iabetes mellitus is a multifactorial disease, which is characterized by hyperglycemia (Ugochukwu et al., 2003), lipoprotein abnormalities (Scoppola et al., 2001), raised basal metabolic rates (Okwu et al., 2006), defect in reactive oxygen species scavenging enzymes and altered intermediary metabolism of major food substances (Unwin et al., 2001). Diabetes being a major degenerative disease is found in all parts of the world and it is becoming the third most lethal disease of mankind and rapidly increasing. It is affecting at least 15 million people and having complications which include hypertension, atherosclerosis and microcirculatory disorders (Saidu et al., 2012).
Diabetes mellitus is a group of metabolic disease caused by a defect in insulin production, insulin action or both.
Type 1 diabetes is caused by a lack of insulin due to the destruction of insulin-producing ? -cells in the pancreas. Type 2 diabetes, the most common form of diabetes is caused by a combination of factors, including insulin resistance, a condition in which the body's muscle, fat and liver cells do not use insulin effectively.
Too much glucose circulating in the blood results in hyperglycemia, one of the major symptoms of diabetes. Hyperglycemia causes many of the health problem associated with diabetes, including eye, kidney, heart disease and nerve conditions.
The World Health Organization (WHO) in its 2014 release repoeted that the prevalence of diabetes has reached epidemic proportions. In 2014 the global prevalence of diabetes was estimated to be 9% among adults aged 18+ years. In 2012, an estimated 1.5 million deaths were directly caused by diabetes. More than 80% of diabetes deaths occur in low-and middleincome countries (WHO, 2014).
Diabetes mellitus is associated with an increase in reactive oxygen species (ROS) generation by mononuclear cells and an increased oxidative load resulting in oxidative damage to lipids, proteins and DNA (Marfella et al., 1995;Giugliano et al., 1997; Paoliso and Giugliano, 1996).
Chronic hyperglycemia and subsequent augmentation of reactive oxygen species (ROS) deteriorate ?-cell functions and increase insulin resistance which leads to the aggravation of type 2 diabetes. In addition, chronic hyperglycemia and ROS are also involved in the development of atherosclerosis which is often observed under diabetic conditions (Kaneto et al., 2010).
It has been shown that ROS are produced in various tissues under diabetic conditions (Baynes and Thorpe, 1999). There are several sources, of ROS in cells such as the nonenzymatic glycosylation reaction, the electron transport chain in mitochondria, and membrane-bound NADPH oxidase (Brownlee, 2001;Harrison et al, 2003;Mohazzab et al, 1994).
Chronic hyperglycemia is a cause of impairment of insulin biosynthesis and secretion. This process is called ?-cell glucose toxicity which is often observed under diabetic conditions (Evans et al, 2003).It is also known that lipotoxicity is also involved in the deterioration of ?-cell function found in type 2 diabetes (Kaneto et al, 2010).
Blighiasapida is a plant belonging to the family of Sapindaceae. It is commonly known as ackee. In Nigeria, it is called Gwanja Kusa (Hausa), Isin (Yoruba) and Okpu (Igbo). It is an evergreen tree of about 33 to 40ft (10-12m) with a dense crown of spreading branches. The leaves are compound with three to five pairs of oblong, ovate-oblong, or elliptical leaflets 1.5-3.0cm long. The seed of the fruit is not edible, whereas the fleshy aril is edible. The fruit is known to contain saponins, which are hemolytic (Aderinola et al., 2007).
Most of the earlier studies on Blighiasapida have been on the nutritional qualities of the root (Abolaji et al, 2007) and the leaves as a dry season feed resource for West African dwarf goats in the Northern savanna zone of Nigeria (Aderinola et al, 2007). The repellant potential of the fruit part components against stored-product insect pests (Khan and Gumbs, 2003) as well as neutropenia and thrombocytopenia effects of the aqueous and lipid extracts of the unripe fruit have been investigated in mice (Gardiner et al, 1996). More recently, the physicochemical properties of the oil from the fruit of the species and toxicological evaluation of the oil -based diet in Wister rats have been investigated (Oladiji et al, 2009).
However, the scanty information on the antioxidant activity of extract of Blighiasapida stem bark and its anti-hyperglycemic effect prompted this study. Tree bark is an important component of African traditional medicine as herbal medicine is still the main source of health care for the majority of Africans and in particular, Nigerians.
There has been increasing demand for the use of plant products with anti-diabetic activity. The prohibitively high cost, unavailability, uncertainty of use of the common anti-diabetic agents during pregnancy and undesirable side effects of these drugs have been some of the factors limiting their use and leading to a preference for anti-diabetic drugs of plants origin. This study is thus aimed at isolation of hypoglycemic agents from readily available Blighiasapida.
Chemicals: All chemicals used were of analytical grade and items are products of BDH and Sigma Chemical Ltd., UK and Accu-chek ® Advantage, Roche Diagnostic, Germany. Animals: Male albino rats (Ratusnorvegicus) weighing between 100g and 120g were used for the experiment. The rats were bred in the animal holding of the Department of Anatomy and Cell Biology, Obafemi Awolowo University, Ile-Ife and were maintained on standard rat pellets (Ladokun feeds, Ibadan, Nigeria), and were given water ad libitum.
Sourcing for the Tree Bark of Blighiasapida: A sizeable quantity of the tree bark of Blighiasapida was obtained from the compound of the Federal Polytechnic, Ado Ekiti, Nigeria.
Identification of Plant: The fruits and leaves of Blighiasapida plant were obtained from the compound of the Federal polytechnic, Ado Ekiti, Ekiti State, Nigeria and were used for the purpose of authentication of the identity of the plant at the Herbarium unit of the Department of Plant Biology, University of Ilorin, Ilorin, Nigeria. The voucher number of identification is UIH624.
Processing of sample and preparation of extract: The sample obtained was air-dried at room temperature for fifty-six(56) days until a constant weight was obtained.
The air-dried tree bark of Blighiasapida was pulverized.
100g of the pulverized sample was extracted with 800ml of distilled water for seventy-two (72) hours in an extractor. The aqueous extract was obtained by filtering with What man filter paper and subsequently freezedried in Armfield freeze-drier for ten (10) days. Induction of experimental diabetes mellitus: After an overnight fasting, rats were induced by intraperitoneal administration of alloxan monohydrate at a dose of 120mg/kg body weight. Alloxan monohydrate was freshly dissolved in distilled water and maintained on ice prior to use. Four days after the administration, the animals were fasted for 16 hours and blood glucose levels were determined in mg/dl using a digital glucometer (Accu-chek ®, advantage, Roche, Diagnostic, Germany) and animals which had basal glycemia levels of 125mg/dl were used in the experiment.
Experimental Design: Randomized Complete Block Design (RCBD) method was used. Eighty male albino rats were grouped as follows: All the animals were fed with vital finisher made up of maize and soya bean mainly. The administration of the extracts as written above was carried out every 24 hours for 21 days.
Analysis of the various parameters stated was carried out weekly after diabetes detection, for three weeks.
The fasting blood glucose levels of all groups were measured and then the extract dissolved in distilled water. The solution of the extract was administered to one of the diabetic groups orally at 100mg/kg body weight once a day for twenty-one (21) days.The diabetic control and untreated (without alloxan induction). Body weight and blood glucose levels of the groups were monitored daily, blood sample was obtained from the tail vein of the animals and their fasting blood glucose level was determined in mg/dl using a digital glucometer (Accu-chek ® Advantage, Roche Diagnostic, Germany). Five animals each were sacrificed from each of the four groups by chloroform anaesthesia and the blood and liver obtained from them. The plasma was obtained from the blood by using centrifuge at 3000g for 15 minutes. The plasma and liver so obtained were stored in phosphate buffer (0.1M, pH = 7.0) maintained below -20 0 C until required for analysis.
In vivoantioxidant assay: Liver tissues were homogenized with cold 1.5% KCl to make a 10% homogenate.
Determination of the activity of Catalase (CAT): Catalase activity was determined in the lysate using Aebi's method (Aebi, 1984).
Determination of the activity of Superoxide dismutase (SOD): This method is well described by Mccord and Fridovich (1969).
Determination of the activity of Glutathione Peroxidase (GPx): Glutathione peroxidase (GPx) was measured by the method described by Rotruck et al. (1973). Determination of reduced glutathione (GSH): Reduced glutathione (GSH) was measured by the method of Beatleret al. (1963). Determination of Malondialdehyde (MDA): Total amount of lipid peroxidation products present in the samples was estimated by the thiobarbituric acid (TBA) method which measures the malondialdehyde (MDA) reactive products according to the method of Ohkawa et al., (1979).
Determination of Protein Carbonyl Content: The protein carbonyl content was assayed according to a previous method of Levine et al (1990). Determination of Protein: Protein determination was carried out according to the method of Lowry et al., (1951) as described by Holme and Peck, (1998).
Statistical Analysis: Data were expressed as mean + S.E.M. of five replicates and subjected to one-way analysis of variance (ANOVA) followed by Duncan's multiple range test to determine significant differences in all the parameters. Values were considered statistically significant at P<0.05.
Blood Glucose Level: The administration of aqueous extract of Blighiasapida stem bark was found to significantly (P<0.05) reduce the blood glucose in diabetic albino rats at the end of the experiment (Table 1). The effect was more rapid in the first week of administration and compared favourably well with metformin-treated diabetic rats.
Weight gain or loss: There was a significant reduction (P<0.05) in the weight gained by the untreated diabetic rats when compared with the metformin-treated and extract-treated groups. Generally, the effect of treatment with 100mg/kg body weight of aqueous extract of B. sapida stem bark compared favourably well with that of metformin hydrochloride which is a known standard drug for diabetes.
Catalase activity: Specific activity of the antioxidant enzyme catalase was found to be increased (P<0.05) plasma and liver following administration of aqueous extract of B. sapida stem bark while the administration of metformin, a standard antidiabetic drug did not seem to have any ameliorative effect on the reduced specific activity of catalase in the plasma and liver of diabetic rats when compared with the untreated diabetic rats (Table 3).
Glutathione peroxidase (GPx) activities: A significant increase (P<0.05) was noticed in the specific activity of glutathione peroxide in the plasma of diabetic rat following administration of aqueous extract of B. sapida stem bark at the later stage of the experiment. On the other hand, the specific activity of glutathione peroxidase in the liver of diabetic rats did not increase but significantly reduced (P<0.05) during the course of the experiment, a result similar to the one obtained for the untreated diabetic rats (Table 4). Superoxide dismutase (SOD) activity: A significant increase (P<0.05) in the specific activity of superoxide dismutase was observed in the plasma and liver diabetic rats administered with aqueous extract of B. sapida stem bark similar to what was observed in those treated with metformin, a standard antidiabetic drug. However, the specific activity of superoxide dismutase in the plasma and liver of untreated diabetic rats was found to reduce significantly (P<0.05) during the course of the experiment (Table 5).
Reduced glutathione: Table 6 shows the effect of administration of aqueous extract of B. sapida stem bark on concentration of reduced glutathione (GSH) in plasma and liver of diabetic rats. A significant increase (P<0.05) in the oxidant was noticed in the plasma and 7). On the other hand changes in the concentration of malondialdehyde in plasma and liver tissues of diabetic rats did not follow a particular pattern following the treatment of diabetic rats with metformin hydrochloride, a standard antidiabetic drug.
Protein carbonyl: A significant reduction (P<0.05) was noticed towards the end of the experiment after an initial increase in the concentration of protein carbonyl in the plasma and liver tissues of diabetic rats following the administration of aqueous extracts of B. sapida stem bark (Table 8). A similar result was obtained for the diabetic rats treated with standard antidiabetic drug, metformin.
The increase in blood glucose concentration is an important characteristic feature of diabetes. Blighiasapida extract produced significant hypoglycemic effect on diabetic rats, and by day 14, the glucose levels tended towards normalcy as found in the control rats. Phytochemical screening of the aqueous extract of the root bark of B. sapida had indicated the presence of saponins (Saiduet al., 2012), which have been reported to possess hypoglycemic activity in diabetic rabbits (Abdel-Hassan et al., 2000).
The marked increase in the body weight in the B. sapida stem bark extract-treated rats could be attributed to the increase in the metabolic activity of their body systems. This clearly indicates that the plant extract increase glucose metabolism which enhanced body weight gain in rats. This observation was reported by Sunmonu and Afolayan (2013). According to these authors, Artemisia afra leaves and stem increased the body weight of diabetic rats. It is interesting to note that the effect of B. sapida stem bark aqueous extract at the dose of 100mg/kg body weight compared favorably well with metformin.
Diabetes mellitus is associated with an increase in reactive oxygen species (ROS) generation by mononuclear cells and an increased oxidative load resulting in oxidative damage to lipids, proteins and DNA. Acute hyperglycemia has been shown to result in an increase in blood pressure, which is prevented by antioxidants; this suggests that acute hyperglycemia probably causes increased generation of ROS.
Chronic hyperglycemia and subsequent augmentation of reactive oxygen species (ROS) deteriorate ? -cell functions and increase insulin resistance which leads to the aggravation of type 2 such as the nonenzymatic glycosylation reaction, the electron transport chain in mitochondria, and membrane-bound NADPH oxidase (Brownlee, 2001; Harrison et al., 2003; Mohazzab et al., 1994). Chronic hyperglycemia is a cause of impairment of insulin biosynthesis and secretion. This process is called ?cell glucose toxicity which is often observed under diabetic conditions. In diabetic state, hyperglycemia and subsequent production of ROS decrease insulin gene expression and finally bring about apoptosis. In addition, ROS are induced and involved in the ? -cell glucose toxicity. ? -cells are rather vulnerable to ROS due to the relatively low expression of antioxidant enzymes such as catalase, glutathione peroxide and superoxide dismutase. Therefore it is likely that ROS are involved in ? -cell deterioration found in diabetes (Evans et al., 2003). The potential mechanism of oxidative stress includes the reduction of antioxidant defense. In general, antioxidants such as phenolic compounds (tocopherols, flavonoids and phenolic acids), nitrogen compounds chlorophyll derivatives, amino acids and amines), carotenoids and ascorbic acid (Hall and Cuppett, 1997;Larson, 1988) compounds inhibit or delay the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing chain reaction. In this study, the levels of catalase, glutathione peroxidase and superoxide dismutase activities in plasma and liver tissues of diabetic group were significantly reduced and treatment with B. sapida stem bark aqueous extract generated the catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities not only on acute experiments but after 21 days of treatment. Decreased levels of CAT, GPx and SOD in the diabetic state may be due to inactivation caused by reactive oxygen species. In treated groups the increased CAT specific activity could be due to higher production of H 2 O 2 . It is possible that CAT activity which in turn would protect SOD inactivation by H 2 O 2 causes an increase in SOD activity. Increase in SOD activity would protect GPx and CAT against inactivation by superoxide anion (Blum and Fridovich, 1985). An increase in the level of reduced glutathione could thus be due to it been spared as a result of the protection offered by superoxide dismutase to glutathione peroxidase.
It is known that lipotoxicity is also involved in the deterioration of ? -cell function found in diabetes. The increase in free radicals in diabetic condition is suggested to be due to the increased lipid peroxidation and the damage to antioxidant defense system. Protein glycation and glucose autoxidation can generate free radicals that catalyze the lipid peroxidation (Altanet al., 2006). Any compound, natural or synthetic, with antioxidant activity might totally or partially alleviate this damage. In this study, direct effects of aqueous extract of B. sapida stem bark on malondialdehyde (MDA) levels in diabetes group were found to be higher than those in control group (P<0.05), indicating free radical generation via lipid peroxidation. Treatment of diabetes with the aqueous extract of B. sapida stem bark caused an eventual reduction in the MDA levels in plasma and liver after 21 days of treatment. Furthermore, direct effects of aqueous extract of B. sapidaextract on protein carbonyl levels in diabetes group were found to be higher than those in control group (P<0.05), indicating increased free radical generation via production of various kinds of glycated proteins such as glycosylated hemoglobin, albumin and lens. Treatment of diabetes with the aqueous extract of B. sapida stem bark caused a reduction in the levels of protein carbonyl in plasma and liver after 21 days of administration.
One of the major findings of this study is that oral administration of aqueous extract of B. sapida stem bark caused anti-hyperglycemic activity in alloxaninduced diabetes in experimental albino rats. The results also revealed that B. sapida stem bark aqueous extract caused a significant increase in the activities of catalase, glutathione peroxidase and superoxide dismutase in the plasma and liver of diabetic rats after 21 days of treatment. It is also observed that aqueous extract of B. sapida stem bark extract possess the capability of inhibiting both lipid and protein peroxidation in diabetes.
| Groups | Serum Glucose Level (mg/dl) | |||||
| 0 day | 7 th day | 14 th day | 21 st day | |||
| Untreated control | 91.50 + 1.94 a | 90.41 + 2.50 a | 90.21+ | 1.61 a | 88.10 + 2.02 a | |
| Diabetic control | 154.80 + 14.00 b | 172.41+ | 17.32 b | 203.50 +11.20 b | 253.00 + 13.20 b | |
| Diabetic + Aqueous extract | 154.80 + 14.00 b | 86.40+5.43 a | 74.00+4.48 c | 58.00 + 6.04 c | ||
| Diabetic + Metformin | 153.84 +10.26 b | 113.75 +5.41 c | 70.75 + 6.50 c | 57.00 + 9.60 | ||
| Groups | Average body weight of animals (g) | ||||
| 0 day | 7 th day | 14th day | 21 st day | ||
| Untreated control | 129.20 +2.30 a | 133.70 +1.09 a | 146.20+ | 1.12 a | 157.00 + 1.16 a |
| Diabetic control | 132.01 + 1.09 a | 125.20 +2.01 b | 112.00 +0.98 b | 98.20 +2.02 b | |
| Diabetic + Aqueous extract | 131.00 +6.06 a | 149.00 +7.12 c | 157. 00 +6.57 c | 124.00 +6.06 c | |
| Diabetic + Metformin | 132.00 + 2.96 a | 116.25 + 5.41 b | 127.50 ± 6.50 d | 147.50 +9.60 d | |
| Values are mean of five determinations + S.E.M. Values with different superscript in the row and column differ significantly | |||||
| (p<0.05) | |||||
| Tissue Group of animal | 0 day | 7 th day | 14th day | 21 st day | |||
| Plasma Untreated control | 5.86+ 0.14 a | 5.91+ 0.20 a | 5.86+ 0.16 a | 5.82+ 0.16 a | |||
| Diabetic control | 3.86+ 0.70 b | 3.52+ 0.20 b | 2.14+0.10 b | 1.27+ 0.80 b | |||
| Diabetic + Aqueous extract | 3.86+ 0.70 b | 4.25+ 0.90 c | 7.81+ 1.80 a | 15.66+ 3.20 c | |||
| Diabetic + Metformin | 5.30+ 0.28 a,a | 6.10+ 0.10 a | 2.10+ 0.07 b | 1.90+ | 0.62 b | ||
| Liver | Untreated control | 178.48+ 4.90 a | 179.32+ 3.20 a | 178.52+ 5.20 a | 176.05+ 5.10 a | ||
| Diabetic control | 92.26+ 6.30 b | 92.52+ 2. 10 b | 79.16+ 5.20 b | 49.56+ 6.20 b | |||
| Diabetic + Aqueous extract | 97.26+ 6.30 b | 195.02+ 3.93 c | 289.36+ 8.30 c | 190.85+ 3.20 a | |||
| Diabetic + Metformin | 97.30+ 0.28 b | 84.30+ 0.28 b | 87.00+ | 0.31 b | 65.80 + 0.63 | ||
| Specific activity of Glutathione peroxidase (Units/mg protein) |
| (XI0? 5 ) |
| Specific activity of superoxide dismutase (SOD (Units/mg protein) (x10 -3 ) | |||||||||
| Tissue Group of animal | 0 day | 7 th day | 14th day | 21 st day | |||||
| Plasma Untreated control | 10.88+ 1.23 a | 19.53+ 1.06 a | 22.11+ 1.00 a | 19.93+ 1.23 a | |||||
| Diabetic control | 7.91+ 1.40 b | 5.25+ 1.30 b | 3.28+ 1.0l b | 1.96+ 0.91 b | |||||
| Diabetic + Aqueous extract | 7.91+ 1.40 b | 27.12+ 4.05 c | 24.03+ 3.56 a | 28.64+ 7: 12 c | |||||
| Diabetic + Metformin | 8.10+ | 1.10 b | 12.90 + | 0.70 d | 17.20 + | 0.30 c | 28.70 + | 0.30 c , | |
| Liver | Untreated control | 55.87+ 9.65 a | 55.87+ 9.65 a | 66.01+ 7.51 a | 51. 78+ 1.20 a | ||||
| Diabetic control | 35.40+ 5.98 b | 19.87+ 4.43 b | 11.35+ 3.25 b | 9.36+ 1.54 b | |||||
| Diabetic + Aqueous extract | 35.40+ 5.98 b | 150.52+ 1.93 c | 161.80+ 2.27 c | 1153.95+ 12.11 c | |||||
| Diabetic + Metformin | 58.70+ 1.10 b | 12.90+ | 0.70 b | 17.20 + | 0.30 d | 28.70+ 0.30 d . | |||
| Values are mean of five determinations + S.E.M. Values with different superscript in the row and column differ significantly | |||||||||
| (p<0.05) | |||||||||
| Concentration of Glutathione (GSH) (mM/mg tissue) |
| Tissue | Group of animal | 0 day | 7 th day | 14 th day | 21 st day | |
| Plasma Untreated control | 1316.99+ 0.16 a | 1428.60+ 0.13 a | 1316.90+ 0.16 a | 1439.80+ 0.12 a | ||
| Diabetic control | 1619.69+ 0.08 a | 2002.10+ 0.07 b | 4698.70+ 0.09 b | 7023.60+ 0.06 b | ||
| Diabetic + Aqueous extract | 1619.69+ 0.08 a | 1372.55+ 0.05 a | 1065.36+ 0.06 a | 1125.82+ 0.04 a | ||
| Diabetic + Metformin | 280.00+0.11 b | 420.00+ | 0.11 c | 380.00+ 0.51 d | 500.00+ 0.17 c | |
| Liver | Untreated control | 1286.70+ 0.01 a | I286.70+ 0.01 a | 1096.40+ 0.01 a | 1193.00+ 0.01 a | |
| Diabetic control | 1513.10+ 0.08 b | 1735.20+ 0.08 b | 2012.30+ 0.08 b | 2523.20+ 0.06 b | ||
| Diabetic + Aqueous extract | 1513. 10+ 0.08 b | 1388.89+ 0.06 a | 1197.71+ 0.06 a | 1040.S5+ 0.05 a | ||
| Diabetic + Metformin | 1720.00+ 0.08 b | 2000.00+ 0.07 b | 1900.00+ 0.08 b | 3200.00+ 0.07 d | ||
| Values are mean of five determinations + S.E.M. Values with different superscript in the row and column differ significantly | ||||||
| (p<0.05) | ||||||
| Tissue | Group of animal | 0 day | 7 th day | 14 th day | 21 st day |
| Plasma Untreated control | 0.56+ 1.05E-05 a | 0.52+ 1.12 E-05 a | 0.59+ 1.05E-05 a | 0.60+ 1.03E-05 a | |
| Diabetic control | 0.46+ 6.66E-07 b | 0.68+ 8.63 E.07 b | 1.26+ 7.65E-07 b | 1.80+ 8.20E-07 b | |
| Diabetic + Aqueous extract | 0.46+ 6.66E-07 b | 1. 09+ 1. 11 E-06 c | 0.98+ 1.15E-06 c | 0.32+ 9.09E-07 c | |
| Diabetic + Metformin | 0.53+ 1.36E-05 a | 0.55+ 1.36 E-05 a | 0.74+ 1.81E-05 a | 0.53+ 1.39E-05 a | |
| Liver | Untreated control | 2.53+ 1.08E-05 a | 2.53+ 1.09 E-05 a | 2.47+ 1.11E-05 a | 2.51+ 1.08E-05 a |
| Diabetic control | 0.78+ 7.80E-07 b | 1.86+ 8.22 E-07 b | 2.60+ 6.11E-07 b | 3.82+ 8.52E-07 b | |
| Diabetic + Aqueous extract | 0.78+ 7.80E-07 b | 2.08+ 1.05E-06 c | 0.94+ 1.05E-06 c | 0.66+ 7.42E-07 c | |
| Diabetic + Metformin | 1.27+ 6.22E-06 c | 2.06+ 6.22E-06 c | 1.97+ 2.71E-05 a | 2.08+ 5.I2E-06 d | |
| Values are mean of five determinations + S.E.M. Values with different superscript in the row and column differ significantly | |||||
| (p<0.05) | |||||
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