Introduction

The increasing prevalence of diabetes mellitus in the present day world is a cause of concern to the mankind. Diabetes mellitus, whether of type I or type II category, is primarily characterized by either lack of insulin or its action which starts with derangement of carbohydrate metabolism to eventually entangle derangement of protein and lipid metabolism1.

Diabetes is a chronic disease characterized by disordered metabolism and in appropriately high blood sugar (hyperglycemia). Type 2 diabetes is sharply increasing globally, including many parts of the developing world, in major part as a consequence of the world wide “epidemic” of obesity, prior to and after the discovery of insulin, medicinal plants have been used to normalize glycemia in diabetic patients2,3,4.

A whole range of pharmacological agents are available to ameliorate the T2DM symptoms by different mechanisms. A reduction in insulin resistance at any stage of T2DM will improve glucose metabolism by allowing the endogenous insulin to be more effective. The use of different insulin sensitizers and secregatogues, either in single therapy or in combination, would help to improve glycemic control, either by increasing peripheral glucose uptake, improving insulin secretion, decreasing hepatic glucose output or reducing the influx of glucose to the body5.

Avandia manufactured by GlaxoSmithkline (GSK), was approved as an adjunct to diet and exercise to improve control of blood sugar levels. Avandia is approved to be used as a single therapy or used in combination with metformin and sulfonylurea, or with other oral anti-diabetes treatments6.

The WHO expert committee on diabetes mellitus recommendations of 19807 included investigation of hypoglycemic agents from plants used in traditional medicine. Nigella sativa oil have been used for treatment of experimentally induced dia­betes in animals based on its combined hypoglycemic and immunopotentiating effects that help in ameliorating the impaired immunity and infections associated with diabetes8,9.

Increased utilization of medicinal plants became a World Health Organization (WHO) policy in 1970. Plants and herbs are chemical factories that directly provide about 25% of currently used drugs and another 25% of drugs comprise chemically altered natural products10.

A number of natural products exhibit properties that could be used as remedies to improve glucose metabolism11 some plants extract can significantly reduce blood glucose levels and lipids, improving insulin sensitivity12.

Traditional antidiabetic plants provide useful source of new oral hypoglycemic compounds for development as pharmaceutical entities, or as simple dietary adjuncts to existing therapies. A scientific investigation of traditional herbal remedies for diabetes mellitus may provide valuable leads for the development of alternative drugs and therapeutic strategies alternative are clearly needed because of the in ability of current therapies for many rural populations, particularly in developing countries13.

One of the important issues regarding silymarin is that it may be accepted as a safe herbal product since no health hazards or side effects are known in connection with the proper administration of designed therapeutic dosages14. We therefore planned to investigate the insulinotropic effects of extract of natural product of N. sativa seeds and Silymarin on blood glucose and insulin levels of serum in adult male albino rats and its role in reducing the side effects of Avandia drug together with studying their effects on some liver and kidney function parameters.

Experimental

This study was carried out on 70 mature male Sprague dowly rats weighing 200-250 gm .b.wt each. They were divided into 7 equal groups (each of 10) as follows:-

Induction of diabetes:

(STZ diabetic groups). After induction of diabetes by injecting rats with STZ I.P in a dose of 50 mg/kg, rats with fasting blood glucose level more than 250mg/dl were considered diabetic.

I- The 1st group (STZ group)

Animals were served as diabetic non treated group for other diabetic group

II- The 2nd group (STZ + Avandia treated group)

Animals were given a daily oral dose of AVA (0.58 mg/100g.b.wt) dissolved in 1 ml Tragacanth gum as suspension for 4 weeks.

III- The 3 r d group (STZ+ Nigella sativa extract treated group)

Animals were received a daily oral dose of Nigella sativa extract (0.25gm/100g b.wt) for 4 weeks.

VI- The 4th group (STZ+ Silymarin extract treated group)

Animals were given dose of Silymarin extract (50mg/kg.b.wt) suspended in 1 ml CMC suspension orally for 4 weeks daily.

V- The 5th group (STZ + AVA + Nigella sativa extract treated group)

Animals were received a daily oral dose of AVA (0.58mg/100g b.wt) as previously mentioned combined

with Nigella sativa extract (0.25gm/100 b.wt), orally for4 weeks.

VI- The 6th group (STZ + AVA + Silymarin extract treated group)

Animals were received a daily oral dose of AVA (0.58mg/100g. b.wt) as prepared as mentioned above with a daily dose of Silymarin extract (50mg/kg.b.wt) orally for 4 weeks.

VII- The 7th group (control group)

Animals were served as normal control group given 1ml citrate buffer (PH=4.5) (The vehicle in which STZ was dissolved) daily orally for4weeks.

Blood sampling

After the end of the experiment, blood samples were collected after the end of 1st, 2nd, 3rd and 4th week post drugs administration from the retro orbital plexus using heparinized microhaematocrit capillary tubes into centrifuge tubes. Serum was harvested from blood without anticoagulant and used for determination of serum ALT & AST15, Lactic dehydrogenase16, Urea17, Creatinine18, Uric acid19, glucose20 and serum insulin was assayed using insulin – I125.

Kit 21 using radioimmunoassay kit supplied by Radioassay system laboratory Inc (England).

Statistical analysis

Data were collected and analyzed using the computer program SPSS / Pc+ (2001). The statistical method used was one way ANOVA test (F-Test) according to22.

Results and Discussion

The results of experiment revealed the following observations.

(1) Effect on some liver function parameters

(A) Effect on serum Alanine amino transferase (ALT) activity

Table (1) revealed that the induction of diabetes in rats by STZ elicited a marked increase in serum ALT along the entire period of the study when compared with control group.

Table 1: Effect of Avandia (0.58 mg/100g.b.wt), Nigella sativa (0.25gm/100g b.wt), Silymarin (50mg/kg.b.wt) and their combinations on serum alanine amino transferase (ALT) concentration (U/mI) in diabetic male albino rats (mean±SE). (N =7).

Means within the same column in each category carrying different litters are significant at (P < 0.05).

The administration of various drugs & their combinations for 28 days to diabetic rats afforded a significant decrease (P< 0.05) in serum ALT activity along the entire period of the experiment when compared with STZ treated group while administration of Avandia elicited highly significant increase in serum ALT activity when compared with control group.

(B) Effect on serum Aspartate amino transferase (AST) activity

Our results revealed that STZ afforded a marked increase in serum AST activity (P< 0.05) along the entire period of the study when compared with control group.

All treatments of diabetic rats elicited a marked decrease (P< 0.05) in serum activities of AST when compared with STZ except treating with Avandia showed marked increase in serum AST activity.

Diabetic rats along the course of the study except groups treated with Silymarin, Nigella sativa and combination of Avandia+ Nigella sativa after 3rd week and group treated with Avandia+ Silymarin after 4th week post drug administration which showed non significant changes (Table 2).

Table 2: Effect of Avandia (0.58 mg/100g.b.wt) ,Nigella sativa (0.25gm/100g b.wt), Silymarin (50mg/kg.b.wt) and their combinations on serum aspartate amino transferase (AST) concentration (U/mI) in diabetic male albino rats (mean ± SE). (N =7).

Means within the same column in each category carrying different litters are significant at (P < 0.05).

(C) Effect on serum lactic dehydrogenase enzyme (LDH) activity

STZ diabetic group showed significant increase in serum LDH activity along the entire period of the experiment when compared with control group. Whereas all treatments of diabetic rats for 28 days afforded a marked decrease (P< 0.05) in serum LDH activity along the course of the study when compared with STZ treated group except group treated with Avandia showed highly significant increase in LDH activity when compared with control group (Table 3 ) .

Table 3: Effect of Avandia (0.58 mg/100g.b.wt), Nigella sativa (0.25gm/100g b.wt), Silymarin (50mg/kg.b.wt) and their combinations on serum lactate dehydrogenase enzyme activity (LDH) (µIU/ml) in diabetic male albino rats (mean ± SE). (N =7).

Means within the same column in each category carrying different litters are significant at (P < 0.05)

(2) Effect on Urea, Uric acid and Creatinine:

Induction of diabetics by STZ induced a significant increase in serum urea, uric acid and creatinine along the entire period of the study when compared with control group.

Treatments of diabetic rats with various treatments elicited a significant decrease in serum urea, uric acid and creatinine along the course of the experiment when compared with STZ diabetic group except group treated with Avandia showed highly significant increase in serum urea, uric acid and creatinine when compared with control group (Table 4, 5, 6).

Table 4: Effect of Avandia (0.58 mg/100g.b.wt) ,Nigella sativa (0.25gm/100g b.wt) and Silymarin (50mg/kg.b.wt) and their combinations on serum urea concentration (mg/dl) in diabetic male albino rats (mean ± SE). (N =7).

Table 5: Effect of Avandia (0.58 mg/100g.b.wt)  ,Nigella sativa (0.25gm/100g b.wt), Silymarin (50mg/kg.b.wt) and their combinations on serum uric acid concentration (mg/dl) in diabetic male albino rats (mean ± SE).

Means within the same column in each category carrying different litters are significant at (P < 0.05).

Table 6: Effect of Avandia (0.58 mg/100g.b.wt) ,Nigella sativa (0.25gm/100g b.wt), Silymarin (50mg/kg.b.wt)  and their combinations on serum creatinine concentration (mg/dl) in diabetic male albino rats (mean ± SE). (N=7).

Means within the same column in each category carrying different litters are significant at (P < 0.05).

Effect on blood glucose levels

(A) Effect on serum Glucose

Concerning the effect of test drugs and their combinations on serum glucose level of diabetic rats, (table 7 ) showed that STZ afforded a marked elevation in serum glucose when compared with control group along the course of the study, whereas all treated groups revealed a significant decrease (P<0.05) in serum glucose level when compared with STZ non-treated group along the entire course of the experiment with the rank order of potency as antidiabetic as follows: Avandia + Silymarin>Silymarin> Avandia + Nigella sativa > Avandia > Nigella sativa. After the end of the experiment (4 weeks).

Table 7: Effect of Avandia (0.58 mg/100g.b.wt) ,Nigella sativa (0.25gm/100g b.wt), Silymarin (50mg/kg.b.wt)  and their combination on serum glucose concentration (mg/dl) in diabetic male albino rats. (mean ± SE). (N=7).

Means within the same column in each category carrying different litters are significant at (P < 0.05).

Table 8: Effect of Avandia (0.58 mg/100g.b.wt), Nigella sativa (0.25gm/100g b.wt), Silymarin (50mg/kg.b.wt) and their combinations on serum insulin levels (IU/ml) in diabetic male albino rats. (mean ± SE). (N=7).

Means within the same column in each category carrying different litters are significant at (P < 0.05)

(B) Effect on serum Insulin

(Table 8) reveals that STZ afforded a marked decrease in serum insulin of rats (P< 0.05) when compared with control group along the 4 weeks of the experiment. Whereas, administration of Avandia for 4 weeks elicited non-significant change in serum insulin level of diabetic rats when compared with STZ diabetic group along the course of the study. The other treatments of the diabetic rats for 4 weeks elicited a significant increase (P< 0.05) in serum level of insulin along the entire period of the experiment except with silymarin after the first week which revealed non-significant increase when compared with STZ treated group.

Discussion

The present study was an attempt to evaluate the hypoglycaemic effect of Avandia, Nigella sativa, silymarin each alone and the combination of Avandia with either N.sativa or silymarin when given to normal and diabetic rats for 28 successive days. Their effects on some on some liver function parameters (ALT, AST, and LDH) were also studied. Some kidney function parameters (serum urea, uric acid and creatinine), lipogram as well as the effect on insulin were also investigated).

Because of low cost, traditional medicinal plants also raise significant interest to prevent morbidity and mortality from chronic diseases where low or middle income populations are important23.

Effect on liver function parameters

Our results showed that Avandia, Silymarin, Nigella sativa and their combination on when given daily for successive 28 days afforded a significant decrease in serum ALT activity along the entire period of the experiment in hyperglycemic rats when compared with STZ group.

Our result seems to be conceivable with that previously reported24, 25. They reported that milk thistle have a protective effects on the liver and greatly improve its function since it is typically used to treat liver cirrhosis and chronic hepatitis (liver inflammation).

In addition, milk thistle extracts both prevent and repair damage from toxic chemicals and medications. Workers who had been exposed to vapors from toxic chemicals for 5 – 20 years were given silymarin for 30 days. They showed significant improvement in liver function tests (ALT & AST) and platelet counts26. The liver regenerating effect induced by silymarin results from stimulation of RNA polymerase enzyme in the nucleus of liver cells. This results in an increase of ribosomal protein synthesis which helps to regenerate hepatocytes27. On a similar ground, silymarin might also affect bone marrow.

Our results were in full agreement with Yadivet al.28. They reported that oral administration of rosiglitazone in diabetic patients significantly increased serum ALT and AST activities 2.5 times more than normal and leads to severe dysfunction, weight gain, oedema and milk dilutional anemia. Induction of diabetes by STZ afforded a significant elevation in serum ALT activity along the entire course of the study. A result which is supported by the results reported before29. She reported that the induction of diabetes by alloxan caused a significant elevation in serum ALT and AST along the course of the study (4 weeks). Treatments other than Avandia caused a significant reduction in the elevated serum transaminases activity along the entire course of the study when compared with STZ group alone. Indicating that these plant drugs succeeded in improving the status of injured liver caused by STZ. It has been reported that thymoquinone, one the active constituents of N.sativa have a hepatoprotective activity. An  in vitro study showed the protective effect against tert-butyl hydroperoxide (TBHP) induced oxidative damage to hepatocytes. The activity was demonstrated by a decreased leakage of ALT, AST and decreased trypan blue uptake30,31.

The hepato protective effect of N.sativa was supported also by the results of El-Dakhakhny et al.,32, and Mahmoud et al., 33. The same previous effects were observed on serum AST level with the exception of effects of N. sativa, Silymarin and their combination with Avandia which showed non-significant changes when compared with STZ group. This may be attributed to the fact that AST is not a liver specific enzyme.

Our results coincide also with Szilard, et al. 26 they reported that milk thistle and both extracts repair damage caused by toxic chemicals and medications. They exposed workers to vapors from toxic chemicals (Toluene and / or xylene) for 5-20 years, and were given either a standardized milk thistle

(80% silymarin) for 30 days. The workers showed significant improvement in liver function tests (ALT & AST) when compared with placebo.

On the same basis, milk thistle is used primarily to treat various liver diseases and dysfunctions including alcoholic cirrhosis, hepatitis (due to viral infection or drug – induced) as well as hepatic problems related to diabetes34-38.

Silymarin has liver regenerative effects by stimulating the enzyme known as RNA Polymerase in the nucleus of liver cells. This results in an increase of ribosomal protein synthesis which helps to regenerate hepatocytes27.

All treatments of diabetic rats for 28 days afforded  a marked decrease in serum  LDH activity along the entire course of the study  when compared with STZ diabetic group.

These effects could be discussed in the same manner as with ALT and AST as previously mentioned.

Effect on Kidney functions parameters

Concerning the effects of the test plant drugs on the kidney function parameters,Our results revealed that serum urea, uric acid and creatinine were significantly elevated in serum of STZ – diabetic rats compared to control group. Our results coincides with Jones et al., 39. They recorded that creatinine is increased in diabetic rats , they attributed this increase due to the deterioration of renal function induced by diabetes. Their findings were supported by the degenerative changes observed in the kidney in this study. While it has been found that serum creatinine is increased significantly in IDDM patients40.

The functional abnormalities in diabetic kidneys due to the increase in glomerular filtration rate (GFR) were attributed to the increase in both glomerular capillary pressure and flow 41. Treatment of diabetic rats with various treatments afforded a significant decrease in serum urea, uric acid and creatinine when compared with STZ diabetic group along the course of the study . Our results were reinforced with the results of Ledi et al., 42. They found that treatment of diabetic rats with rosiglitazone (Avandia) showed a significant decrease in urea, uric and creatinine by decreasing creatinine kinase. The other drugs may produce their effect also through this mechanism.

Effect on Glucose level

Concerning the effect of the test plant drugs on serum glucose level in diabetic rats. It is a well known fact that diabetes mellitus is a syndrome characterized by chronic hyperglycemia and disturbances of carbohydrate, fat and protein metabolism associated with absolute or relative deficiencies in insulin secretion or insulin action43.

The STZ treated group showed a marked increase in serum glucose level along the entire period of the experiment when compared with control group. These results were supported by the decreased insulin level in the STZ treated group and the significant increase in insulin level in all treated groups along the course of the study when compared with diabetic non-treated groups.

It has been reported that insulin is a hormone that has extensive effects on metabolism and other body functions, such as vascular compliance. Insulin causes cells in the liver, muscle and fat tissue to take up glucose from the blood storing it as glycogen in the liver and muscle and supporting use of fat as an energy source. When insulin is absent (or low), glucose is not taken up by body cells, and the body begins to use fat as an energy source for example by transfer of lipids from adipose tissue to the liver for mobilization as an energy source. As its level is considered control metabolic control mechanism, its status is also used as a control signal to other body systems (such as amino acid uptake by body cells). It has several other anabolic effects throughout the body. When control of insulin levels fails, diabetes mellitus results(44).

Our results were also in accordance with Guyton and Hall 45. They reported that most of the pathology of diabetes can be attributed to one of the following 3 major effects of insulin lack.

(a) Decreased utilization of glucose by the body cells, with a resultant increase in blood glucose concentration 300–1200 mg/dl.

(b) Markedly increased mobilization of fats from the fat storage areas, causing abnormal fat metabolism as well as deposition of lipids in vascular walls to cause atherosclerosis. A fact which is supported by the increased serum level of triglycerides and total cholesterol and its fraction LDL-C and VLDL-C.

(c) Depletion of protein in the tissues of the body.

Moreover, it has been found that rosiglitazone treatment decreased blood glucose concentration, increased plasma insulin concentration and preserved pancreatic islet mass46.

Thiazolidinediones increase glucose transport into muscle and adipose tissue by enhancing the synthesis and translocation of specific forms of the glucose transporter proteins47, They added also that thiazolidinediones exert their principal effects by lowering insulin resistance in peripheral tissue, but an effect to lower glucose production by the liver has been also reported.

It has been recorded also that Rosiglitazone a thiazolidinedione with a different side chain from those of troglitazone and pioglitazone, reduces plasma glucose levels and glucose production and increases glucose clearance in patients with type 2 diabetes mellitus. Insulin sensitivity, pancreatic beta cell function and surrogate markers of cardiovascular risk factors are significantly improved by rosiglitazone (48). Our results were reinforced also by Wagstaff and Goa49. They recorded that rosiglitazone 4mg/day provides significant antihyperglycemic efficacy, and generally tolerate, both as monotherapy and in combination with other antihyperglycemic agents, in patients with type 2 diabetes mellitus who do not have active liver disease.

Furthermore, it has been shown that sulphonylureas and rosiglitazone significantly improved long term glycemic control by restoring insulin secretion and reduced postprandial hyperglycemia in peripatetic subjects and similarly effective in elderly by and non elderly populations with type 2 diabetes. In the same direction50. Rosiglitazone enhanced the insulin sensitivity in the liver, skeletal muscle and adipose tissue in type 2 diabetes47,51.

TZD enhance insulin action and improve glycemic control by increasing peripheral glucose disposition and reducing hepatic glucose output through activation of PPAR-Y 52.

The hypoglycaemic effect of N. sativa observed in this study in diabetic rats together with the increased level of insulin was in full agreement with Al-Awadi et al., 53, Al-Haderet al.8. Their results indicated that the volatile oil of N.sativa afforded a hypoglycaemic effect in diabetic rats and rabbits.

It has been also reported that treatment of STZ diabetic rats with the plant extracts (N.sativa) and (Silymarin) produce a significant increase of serum insulin level54. Recently, Benhaddou–Andaloussi et al., 55 used in vitro bioassays to identify target tissues and demonstrate the insulinotroipic and insulin – like activities of an ethanol extract of Nigella sativa. Haddad et al., 56, reported that N. sativa has been found to rank high among the antidiabetic plants, and most recommended by traditional practitioners. It has been also reported that N. sativa possesses a significant hypoglycemic activity which is though to be due to the essential oil present (30&31). Eskander et al.,57 recorded the hypoglycemic effect of herbal formulation of N. sativa plant in alloxan induced diabetic rats.

The hypoglycemic effect of the volatile oils of N. sativa was confirmed before58. They found that oral administration of the volatile oil of N. sativa to STZ – diabetic rats afforded a significant elevation in serum insulin level.

The hypoglycemic effect of Milk thistle (Silymarin) observed in this study in STZ diabetic rats is supported with the findings of (59). They stated that the extract of milk thistle (Silymarin) can help people to lower the amount of sugar bound to haemoglobin in blood, as well as reducing fasting blood sugar level. Silymarin is also effective in improving glycaemic profile in patients with type II diabetes.

Conclusions

From the obtained results, we report that Avandia drug is not an ideal antidiabetic drug, since it showed many side effects represented by high level of AST, ALT, Urea, Uric acid,Creatinine. Moreover, the combination of Avandia and Silymarin gave the best results on blood sugar level.

Recommendations

So we recommended the use of the combination of Avandia and Silymarin which is known as a hepatoprotective drug in treatment of diabetic patients to avoid the proven hazardous effect of Avandia on cardiovascular system and to overcome the side effects of Avandia on liver as well as on male and female fertility.

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