiving aqueous extract of Viscum album (200 mg/kg body weight).

After 14 days of treatment, all the rats were decapitated after fasting for 16 hours. Blood was collected in two different tubes i.e. one with fluoride oxalate anticoagulant for plasma separation and another without anticoagulant to separate serum for various biochemical estimations. The livers were dissected out and cleared off blood. They were immediately transferred to ice-cold containers containing 0.9% Nacl and homogenized in 0.1N Tris-Hcl buffer (pH 7.4), and used for the estimation of malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activity.

The body weight and blood glucose levels were measured at the beginning and end of the experiment. Blood samples were obtained by tail vein puncture of both the normal and STZ-induced diabetic rats. Blood glucose levels were determined using a glucometre (Lifescan Johnson and Johnson Company, Milipitas, CA).

The acute toxicity of the extract was tested using 30 Wistar rats divided into 5 groups of 6 rats each, with each group receiving graded dose (200-1000 mg/kg body weight, intraperitoneally) of the aqueous extract of Viscum album as described by Ghosh, (1984). After administration of the extract the rats were observed for toxic effects after 48 hours treatment. The toxicological effects were observed in terms of mortality expressed as LD₅₀. The number of animals dying during a period was noted. The LD₅₀ of the extract was estimated from the graph of percentage (%) mortality (converted to probit) against log-dose of the extract, probit 5 being 50% (Litch field, et al, 1959).

Extraction of lipids from serum carried out according to the procedure of Folch et al, (1957) by using chloroform-methanol (2:1 v/v) mixture. From this total cholesterol LDL- and HDL – cholesterol triacylglyceroles and phospholipids were estimated as described earlier by us (Nwanjo, 2005; Nwanjo and Ojiako, 2005,).

Lipid peroxidation in plasma and liver was estimated colorimetrically by measuring malondialdehyde (MDA) by the method of Nichans and Samualson (1968) in brief, 0.1ml of plasma was treated with 2ml of (1:1:1 ratio) TBA – TCA – HCL reagent (TBA 0.37%: 0.25N HCL: 15% TCA) and placed in water bath for 15min, cooled and centrifuged and then clear supernatant was measured at 535 nm against reference blank.

Superoxide dismutase (SOD) activity was determined by the modified method of NADH-phenazinemethosulphate-nitrobluetetrazolium formazon inhibition reaction spectrophotometrically at 560 nm (Kakkar et al, 1984). A single unit of enzyme was expressed as 50% inhibition of NBT (Nitroblue tetrazolium) reduction /min/mg protein.

Catalase (CAT) was assayed calorimetrically as described by Sinha (1972) using dichromate-acetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed in 1.3 ratio). The intensity was measured at 620nm and the amount of hydrogen peroxide hydrolyzed was calculated for the catalase activity.

Reduced glutathione (GSH) was determined by the method of Ellman (1959). 1ml of supernatant (0.5ml plasma/0.5ml liver homogenate precipitated by 2ml of 5% TCA) was taken and 0.5ml of Ellman's reagent (0.0198% DTNB in 1% sodium citrate) and 3ml of phosphate buffer (pH 8.0) were added. The colour developed was read at 412nm.

Vitamin C (ascorbic acid) concentration was measured by Omaye et al, (1979) method. To 0.5ml of plasma/0.5ml liver homogenate, 1.5ml of supernatant, 0.5ml of DNPH reagent (2% DNPH) and 4% thiourea in 9N sulphuric acid) was added and incubated for 3 hours at room temperature. After incubation 2.5ml of 8.5% sulphuric acid was added and colour developed was read at 530nm after 30 min.

Vitamin E (α-tocopherol) was estimated by the method of Desai (1984). Vitamin E was extracted from plasma/liver homogenate by addition of 1.6ml ethanol and 2.0ml petroleum ether to 5.0ml plasma and centrifuged. The supernatant was separated and evaporated. To the residue, 0.2ml of 0.2% 2, 2, - dipyridyl, 0.2ml of 0.5% ferric chloride was added and kept in dark for 5min, an intense red colour layer obtained on addition of 4ml butanol was read at 520nm.

Statistical evaluation of data was performed by using one-way analysis of variance ANOVA followed by Duncan's multiple range test (DMRT) (Duncan 1957).

The results of blood glucose levels and body weight changes in normal, STZ-induced diabetic rats and Viscum album treated diabetic rats were shown in Table 1. There was a significant (p< 0.01) increase in blood glucose levels in STZ-induced diabetic rats (Group II) when compared with normal rats. Administration of aqueous extract of Viscum album at a dose of 100 and 200 mg/kg body weight significantly (p<0.05) decreased blood glucose in STZ induced rats (Group III and IV). The results were found to be in a dose dependent manner.

The body weight changes in diabetic group was significantly decrease (p<0.05) when compared with the normal control which then returned to near normal in diabetic rats treated with aqueous extract of Viscum album at a dose of 100 and 200 mg/kg body weight

The changes in the levels of serum lipids in control and experimental rats are illustrated in Table 2. The total-cholesterol, LDL-Cholesterol and triacylglycerol significantly increased and HDL-Cholesterol and phospholipids significantly decreased in STZ-induced diabetic rats (group II) (P< 0.05) when compared with the normal (group I) rats. The aqueous extract of Viscum album (100 mg/kg and 200 mg/kg body weight) offered a significant protection against alteration in the serum lipids of diabetic rats. The results were also dose dependent.

The levels of MDA in plasma and liver were significantly (p<0.05) increased in STZ-induced rats as compared to normal rats. Treatment with aqueous extract of Viscum album resulted in a significantly decrease in the levels of lipid peroxidation products (MDA) in diabetic rats.

A significant decrease (P< 0.05) in the activities of enzymic antioxidants such as superoxide dismutase (SOD) and catalase (CAT) of the liver was noted in STZ-induced diabetic rats when compared with the normal rats. Upon administration of 100 and 200mg/kg body weight of aqueous extract of Viscum album, the activities of both SOD and CAT were significantly reversed to near normal.

The levels of plasma and liver vitamins C, E and reduced glutathione (GSH) were significantly depleted in STZ – induced diabetic rats. Treatment with aqueous extract of Viscum album significantly increased the levels of these non-enzymic antioxidants in diabetic rats.