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130
Journal of Stress Physiology & Biochemistry, Vol. 6 No. 4 2010, pp. 28-41 ISSN 1997-0838 Original Text Copyright © 2010 by OmAli Y. El-Khawaga; Abou-Seif M.A; El-Waseef A. and Negm A.A
ORIGINAL ARTICLE
Hypoglycemic, Hypolipidemic and Antioxidant Activities of Cleo-me droserifolia in Streptozotocin-Diabetic Rats
OmAli Y. El-Khawaga1* ; Abou-Seif M.A1.; El-Waseef A.1 and Negm A.A1.
1 Biochemistry Department, Faculty of Science, Mansoura University, Mansoura, Egypt.
* Corresponding Author: dr_elkhawaga@yahoo. com
*Current address: Faculty of Community, King Khalid University, Rejal Almaa 61956, P. Box.
76, Kingdom of Saudi Arabia,
Fax: 0096672580857
Received September 28, 2010
Background: Diabetes mellitus (DM) is characterized by absolute or relative deficiencies in insulin secretion and/or insulin action associated with chronic hypoglycemia clinical research has confirmed the efficacy of several plant extracts in the amelioration of diabetic disorders. Aim of the work hypolipidemic and antioxidant activities of the aqueous extract of Cleome droseriloia was investigated in streptozotocin (STZ)-induced diabetic rats.
Methods and Results: A single dose of STZ (60mg/Kg body weight) produced a decrease in hepatic glycogen and GSH contents, hepatic glucose-6-phosphate dehydrogenase, superoxide dismutase, activities and HDL- cholesterol, whereas, hepatic glucose-6-phosphatase activity and level of MDA, cholesterol, triglyceride, LDL-cholesterol and VLDL-cholesterol were increased. An aqueous extract of Cleome droseriloia (500, 750 and 1000mg/Kg or Gliclazide (100mg/Kg) was administered orally once daily for two weeks to STZ-induced diabetic rats ameliorated hyperglycemia, improved lipid profile, amelioration of antioxidants and restored the metabolic enzymes of glucose to the normal value in the liver of STZ-treated rats. In addition, the administration of Cleome droserifolia induced the secretion of insulin from pancreatic rats. Furthermore, the hypoglycemic efficacy of one dose of aqueous extract Cleome droserifolia has extended to 12 hours. The effects produced by Cleome droserifolia extract were found to be comparable with that of gliclazide.
Conclusion: The present results suggested that Cleome droserifolia could be used as antidiabetic complement of diabetes mellitus. This may be related to its insulin-induction action.
key words: Cleome droserifolia, streptozotocin, insulin, glucose, lipids, metabolic enzymes, Antioxidants.
ORIGINAL ARTICLE
Hypoglycemic, Hypolipidemic and Antioxidant Activities of Cleo-me droserifolia in Streptozotocin-Diabetic Rats
OmAli Y. El-Khawaga1*; Abou-SeifM.A1.; El-Waseef A.1 andNegm A.A1.
1 Biochemistry Department, Faculty of Science, Mansoura University, Mansoura, Egypt.
* Corresponding Author: dr_elkhawaga@yahoo. com
* Current address: Faculty of Community, King Khalid University, Rejal Almaa 61956, P. Box.
76, Kingdom of Saudi Arabia,
Fax: 0096672580857
Received September 28, 2010
Background: Diabetes mellitus (DM) is characterized by absolute or relative deficiencies in insulin secretion and/or insulin action associated with chronic hypoglycemia clinical research has confirmed the efficacy of several plant extracts in the amelioration of diabetic disorders. Aim of the work hypolipidemic and antioxidant activities of the aqueous extract of Cleome droseriloia was investigated in streptozotocin (STZ)-induced diabetic rats.
Methods and Results: A single dose of STZ (60mg/Kg body weight) produced a decrease in hepatic glycogen and GSH contents, hepatic glucose-6-phosphate dehydrogenase, superoxide dismutase, activities and HDL- cholesterol, whereas, hepatic glucose-6-phosphatase activity and level of MDA, cholesterol, triglyceride, LDL-cholesterol and VLDL-cholesterol were increased. An aqueous extract of Cleome droseriloia (500, 750 and 1000mg/Kg or Gliclazide (100mg/Kg) was administered orally once daily for two weeks to STZ-induced diabetic rats ameliorated hyperglycemia, improved lipid profile, amelioration of antioxidants and restored the metabolic enzymes of glucose to the normal value in the liver of STZ-treated rats. In addition, the administration of Cleome droserifolia induced the secretion of insulin from pancreatic rats. Furthermore, the hypoglycemic efficacy of one dose of aqueous extract Cleome droserifolia has extended to 12 hours. The effects produced by Cleome droserifolia extract were found to be comparable with that of gliclazide.
Conclusion: The present results suggested that Cleome droserifolia could be used as antidiabetic complement of diabetes mellitus. This may be related to its insulin-induction action.
key words: Cleome droserifolia, streptozotocin, insulin, glucose, lipids, metabolic enzymes, Antioxidants.
Diabetes mellitus is probably the fastest growing metabolic disorder in the world and it is a major source of morbidity in developing countries. Once regarded as a single disease entity, diabetes mellitus is now regarded as a heterogeneous group of diseases characterized by a state of chronic hyperglycemia, which causes a number of secondary complications like cardiovascular, renal, neurological and ocular disorders (Miller, 1991). Diabetes mellitus is characterized by hyperglycemia together with biochemical alterations of glucose and lipid metabolism (Mann,
1998). Liver tissues are insulin dependent tissues, which play a pivotal role in glucose and lipid homeostasis and are severally affected during diabetes mellitus (Seifter, 1982). Liver tissues participate in the uptake, oxidation and metabolic conversion of free fatty acids, synthesis of cholesterol and triglycerides . During diabetes mellitus, a profound alteration in the concentration and composition of lipid occurs (Sochor et al.,1985). Decreased glycolysis, impeded glycogenesis and increased gluconeogenesis are some of the changes of glucose metabolism in the diabetic liver (luzi,1998).
There is increasing evidence that complications related to diabetes mellitus are associated with oxidative stress induced by the generation of free radicals[6]Garg et al.1996. Free radicals result in the consumption of antioxidant defenses which may lead to disruption of cellular functions and oxidative damage to membranes and enhance susceptibility to lipid peroxidation. Increased generation of reactive oxygen species (ROS) and lipid peroxidation have been found to be involved in the pathogenesis of many diseases of known and unknown etiology and in the toxic actions of many compounds (Andallu and Varadacharyulu, 2003).
In consequence, antioxidants play an important role to protect the human body against damage caused by reactive oxygen species (Giugliano et al. 1996). In diabetes mellitus, oxidative stress has been found to be mainly due to an increased production of oxygen free radicals and a sharp reduction of antioxidant defences (Laaksonen and Sen 2000).
Streptozotocin is one of the most commonly used substances to induce diabetes in the rat. This toxin causes the death of pancreatic p-cells by alkylation of DNA resulting in reduced synthesis and release of insulin. Furthermore, it has been shown to be involved in the fragmentation of DNA by means of production of reactive oxygen species (). The diabetes induced by streptozotocin is associated with polydipsia and loss in body weight. The sulfonylurea, glibenclamide (GLB) and the semi-essential amino acid taurine (TR), shown to have hypoglycemic and hypocholesterolemic effects in animal models of diabetes (Pari and Venkateswaran, 2004), were used as reference
compounds. Hence, compounds with both
hypoglycemic and antioxidant properties would be useful as antidiabetic agents (Opara, 2002). Many herbal plants are considered useful means to prevent and/or ameliorate certain disorders, such as diabetes mellitus atherosclerosis and other
complications (Scartezzini and Speroni, 2000). Among these herbal resources, Cleome droseriloia are selected for the present study. The aqueous and chloroformic extracts has been used as hepatoprotective, hypoglycemic and antimicrobial agents. Moreover the ethanolic extract has
antihistaminic, relaxant and tranguilizing effects (El-Askary, 2005).
Therefore, this context was aimed to investigate the activity of an aqueous extract of the Cleome droseriloia as hypoglycemia, hpolipidemic and
antioxidant substance in streptozotocin-induced diabetic rats as a model for type II diabetes .
MATERIALS AND METHODS
• Preparation of plant extracts:
Cleome droseriloia was purchased from herbalists at local commercial sources in Mansoura, Egypt. The Cleome droseriloia herb was spread over the bench and left for drying in the shade, then reduced to a powder. Decoction of the plant material was prepared by boiling 100 g of the dry plant material in 1.5 liters of tap water for 2 minutes and then filtered after 10 minutes. To minimize the volume of the decoction, it was left to dry in a wide container over a boiling water bath (Kamal et al., 1991).
• Chemicals:
Streptozotocin, thiobarbituric (TBA), phenazin methosulphate (PMS), nitrobluetetrazolium (NBT), NADH, NADPH and glucose-6-phosphate were purchased from Sigma chemical Company
(St.Louis, MO, USA). Gliclazide (Servier
Laboratories, France) was purchased from local pharmacies in Mansoura, Egypt. All other
chemicals were of analytical grade.
1. Experimental animals:
All experimentals were performed using adult male albino rats, with an average body weight of 100 to 120 g purchased from Theodore Bilharz Research Institute, Giza, Egypt. The rats were housed in steel mech cage and provided with commercial standard diet and tap water ad libitum.
Induction of Experimental Diabetes.
The rats were fasted for 12 hours before the induction of diabetes with STZ. The rats were injected intraperitoneally with freshly prepared solution of STZ (60 mg STZ/kg body weight)
(Montilla et al., 2004). Seventy two hours after diabetes induction, blood samples were collected from the tail vein for measuring blood glucose levels by One-Touch blood glucose meter from Lifescan (Johnson& Johnson Company, USA).
2. Experimental Procedure
I.Toxicitv test of an aqueous extract of the Cleome droseriloia
Fifty male rats were divided into five groups of 10 each and were administered orally with aliquot doses of the aqueous extracts of Cleome droseriloia (250-1500 mg/Kg). Mortality was observed after 72 hours Litchfield and Wilcoxon, 1949.
II. Hypoglycemic action of Cleome droseriloia
A total of 50 rats were divided into 10 groups of 5 rats each as follows: group 1. normal control rats; group 2-4, normal rats treated with Cleome droseriloia extract (500, 750, 1000 mg/kg
consequently) daily by an intragastric tube for two weeks. group 5, normal control Gliclazide (100mg/ kg body weight); group 6, STZ-diabetic control rats; group 7-9, STZ-diabetic rats treated with Cleome droseriloia extract (500 , 750, 1000 mg/kg consequently) daily by an intragastric tube for two weeks ,group 10, STZ-diabetic rats treated with gliclazide (100mg/ kg body weight) daily by an intragastric tube for two weeks. At the end of two weeks, the rats were deprived of food overnight and sacrificed by decapitation. Blood samples were collected and the livers were immediately dissected out, washed in ice-cold saline, blotted dry and weight for measuring various biochemical parameters.
III. Preparation of homogenates.
An accurately weight piece of liver tissue homogenized in ice-cold 0.9 % saline using a
Teflon pestle connected to a homogenizer motor. The liver homogenate was diluted to yield 5%(w/v) liver homogenate. The homogenate was centrifuged at 5000 rpm for 30 minutes at 4 0C to remove cell debris and nuclei. The resulting supernatant was used for biochemical analysis.
IV. Biochemical Analysis.
Serum glucose concentrations were estimated by the method of Trinder (1969) using a commercial available diagnostic kit (Diamond Diagnostics, Egypt). Glycogen content in tissue homogenates was determined as described by the method of Damsbo et al.(1991). Hepatic glucose-6-phosphate dehydrogenase activity was measured by applying the method of Chan et al. (1965). Hepatic glucose-6-phosphatase was assayed according to the method of Rossetti et al. (1993). Superoxide dismutase (SOD) activity was determined using the method of Nishikimi et al. (1972). Reduced glutathione (GSH) content in the liver homogenate was estimated by the method of Beutler et al. (1963). Lipid peroxidation in the liver tissues was estimated by measuring the formation of thiobarbituric acid reactive substance (TBARS) as an index of malondialdehyde (MDA) production according to the method of Draper and Hadley (1996). Cholesterol content was quantified in the liver homogenate by the method of Ellefson and Caraway (1976) using a commercial available diagnostic kit (Spectrum Diagnostics, Egypt), triglycerides content was determined by the method of Bucolo and David (1973) using a commercial available assay kit (Spectrum Diagnostics, Egypt) and HDL-cholesterol content was determined by applying the method of Gordon et al(1977).
Statistical analysis: The results are expressed as means ± SD. Statistical analysis was performed according to the method of Murray (1982). Data
were analyzed using unpaired Students t-test. P values of < Q.Q5 were considered to be statistically significant.
RESULTS
The acute oral toxicity of Cleome droseriloia extract showed neither toxicity nor mortality up to 15QQ mg/kg. Thus, the maximum tolerated dose of the extract was found to be 15QQ mg/kg body weight. Also no toxic signs observed over 72 hours of administering the extract. Table 1 demonstrates serum blood glucose levels in normal-control, normal STZ-treated and normal- Cleome droseriloia treated rat groups after two weeks of treatment. A highly significant increase in blood glucose level is observed in STZ-control untreated rats compared with that of normal rats. In contrast, a highly significant decrease in the mean value of blood glucose level is observed in either STZ-treated with gliclazide or Cleome droseriloia extract (5QQ, 75Q, 1QQQmg/kg body weight) respectively as compared to STZ-control rats without treatment. Also, table 1, shows serum insulin levels in the STZ-treated groups with gliclazide or Cleome droseriloia extract. It has shown that, the insulin level of STZ-diabetic rats is 1.1 ^U/ml while the insulin levels of STZ-diabetic rats treated with (5QQ, 75Q, 1QQQ mg/kg body weight) Cleome droseriloia and gliclazide are 1.3, 1.5, 1.8 and 1.9 ^U/ml respectively. Table 2, illustrates hepatic glycogen content, glucose-6-phosphate dehydrogenase and glucose-6-phosphatase activities of different experimental rat groups. There are no significant change in glycogen content and glucose-6-phosphate dehydrogenase and glucose-6-phosphatase activities in normal treated with 5QQ mg/kg as compared to normalcontrol group . A highly significant increase in the hepatic glycogen content and glucose-6-phosphate
dehydrogenase activity are observed in Cleome droseriloia extract (75Q, 1QQQmg/kg body weight) normal treated rats as compared to normal-control rats. A highly significant increase in hepatic glycogen content, glucose -6-phosphate dehydrogenase activity are observed in either gliclazide or Cleome droseriloia extract (5QQ, 75Q, 1QQQ mg/kg) STZ-treated rats as compared to STZ-control rats without treatment. No significant change is observed in the activity of glucose-6-phosphatase of normal treated rats with either gliclazide or (5QQ, 75Q, 1QQQ mg/kg body weight) of Cleome droseriloia extract compared to the corresponding value of normal- control rat group although a highly significant decrease in its activity was observed in either gliclazide or Cleome droseriloia extract (5QQ, 75Q, 1QQQmg/kg body weight) STZ-treated rats as compared to STZ-control rats without treatment. Table 3 represents superoxide dismutase activity and reduced glutathione and malondialhyde contents in the liver of different experimental groups. The activity of superoxide dismutase is highly significant increase in the liver of normal rats treated with (5QQ, 75Q, 1QQQmg/kg body weight) of Cleome droseriloia extract compared to normal-control rat group. On the other hand, no significant changes were observed in the contents of hepatic GSH and MDA of rats treated with either (5QQ, 75Q, 1QQQmg/kg body weight) of Cleome droseriloia extract or gliclazide but a significant increase in the content of hepatic GSH and a significant decrease in the level of MDA of normal treated rats by 1QQQmg/kg
Cleome droseriloia compared to normal-control group. Moreover, there are a highly significant increase in the activity of SOD and GSH content but a highly significant decrease of the level of MDA are observed in the groups treated with either (5QQ, 75Q, 1QQQmg/kg body weight) of Cleome droseriloia extract or gliclazide STZ-diabetic groups compared to STZ- control groups without treatment. Table 4, shows the lipid profiles as total cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol contents in the liver of different experimental rat groups. A highly significant decrease in total cholesterol, triglycerides, LDL-cholesterol contents but a significant increase in HDL-cholesterol content are observed in hepatic homogenates of STZ-diabetic rats treated with either (5QQ, 75Q, 1QQQmg/kg body weight) of Cleome droseriloia extract or gliclazide as compared to STZ-diabetic control group. No significant changes is observed in total cholesterol, HDL- cholesterol in the liver of normal rats treated with (5QQ, 75Q, 1QQQmg/kg body weight) of Cleome droseriloia extract or gliclazide as compared to normal-control group. Also, no significant changes is observed in the contents of triglyceride and LDL-cholesterol of normal rats treated with Cleome droseriloia extract (5QQ mg/kg) compared to normal rats. On the other hand, a significant decrease is observed in the level of triglycerides and LDL-cholesterol of treated rats with Cleome droseriloia extract (75Q, 1QQQ mg/kg) or gliclazide as compared to normal-control group.
Table і: Effect of two weeks treatment with Cleome droseriloia extract on blood glucose and insulin levels of normal and STZ-diabetic rats.
Groups Blood glucose (mg/dl) Insulin level (^U/ml)
Normal Diabetic Normal Diabetic
Control 1Q3.48±9.75 4Q8.Q7±27.9 1.1
Treated with Cleome droseriloia (5QQmg/kg) 85.92±5.64** 232.53±2Q.33++ 1.3
Treated with Cleome droseriloia (75Qmg/kg) 76.Q7±7.99 ** 175.Q6±23.22++ 1.5
Treated with Cleome droseriloia (1QQQmg/kg) 75.75±6.96 ** 144.15±5.315++ 1.8
Treated with Gliclazide 8Q.17±5.16** 165.Q6±23.9++ 1.9
The results are expressed as mean ± SD for five rats in each group. ** highly significant compared to normal-control groups.
++ highly significant compared to diabetic-control groups.
Table 2: Effect of two weeks treatment with Cleome droseriloia extract on hepatic glycogen content, glucose-6-phosphate dehydrogenase(G6PD) and glucose-6-phosphatase(G6Pase) activities in the liver of normal and STZ-diabetic rats.
Groups Glycogen content (mg/g wet tissue) G6Pase (^mole/min/g wet tissue) G6PD (U/g wet tissue)
Normal Diabetic Normal Diabetic Normal Diabetic
Control 12.17±2.53 4.58±Q.619 2.38±Q.48 5.62±Q.51 16.26± 1.85 8.3±.Q91
Treated with Cleome droseriloia(5QQmg/kg) 18.Q2±6.36NS 6.Q7±Q.558++ 2.29±Q.62NS 3.49±Q.39++ 18.76±3.Q1NS 11.6±2.54+
Treated with Cleome droseriloia (75Qmg/kg) 22.43±2.79 ** 8. 7±1.Q47++ 2.Q1±Q.431 NS 3.Q6±Q.34++ 2Q.84±1.31** 13.37±3.67+
Treated with Cleome droseriloia1QQQmg/kg) 22.98±2.57 ** 9.73±Q.689++ 1.78±Q.418 NS 2.4Q±Q.39++ 22.76±Q.5Q** 15.92±4.Q9++
Treated with Gliclazide 18.71±2.79** 6.11. ±Q.596++ 2.Q9±Q.6Q9 NS 3.26±Q.67++ 18.52±1.73NS 1Q.9Q±1.54++
The results are expressed as mean ± SD for five rats in each group.
** highly significant compared to normal-control groups. ++ highly significant c
Table 3: Effect of two weeks treatment with Cleome droseriloia extract on superoxide
dismutase(SOD) activity, reduced glutathione (GSH) and malondialdehyde (MDA) contents in the liver of normal and STZ-diabetic rats.
Groups SOD (U/g wet tissue) GSH (mmole /g wet tissue) MDA (nmole/g wet tissue)
Normal Diabetic Normal Diabetic Normal Diabetic
Control 2631±78.21 973±73.74 1.62±Q.299 Q.688±Q.Q93 2Q9.31±1.85 3Q5.7±27.Q2
Treated with Cleome droseriloia( 5QQmg/kg) 3Q52±167.5** 2Q16±199.Q3++ 1.73±Q.17NS 1.16±Q.14++ 173.4±33.1NS 23Q±1Q.86++
Treated with Cleome droseriloia (75Qmg/kg) 3Q84±1Q8.3 ** 2345±93.56++ 1.9±Q.167 NS 1.19±Q.16++ 171.6±48.7NS 19Q.6±8.96++
Treated with Cleome droseriloia(1QQQmg/kg) 3245±119.25 ** 2484±135.16++ 2.15±Q.212* 1.413±Q.Q9++ 14Q.Q±4Q.59* 165.8±16.4++
Treated with Gliclazide 2997±91.69** 2Q54 ±85.34++ 1.77±Q.15NS Q.894±Q.Q9++ 177.1±29.2NS 189.4±14.5++
The results are expressed as mean ± SD for five rats in each group.
** highly significant compared to normal-control groups. ++ highly significant compared to diabetic-control groups. NS not significant
Table 4: Effect of two weeks treatment with Cleome droseriloia extract on total cholesterol,
triglycerides, HDL-cholesterol and LDL-cholesterol contents in the serum of normal and STZ-diabetic rats.
Groups Cholesterol (mg/g dl serum) Triglycerides (mg/g dl serum) HDL-Cholesterol (mg/g dl serum) LDL-Cholesterol (mg/g dl serum)
Normal Diabetic Normal Diabetic Normal Diabetic Normal Diabetic
Control 144±15 2Q4±27 228±29. 4 281±14.1 68±8.9 38±6 38±11 1Q9±3Q.3
Treated with Cleome droseriloia (5QQmg/kg) 139±15NS 17Q±8.6+ 2Q4±22NS 246±8.5++ 7Q.4±3 NS 43±3 NS 27±14NS 77±1QNS
Treated with Cleome droseriloia (75Qmg/kg) 118±12 NS 154±11++ 184±5.7 * 233±14.Q3++ 73±6 NS 49±5+ 15±4.1** 58±14++
Treated with Cleome droseriloia (1QQQmg/kg) 1Q6±4.5 NS 139±17++ 138±11** 222±12++ 74±4.8 NS 53±4.6++ 7.4±3** 41.16±19++
Treated with Gliclazide 131±6.6NS 164.±1Q + 192±13* 244±14.5++ 71±6 NS 45±4+ 22±8* 69±12+
The results are expressed as mean ± SD for five rats in each group.
** highly significant compared to normal-control groups.
++ highly significant compared to diabetic-control groups.
NS not significant
DISCUSSION
LHypoglycemic and hypolipidemic effects of the Cleome droseriloia extract:
STZ injection resulted in diabetes mellitus which is probably due to the destruction of ß -cells of islets of langerhans as proposed by many authors (Beppu et al., 2QQ6). It is generally accepted that severe diabetes (SD) is of IDDM type and mildly diabetes (MD) is of NIDDM type (Maiti et al., 2QQ5). This effect is being depicted by the high level of blood glucose in animals. The discovery and development of new and more effective diabetic treatments from plants is one of the main goals of the present day and chemical research (Tiwari and Madhusudana, 2QQ2). In the present study, a detailed account has been given to the hypoglycemic and hypolipidemic effects of the Cleome droseriloia in STZ- induced diabetic rats. The expanded hypoglycemic effect of Cleome droseriloia till two weeks may be attributed to its long efficacy on glucose uptake by the lived cells when compared to the effect of gliclazide. In diabetes mellitus, the disorders of carbohydrates and lipid metabolism play predominant role in diabetic complications (Sridhar et al., 2QQ5). This fact has been observed in the present results in which decreased the levels of hepatic glycogen content, glucose-6-phosphate dehydrogenase activity, HDL-cholesterol content and increased the concentrations of hepatic glucose-6-phosphatase activity, total cholesterol, triglycerides and LDL-cholesterol when compared to normal-control group. The hepatic glycogen contents are significantly lowered in STZ-control group when compared to normal-control group. The decrease in glycogen contents in the liver homogenates of STZ-diabetic group may be attributed to the depression of glycogenesis pathway in the liver of STZ-diabetic group (Ghosh et al., 2QQ4). In the present
study, it was observed that the activity of G6PD was significantly decreased in the liver homogenates of STZ-diabetic rats when compared to the corresponding activity of that in the normal control group. The deficiency of the hepatic G6PD in STZ-diabetic rats may be attributed to the decrease in blood insulin levels which enhances the glucose uptake by the liver cells leading to stimulate glucose oxidation by pentose phosphate pathway. These findings are in agreement with the previous studies which reported that, G6PD activity was decreased in STZ-induced diabetic rats (Maiti et al., 2004). Further more, the deficiency of G6PD activity may participate in the building up of glucose and consequently increase the susceptibility of type 2 diabetes mellitus as a result to the reduction in blood insulin levels (Gaskin et al., 2001). Glucose-6-phosphatase(G6Pase), an enzyme located mainly in the liver and catalyzes the terminal step in both gluconeogenesis and glycogenolysis. Thus, an increase in G6Pase activity in the liver homogenates of STZ-diabetic rat cells may cause a marked increase in the rate of glucose formation and also decreases glucose usage (Aistonelal., 1999). Therefore, the elevated G6Pase activity may become a compensatory pathway for building up glucose to compensate the requirements of liver cells for energy from glucose (Ashokkumar and Pari, 2005) . The present results demonstrate that, STZ-induced diabetic rats showed significant increase in the hepatic levels of cholesterol, triglycerides and LD-cholesterol with a significant decrease in hepatic HDL-cholesterol level. These results are in accordance with those of other investigators (Pari and Latha, 2002). The elevation of lipid profiles in STZ-diabetic rats may be attributed to an increase in the rate of lipolysis with a decrease in lipogensis leading to release more fatty acids into the blood circulation (Agardh et al.,
1999). Further more, the increased activity of G6Pase caused formation of excess NADPH which is important for fatty acid biosynthesis and the synthesis of fats from carbohydrates. The elevation of fatty acids concentrations may contribute to triglycerides biosynthesis (Seifter, 1982). In addition, insulin deficiency will lead to a decrease in the activity of lipoprotein lipase and an increase in the metabolism of free fatty acids from peripheral fat depots (Ahmed et al., 2001).The present results demonstrated that, the treatment with Cleome droseriloia significantly ameliorated the adverse influence of streptozotocin, such as: a lowered blood glucose levels, an increased hepatic glycogen contents, an improved lipid profiles and a regulation in the metabolic enzymes of glucose utilization as compared to rats administered with streptozotocin alone. These results are in consistent with those of other studies using different other hypoglycemic plants (Prince et al., 2004).
2. Antioxidant activity of the Cleome _ droseriloia extract:
Insulin deficiency in the diabetic patients results in the impairment of glucose utilization leading to an increased generation of oxygen free radicals a reduction of antioxidants which may play an important role in both oxidative stress and etiology of diabetic complications (Opara, 2002). In our experimental model of diabetes mellitus it was observed that STZ injection produced a significant decrease in hepatic superoxide dismutase (SOD) activity and glutathione (GSH) content accompanied by a significant increase in MDA content in comparison to the normal control group. These finding are in agreement with the previous studies which have reported on the reduction of hepatic SOD activity and GSH content with the elevation of MDA in STZ-induced diabetic rats (Zhang and Tan, 2000). The elevation of mean
concentration of MDA in STZ-injected group may be due to the hypoinulinemia that increases the activity of fatty acyl coenzyme -A -oxidase, which initiates p-oxidation of fatty acids, resulting in lipid peroxidation. Increased lipid peroxidation impairs membrane functions by decreasing membrane fluidity and changing the activity of membrane bound enzymes and receptors (Baynes, 1991). These lipid peroxidation products are more cytotoxic and stable than reactive oxygen species (ROS) that reacts with cellular constituents (Esterbauner et al., 1991). Besides these negative effects, MDA is a modulator of signal transduction pathways that disturb cellular activities (Grune et al., 1997). The increase of ROS and/ or lipid peroxidation products (MDA) could inactivate and reduce the hepatic SOD activity in STZ-diabetic rats. Furthermore, the decrease of SOD activity might be attributed to the following reasons: i) Hyperglycemia activates various biochemical pathways such as glucose auto-oxidation, and activation of protein kinase C which in turn overproduce oxidants like superoxide (O2) and hydroxyl (-OH) radicals as well as hydrogen peroxide (H2O2) (Abou-Seif and Youssef, 2004). ii) The increase of glycsylated SOD that leads to the inactivation of this enzyme (Aria et al., 1989). Reduced glutathione (GSH) participates in the cellular defense system against oxidative stress by scavenging the free radicals and reactive oxygen intermediates and it is important in the regulation of the cellular redox state. A decline in its cellular level in diabetes has been considered to be indicative of increased oxidative stress (Mclennan et al., 1991). Thus, the decrease in hepatic GSH levels in STZ-diabetic rats might be in part, attributed to the inhibition of glutathione reductase (GSSG-R) activity which is responsible for regeneration of GSH from its oxidized form. Blum and Fridovich (1985) showed that GSSG-R is
inactivated by superoxide anion (O2-). Therefore, hepatic containing reduced SOD activity might have enhanced flux of (O2) that potentially could damage GSSG-R and decrease GSH content in the liver tissues. In addition, the decrease in GSH level might reflect a direct reaction between GSH and free radicals generated by STZ (Ananthan et al., 2QQ4). The present results showed that the treatment of STZ-diabetic rats with Cleome droseriloia enhanced the activity of G6PDH. G6PDH plays an important role in glutathione synthesis that according to the following reports: G6PDH maintains the levels of the NADPH. In turn, NADPH maintains the level of GSH in liver cells and GSH protects cells against the oxidative damage of ROS (Mehta et al., 2QQQ). Finally, Cleome droseriloia induced an increase in hepatic GSH content which might enhance the GSH/GSSG ratio and decrease hepatic lipid peroxidation (MDA) and therefore, improve glucose regulation. It has been illustrated that the GSH/GSSG ratio plays a critical role in glucose homeostasis of diabetes mellitus because thiol groups are important in intracellular and membrane redox state (Soto et al., 1998).
Conclusion: the improvement of SOD activity, GSH content and decreased lipid peroxidation recorded after Cleome droseriloia treatment of STZ-diabetic rats might suggest a treating influence of Cleome droseriloia against STZ action that might be mediated through neutralization of oxygen free radicals produced by STZ. In addition, the aqueous extract of Cleome droseriloia stimulates insulin secretion from ß-pancreatic cells of rats. Therefore, the hypoglycemic action of Cleome
droseriloia may be attributed to its
stimulation of ß-cells for insulin secretion and its antioxidant properties. Further
clinical investigations will be conducted prior utilization as a safe oral
hypoglycemic agent.
REFERENCES
Abou-Seif, MA. And Youssef, AA. 2004: Evaluation of some biochemical changes in the diabetic patients. Clin Chim Acta, 346: 161-170.
Agardh, CD.; Bjorgell, P. and Nilson, EP. 1999: The effect of tolbutamide on lipoproteins and lipoprotein lipase and hormone sensitive lipase. Diab Res Clin Pract, 46: 99-108.
Ahmed, I.; Lakhani, MS.; Gillett, M.; John, A. and Raza, H. 2001: Hypotriglyceridemic and hypocholesterolemic effects of antidiabetic Momordica charantia fruit extract in streptozotocin induced diabetic rats. Diabetes Res. Clin. Pract. 51(3): 155-161. Aiston, S.; Trinh, KY.; Lang, AJ; Newgard, CB. And Agius, L. 1999: Glucose-6-
phosphatase overexpression lowers glucose-6-phosphate and inhibits glycogen synthesis and glycolysis in hepatocytes without affecting glucokinase translocation. Evidence against feedback inhibition of glucokinase. J Biol Chem, 274: 24559-24566.
Ananthan, R.; Latha, M.; Ramkumar, KM.; Pari, L.; Baskar, C. and Narmatha, BV.2004: Antidiabetic effect of Gtmnema montanum leaves. Nutrition, 6: 379-386.
Andallu, B. and Varadacharyulu, N.C 2003: Antioxidant role of mulberry leaves in streptozotocin-diabetic rats. Clin Chim Acta, 338: 343.
Aria, K.; Lizuka, S.; Tada, Y. ; Oikawa, K. and Taniguch,N. 1989: Increased in the
glycosylated form of erythrocyte Cu, Zn-superoxide dismutase in diabetes and else association of the nonenzymatic glucosylation with the enzyme activity. Biochem Biophys Acta, 924: 292-296.
Ashokkumar, N. and Pari, L. 2005: Effect of N-benzoyl-D-phenylalanine and metformin on carbohydrate metabolic enzymes in neonatal streptozotocin diabetic rats. Clin Chim Acta, 351 (1-2): 105-113.
Baynes, JW. 1991: Role of oxidative stress in development of complications in diabetes. Diabetes 40: 405-412.
Baynes, JW. 1995: Reactive oxygen in the etiology and complications of diabetes. In Ioannides, C. and Flatt, PR.(eds.). Drug, diet and disease. Volume 2: mechanistic approach to diabetes. Hertfordshire: Ellis Horwood Limited 203-231.
Beutler, E.; Duron, O. and Kelly, MB 1963: Determination of blood glutathione. J Lab Clin Med, 61: 882-890.
Blum, J. and Fridovich, I. 1985: Inactivation of glutathione peroxidase by superoxide radical. Arch Biochem Biophys, 240: 500508.
Bucolo, G. and David, H. 1973: Enzymatic determination of serum triglycerides quantitatively. Clin Chem, 19: 475-481.
Chan TK.; Todd, D and Wong, CC. 1965: Tissue levels in erythrocyte glucose-6-phosphate dehydrogenase deficiency. J Lab Clin Med, 6: 936-940.
Damsbo, P.; Vaag, A.; Hother-nielsen, O. and Beck-Nielsen, H.1991: Reduced glycogen synthase activity in skeletal muscle from obese patients with and without type 2 diabetes mellitus. Diabetologia 34(4): 239245.
Draper, W. and Hadley, M. 1990: Indirect determination of oxygen free radical. Methods Enzymol, 186: 421-431.
Ellefson, RD. and Caraway, WT. 1976: Fundamentals of clinical chemistry. Tietz, N.W. (ed.), pp 506-576.
Esterbauner, H.; Schaur, RJ. And Zollner, H. 1991: Chemistry and biochemistry of 4-hydroxynonenal, malondialehyde and related aldehydes. Free radical Biol Med, 11: 81-128.
Garg MC; Ojha, S. and Bansal, D.D 1996: Antioxidant status of streptozotocin-diabetic rats. Indian J Exp Biol, 34: 264271.
Gaskin, RS.; Estwick, D. and Peddi, R. 2001: G6PD deficiency: its role in the high prevalence of hypertension and diabetes mellitus. Ethn Dis, 11: 749-754.
Ghosh, R.; Sharatchandra, KH.; Rita, S. and Thokchom, IS.2004: Hypoglycemic
activity of Ficus hispida in normal and diabetic albino rats. Indian J of pharmacology, 36(4):222-225.
Giugliano, D.; Ceriello,A. and Paolisso, G. 1996: Oxidative stress diabetic vascular complications. Diabetes Care, 19:257-267.
Gordon, T.; Castelli, WP.; Hjortland, MC, Kannel, WB and Dawber, TR. 1977: High density lipoprotein in protective factor against coronary heart diseases, the Framingham study. Amer J Med, 62: 707-714.
Grune, T.; Siems, WG. Ad Petras, T. 1997: Identification of metabolic pathways of the lipid peroxidation product, 4-hydroxynonenal, in in situ perfused rat kidney. J Lipid Res, 38(8): 1660-1665.
Kamel, MS.; Ohtanti, K.; Kurokawa, T.; Assaf, MH.; El-Shanawany, MA.; Ali, AA.; Kasai, R. Ishibashi, S. and Tanaka,
O.1991: Studies on Balanites aegyptaca fruits, an antidiabetic Egyptian folk medicine. Chem Pharm Bull, 39(5): 12291233.
Laaksonen, D.E. and Sen, C.K.2000: Exercise and oxidative stress diabetes mellitus. In: Handbook of oxidants and antioxidants in exercise, Sen, CK.; Packer, L. and Hanninen, O. (eds.), Amsterdam, Elsevier, pp 1105-1136.
Litchfield, JT and Wilcoxon, FA. 1949: Simplified method of evaluating dose effect experiments. J Pharmacol Exp Ther 96: 99-133.
Luzi, L. 1998: Pancreas transplantation and diabetic complications. New England J Med, 339: 115-117.
Maiti, R.; Jana, D.; Das, UK. And Ghosh, D. 2004: Antidiabetic effect of aqueous extract of seed of Tamarindus indica in streptozotocin -induced diabetic rats. J Ethnopharmacol, 92(1): 85-91.
Mclennan, SV.; Heffernan, S.; Wright, L.; Rae, C.; Fisher, E. ;Yue, DK. And Turtle, J.1991: Change in hepatic glutathione metabolism in diabetes. Diabetes, 40: 344-348.
Mehta, A.; Mason, PJ. And Vulliamy, TJ. 2000: Glucose-6- phosphate dehydrogenase deficiency. Baillieres Best Res Clin Haematol, 13: 21-38.
Miller, G.T. 1991: Coronary heart disease and associated characteristics in tropical development community. Trans-R. Soc. Trop.Med. Hyp.,85: 324-335.
Montilla, p.; Barcos, M.; Munoz, MC; Munoz-Castaneda JR; Bujalance,I. and Tunez, I. 2004: Protective effect of Montilla-Moriles appellation red wine on oxidative stress
induced by streptozotocin in the rat. J Nutr Biochem 15(11): 688-693.
Murray, RS. 1982: Schaums outline series of theory and problems of probability and statistics, Singapore, McGraw-Hill Book Company, vol, 8: pp 265-298.
Nishikimi, M.; Roa, NA. and Yogi, K. 1972: The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophy Res Commun, 46: 849854.
Pari, L. and Latha, M. 2002: Effect of Cassia auriculata flowers on blood sugar levels, serum and tissue lipids in streptozotocin diabetic rats. Singapore Med J 43: 617621.
Prince PS.; Kamal, N. and Menon, VP. 2004: Antidiabetic and antihyperlipidaemic effect of alcoholic Syzigium Cumini seeds in alloxan induced diabetic albino rats. J Ethnopharmacol, 91: 209- 213.
Rossetti, L.; Lee, YT; Ruiz, J.; Aldridge, S.
;Shamoon, H and boden, G. 1993:
Quantitation of glycolysis and skeletal muscle glycogen synthesis in humans. Am J Physiol, 295: 761-769.
Scartezzini, P.; speroni, E. 2000: Review on some plants of Indian traditional medicine with antioxidant activity. J. Ethnopharmacol. 71: 23-43.
Seifter,S. and England, S. 1982: Energy
metabolism. In: The liver; biology and pathology, Arias, I.;Papper, M. and
Schacter, D. (eds.), New York, Reven Press, pp 219-249.
Sochor, M.;Baquer, NZ. And McLean, P.1985:Glucose over and under untilization in diabetes: comparative
studies on the change in activities of enzymes of glucose metabolism in rat kidney and liver. Mol Physiol. 16:51-68.
Soto, CP.; Perez, BL.; Favari, LP.And Reyes, JL. 1998: prevention of alloxan-induced
diabetes mellitus in the rat by silymarin. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 119(2): 125-129.
Sridhar, SB.; Sheetal, UD.; Pai, MR. and Shastri, MS. 2005: Preclinical evaluation of the antidiabetic effect of Eugenia jambolana seed powder in streptozotocin-diabetic rats. Brazilian J Med Biol Res, 38(3): 463470.
Tiwari, AK. And Madhusudana, RJ, 2002: Diabetes mellitus and multiple therapeutic approaches of phytochemicals: present status and future prospects. Current science, 83: 30-38.
L. Pari and S. Venkateswaran, Protective role of Phaseolus vulgaris on changes in the fatty acid composition in experimental diabetes, Journal of Medicinal Food 7 (2004), pp. 204-209.
T.E. Tenner Jr., X.J. Zhang and J.B. Lombardini, Hypoglycemic effects of taurine in the alloxan-treated rabbit, a model for type 1 diabetes, Advances in Experimental and Medical Biology 526 (2003), pp. 97-104.
Trinder, P. 1969: Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem, 6: 24-27.
Zhang, XF. And Tab, BK. 2000: Antihyperglycemic and antioxidant properties of andrographis paniculata in normal and diabetic rats. Clin Exp Pharmacol physiol, 27: 358-363.
Beppu, H., Shimpo, K., Chihara, T., Kaneko, T., Tamai, I., Yamaji, S., Ozaki, S., Kuzuya,
H., Sonoda, S., 2QQ6. Antidiabetic effects of dietary administration of Aloe arborescens Miller components in multiple low-dose streptozotocin-induced diabetes in mice: Investigation on hypoglycemic action and systemic absorption dynamics of aloe components. J. Ethnopharmacol. 1Q3, 46S-477.
Maiti, R., Das, U.K., Ghosh, D., 2QQ5. Attenuation of hyperglycemia and hyperlipidemia in streptozotocin induced diabetic rats by aqueous extract of seed of Tarmarindus indica. Biol. Pharm. Bull. 2S (7), 11721176.
Montilla, M. Barcos, M.C. Munoz, J.R. Munoz-Castaneda, I. Bujalance and I. Tunez, Protective effect of Montilla-Moriles appellation red wine on oxidative stress induced by streptozotocin in the rat, The Journal of Nutritional Biochemistry 15 (2QQ4), pp. 688-693.
Roy, R. Sehgal, B.M. Padhy and V.L. Kumar, Antioxidant and protective effect of latex of Calotropis procera against alloxan-induced diabetes in rats, Journal of Ethnopharmacology 102 (2QQ5), pp. 47Q-473
