Free Radicals Scavenging Potential Of The Aqueous Extract Of Viscum Album (Mistletoe) Leaves In Diabetic Wistar Rats Hepatocytes
diabetic rats, free radical, viscum album
H Nwanjo. Free Radicals Scavenging Potential Of The Aqueous Extract Of Viscum Album (Mistletoe) Leaves In Diabetic Wistar Rats Hepatocytes. The Internet Journal of Nutrition and Wellness. 2006 Volume 3 Number 2.
The aqueous extract of
Diabetic mellitus and other numerous pathological events such as atherosclerosis and inflammation processes are associated with the generation of relative oxygen species (ROS), and consequently the induction of several chain reactions among them, lipid peroxidation and others (Cross
Recently, some synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have been suspected to be dangerous to human health (Safer
The effectiveness of the plant to remedy ailment conditions in trado-medical practices may not be unconnected with the natural products present in the plant and their abilities to act as radical scavengers. For this reasons it was therefore reasonable to investigate the free radical scavenging activities of
Materials And Methods
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).
Preparation of the Aqueous Plant Extract
The method for leaf process was carried out according to (8). Fresh leaves of
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)
Induction of Diabetes in Rats
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
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
Group IV: Diabetic received STZ (65 mg/kg body weight) once before receiving aqueous extract of
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.
Measurement of Blood Glucose Levels
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).
Acute Toxicity Tests
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
Estimation of Serum Lipids
Extraction of lipids from serum carried out according to the procedure of Folch
Estimation of Lipid Peroxidation
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.
Estimation of Enzymic Antioxidants
Superoxide dismutase (SOD) activity was determined by the modified method of NADH-phenazinemethosulphate-nitrobluetetrazolium formazon inhibition reaction spectrophotometrically at 560 nm (Kakkar
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.
Estimation of Non-Enzymic Antioxidants
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
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).
Effect of Extract of Viscum album on Blood Glucose Levels and Body Weight Changes
The results of blood glucose levels and body weight changes in normal, STZ-induced diabetic rats and
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
Effect of Aqueous Extract of Viscum album on Serum Lipids
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
Effects of Viscum album Aqueous Extract on MDA and Some Enzymic and Non-Enzymic Antioxidant Status of Control and Experimental Rats
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
Effects of Viscum album Aqueous Extract on Enzymic Antioxidant Status of Control and Experimental 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
Effects of Viscum album Aqueous Extract on Non-Enzymic Antioxidant Status of Control and Experimental Rats
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
The aqueous extract of
Membrane lipids succumb easily to deleterious actions of (reactive oxygen species) ROS (Reiter, 1995). STZ is toxic to pancreatic β-cells and is thus widely used for induction of experimental diabetes mellitus in animals, resulting in the production of ROS (Mazunder
In the present study the increased level of malondialdehyde (MDA) levels in plasma and liver of rats treated with STZ reflected the lipid peroxidation as the consequence of oxidative stress caused by STZ. Oxidative stress is associated with the peroxidation of cellular lipids, which is determined by measurement of TBA- reactive substances. The concentration of lipid peroxidation products may reflect the degree of oxidative stress in diabetes. It has been reported previously (Raynes, 1991, Kakkar
Administration with aqueous extract of
Reduced level of non-enzymic antioxidants such as reduced glutathione in (GSH), vitamin C and vitamin E on STZ administration were observed in this study. Administration of aqueous extract of the
It is well established that GSH in blood keeps up the cellular levels of the active forms of vitamins C and E by neutralizing the free radicals. When there is a reduction in the GSH the cellular levels of vitamin C is also lowered, indicating that GSH, vitamin C, and vitamin E are closely interlinked to each other (Paris and Amali 2005).
The enzymic antioxidant defense systems are the nature protector against lipid peroxidation. Superoxide dismutase (SOD) scavenges the superoxide ions produced as cellular by products. SOD is a major defense for aerobic cells in combating the toxic effects of superoxide radicals (McCrod
This investigation shows that the aqueous extract of
Harrison Ugo Nwanjo,Ph.D, Department Of Medical Lab. Science, Imo State University, Owerri, Nigeria. E-Mail: Harrisonnwanjo@Yahoo.Co.Uk GSM-08033525389