Effect of Gundelia tournefortii on some biochemical parameters in dexamethasone-induced hyperglycemic and hyperlipidemic mice

The aim of this study was conducted to evaluate the effect of Gundelia tournefortii on some biochemical parameters in hyperglycemic and hyperlipidemic mice. Male albino mice were induced hyperglycemic and hyperlipidemic by daily injection of dexamethasone 1 mg/kg of body weight intramuscularly (i.m.), the mice randomly divided into five groups (6-8 mice in each group). The group 1: served as negative control group; the group 2: injected with dexamethasone at dose 1 mg /kg.b.w.i.m and served as positive control group; the groups 3, 4, 5: treated with extract of G. tournefortii at doses: 75, 150, 300 mg/kg.b.w. orally respectively companied with injection of dexamethasone 1 mg/kg.b.w.i.m. All treatment were once daily for 22 days. Dexamethasone treatment lead to significant increase in levels of glucose, cholesterol, and triglyceride, and significant decrease of body weight, without any effect on level of total protein. G. tournefortii extract treatment at doses: 75 mg/kg.b.w. resulted significant decrease levels of glucose, and body weight. Beneficial effect were seen when mice treated with G. tournefortii at dose of 300 mg/kg.b.w. that lead to significant decrease in levels of glucose, triglyceride, and cholesterol. These results indicate the usefulness of G. tournefortii extract as hypoglycemia and hypolipidemia in dexamethasone treated mice.


Introduction
Diabetes mellitus is a metabolic disorder characterized by resistance to the action of insulin, insufficient secretion, or both (1).The major clinical manifestation of the diabetes state is hyperglycemia, however, insulin deficiency and/or insulin resistance also are associated with disturbance in lipid and protein metabolism (2).WHO indicates that diabetes mellitus is one of the major killers of humans in our time (3).Management of diabetes without any side effect is still a challenge to the medical system, this had led to an increasing demand for natural products with antidiabetic activity and fewer side effects (4).
Dexamethasone a synthetic glucocorticoids and a wide spread anti-inflammatory drug, and induce a decrease in insulin sensitivity (5), or insulin resistance and an elevation in the level of serum glucose and lipids (6).So this drug is used for the induction of hyperglycemia and hyperlipidemia in mice and rats as model for type -2 diabetes mellitus (7,8).
Throughout history, humans have derived many uses and benefits from the plants found in their own region (9).Gundelia tournefortii GT (kuub; Arabic and kanger; Kurdish names) from the Asteraceae (compositae) family.It is an nature born plant, native to Asian -temperate zones of western Asia, manly Cyprus, Egypt, Jordan, Turkey, Azerbaijan, Turkmenistan and Iraq, its leaves, seeds and stems are used as food sources (10).It is recorded that the water extracts of G. tournefortii roots were containing phenols, glycosides, tannins, flavonoids, carbohydrates, proteins, alkaloids and nitrate (11,12), and saponins (13).It is used to enhance gingivas and as an appetizer (14), also fresh seeds of it are used in pikles and also are effective diuretics (15), and inhibition of α-amylase activity (16).G,tournefortii also have an effect on platelet aggregation (17), and can decrease some cardiovascular risk factors, and decrease atherosclerosis (18).This study was conducted to evaluate the effects of G,tournefortii roots hot water extract in dexamethasone induced hyperglycemic and hyperlipidemic mice.

Plant materials:
Naturally grown G. tournefortii plants were bought from vegetable market in Duhok city.The plant was classified at faculty of Agriculture and forestry / Duhok University, fresh roots were used in this study.

Plant extract
Boiled water extract was prepared by weighing and cutting up a certain weight of plant roots into small pieces, plant pieces were put in conical flask and submerged by 1cm of distilled water, the mixture heated in shaking water bath at 95C • for 30 minutes (19).The mixture was filtrated though a piece of cotton and several layers of gauze which put on the funnel mouth and left for 24 hr to complete the filtration process and the filtrate stored in dark bottles in the freezer.Dry matter was 0.56 gm per 50 ml of extract which is determined by using (Freeze dryer with shell freezer-LFD-55085-Daihan lab tech Co. LTD Korea).

Animals
Male albino mice weighing between 24.6 and 33.2 gm were procured from Veterinary college /Duhok University, they were housed under standard conditions of temperature (22 ±3C • ) with a 14:10 dark light cycle.The animals were fed with standard diet and water ad libitum.

Induction of hyperglycemia
Dexamethasone sodium phosphate ampoules concentration 8 mg/2 ml (H-tech international enterprises Co.ltd) were used for inducing hyperglycemia at a dose (1 mg/kg of body weight intramuscularly diluted by normal saline and volume of injection was 5 ml/kg.b.w.) daily for 22 days (7).

Experimental design
Animals randomly were divided into five groups, each consisting of 6-8 mice.Group 1: Served as negative control group; Group 2: received dexamethasone 1.0 mg/kg of body weight and served as positive control group; Group 3: treated with dexamethasone 1.0 mg/kg of body weight.plus plant extract containing dry matter 75 mg/kg of body weight; Group 4: received dexamethasone 1.0 mg/kg of body weight plus plant extract containing dry matter 150 mg/kg of body weight; Group 5: treat with dexamethasone 1.0 mg/ kg of body weight plus plant extract containing dry matter 300 mg/kg of body weight.
Dexamethasone were given by intramuscular rout and volume of injection 5 ml/kg.,While G. tournefortii extract were given orally at 5 ml/kg.b.w. by gavages needle.All treatment was once daily and lasted for 22 days.

Sample collection
Blood samples were collected at zero,11 and 22 days after overnight fasting from the orbital plexus of vein into clean dry centrifuge tubes allowed to clot, serum was separated after centrifugation at 1500 rpm for 15 minutes (20), Glucose, total cholesterol, triglyceride and total protein were estimated calorimetrically using (Biolabo standard kits, France).

Statistical analysis
All data analyzed by one way analysis of variance, the specific group differences were determined using duncan multiple range test; the accepted level of significance was P<0.05 (21).

Results
As shown from table 1 the results reflect the effect of dexamethasone treating alone or in combination with different doses of G. tournefortii extract on the body weight in mice, the results show that dexamethasone causes a significant decrease in body weight after 22 days when compared with zero time in the same group, also dexamethasone combination with G. tournefortii extract causes significant decrease in body weight in dose 75 mg/kg.b.w.when compare with dexamethasone treated group, and the doses 150 and 300 mg /kg.b.w lead to significant decrease in body weight when compared with zero time in same groups.(6)(7)(8) in each group, Data is the mean ± SEM.Different letters indicate significant differences between groups horizontally and vertically at P < 0.05.
The results in table 3 show that dexamethasone treating in mice cause a significant elevation in the serum triglyceride level when compared with the normal group,but in the groups that received G. tournefortii extract doses 150,300 mg/kg.b.w.there was a significant reduction in serum triglyceride level after 22 days of treatment when compared with dexamethasone treated group.
The level of total serum cholesterol as shown in table 4 were increased in the group of mice which received dexamethasone when compared with the normal, also this table indicates that treatment with G. tournefortii extract cause a reduction in serum cholesterol levels after 11 days in the group that received 75 mg/kg B.W. and at 11,22 days of treatment in the other groups when compared with dexamethasone treated group, the more significant result was in the group of mice which received 300 mg/kg b.w. of the extract.There is no significant changes in serum total protein level in dexamethasone treated group when compared with the normal group,also in the groups of mice which treated with all doses of G. tournefortii (Table-5).

Discussion
The present study demonstrated that dexamethasone treatment produced a significant decrease in body weight when compare with zero time, this result agree with other studies in normal rats (22,23).This may be due to that dexamethasone stimulate production of myostatin leading to muscle atrophy and decrease of body weight (22), (23) reported that dexamethasone have an ability to decrease body weight by prevent enter of glucose to the cells leading to loss of calories.
Dexamethasone produced significant increases in serum glucose, and this result agree with (5,7,24,25) in mice, and (8) in rats, but disagree with (26) in rats.The increase of serum glucose level may be due to decrease insulinstimulated glucose uptake in muscle (27), or dexamethasone disrupts insulin-mediated recruitment of glucose transporters at the cell surface (28,29).Also dexamethasone stimulate gluconeogenesis in liver cells by stimulate glucose-6-phosphatase (30,31).
Dexamethasone lead to significant increase in serum cholesterol, similar result reported by (32) in normal and diabetic rats, and disagree with (33) in human.The increase of serum cholesterol level may be due to that dexamethasone inhibit nitric oxide synthesis which have a role in regulation of lipid levels in blood (26).(6)(7)(8) in each group, Data is the mean ± SEM.Different letters indicate significant differences between groups horizontally and vertically at P < 0.05 Triglyceride levels also increased significantly when we use dexamethasone to induce hyperglycemia and hyperlipidemia, (8,34) reported similar results in rats, and (35) in human.This may be due to that dexamethasone stimulate production and secretion of lipoproteins mainly very low density lipoprotein from liver that rich in triglyceride (36), or may dexamethasone cause insulin resistance which decrease effect of insulin on liver and adipose tissue leading to secret triglyceride from liver and prevent ability of tissue to remove lipoproteins from blood (37).
In our study, treatment mice with G. tournefortii leads to significant decrease in serum glucose levels in all three doses when compare with dexamethasone group.The possible mechanism is due to presence of flavonoide in it (11) which stimulate secretion of insulin from pancreas leading to increase its level in blood (38), or flavonoide may increase the insulin sensitivity (39), also flavonoide activated peroxisoe-proliferator-activated-receptor (PPAR) that regulate the transcription of gene involved in lipid and glucose homeostasis and metabolism within the cell (40), this effect of flavonoide may be like flavonoide of soybean (38).Also may flavonoide of G. tournefortii decrease glucose absorption from intestine through decrease processes of glucose transports depended on calcium (41), flavonoide can acts as antioxidant by increasing antioxidant enzymes (42), similar observation reported by (43) when flavonoide of trifolium alexandrium acts as antioxidant in streptozotasin induced diabetic rats.
Also decreases of serum glucose level may be due to inhibition of α-amylase by G. tournefortii (16) that found in saliva and pancreatic secretions which degrade starch first to oligosaccharide and then to maltose and glucose, (44) that phaseolus acutifolius A. Gray, pistacia atlantica, and paranychia argentea have hypoglycemic activity through α-amylase inhibition.as wall as may the hypoglycemic activity of G. tournefortii is due to presence of sterol in it (17) and in study of (45) shown that extract of Anacandina occidentale that contain sterols cause significant decrease in blood glucose level in normal dogs, and (46) referred that sterol presence in extraction of centaure serdis L.varmentime lge leaves cause significant decrease of blood glucose level in rats.Sterol acts by increase blood insulin levels through stimulation its secretion from β-cells in pancreas (47).
A significant decrease in blood serum cholesterol levels was observed in G.tournefortii treatment in 150,300 mg/kg.b.w. and this results agree with (18) in rabbit.Possible explanation of lowering cholesterol is may due to presence of flavonoids that have antioxidant and hypolipidemic activity which act by inhibition of lipoprotein oxidation and increasing low density lipoprotein receptor activity (48), similar result showed by (49) in mice when treated by soybean seeds that contain flavonoide.Also may be the lowering of cholesterol levels due to presence of saponin in G. tournefortii (13) report that treatment by seeds of fenugreek that contain saponin lowers cholesterol levels in human with type 2 diabetes.The effect of saponin may be due to hydrolysis of saponin to sapogenin in gastrointestinal tract which stimulate secretion of bile acids by liver, also saponin decrease absorption of cholesterol in intestine (50), or effect of saponin may due to stimulate liver to convert cholesterol to bile acids (51).The other mechanisms of lowering cholesterol is that sterol of G. tournefortii (17) acts to decrease estrification of cholesterol in gastric cells and reveres the unestrified cholesterol again to intestine (52).
Significant decrease of blood triglyceride levels were observed in mice treated with G.tournefortii, our result agree with (18) in rabbit, the decrease of blood triglyceride levels may due to flavonoide of G. tournefortii have antioxidant effect that increase insulin's activity as hypolipidemic (53), or flavonoide may affect in cellular lipid homeostasis by the down regulation of sterolregulatory-element-binding-protein (SREBP) and its target genes in the liver which are involved in the synthesis of triglyceride (54).
All treatment doses of G.tournefortii with dexamethasone lead to significant decrease of body weight when compare with zero time in same group, our result may be due to the non significant lowering of serum total protein level.The decrease in body weight observed might be the result of protein wasting due to unavailability of carbohydrate for utilization as an energy source (55).
Our study indicate the usefulness of G. tournefortii extract as hypoglycemia and hypolipidemia in dexamethasone treated mice.

Table ( 1
): Effect of Gundelia tournefortii extract on body weight (g) in dexamethasone treated mice.

Table 2
(2)indicating that dexamethasone treatment causes a significant increase in serum glucose level in mice when compared with the normal group,also the table show that treatment with G. tournefortii extract at doses 75,150, 300 mg/kg b.w.causes a significant decrease in glucose level when compared with dexamethasone treated group,the more significant effect was in the group of mice which received 75, 300 mg/kg.b.w. after 22 days and it was close to the normal level.Table(2): Effect of Gundelia tournefortii extract on serum glucose level (mg/dl) in dexamethasone treated mice.

Table ( 5
): Effect of Gundelia tournifortii extract on serum total protein (g/dl) in dexamethasone treated mice.