Introduction

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 [1]. Diabetes mellitus is characterized by hyperglycemia together with biochemical alterations of glucose and lipid metabolism [2].

Liver tissues are insulin dependent tissues, which play a pivotal role in glucose and lipid homeostasis and are severely affected during diabetes mellitus [3] .

Decreased glycolysis, impeded gl\coueneis increased gluconeogenesis are “some of the changes of -(-11L1C0SL metabolism in the diabetic liver [4].

Many medicinal plants are considered useful means to prevent and/or ameliorate certain disorders,  such as diabetes mellitus,atherosclerosis and other metabolic disorders [5].

Among these plant resources, the green leaves of morns alba are selected for the present study. The morns alba are cultivated in Egypt.

Therefore, this study was thus initiated with the aim of evaluating the possible hypoglycemic activity of an aqueous extract of the green leaves of morns alba in streptozotocin-induced diabetic rats.

Materials and Methods

Preparation of Plant Extract

The green leaves of mores as1ba were left for drying in the shade Lind then reduced to a powder. A total of 200 g of the dried plant material was extracted with 05 liter of distilled water by the method of continuous hot extraction. The extract was evaporated to dryness in a wide container over a boiling water bath in front of an air current. A semisolid residual extract was obtained (3.7 g). It was stored at 0-4 ‘C until used. When needed, the residual extract was dissolved in distilled water and used in the study [6].

Drugs and Chemicals used

Streptozotocin, thiobarbituric acid (TEA), phenazin methosulphate (PMS), nitroblue tetrazolium (NET), NADH, NADPH and glucose-6­phosphate were purchased from Sigma Chemical Company (St. Louis. MO, USA). GI 1clazide (Servier Laboratories, France) %v as purchased

from local pharmacies. All other chemicals were of high purity grade.

Experimental Animals

All experiments were performed using adult male albino rats, with

an average body weight of  50 to 180 g. purchased from  animal house of faculty of medicine , seven of October University, Misuratah, Libya. The rats were housed in steel mesh 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; STZ was dissolved in 0.05 M sodium citrate buffer, pH 4.5) [14]. 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).

Experimental Procedure

Toxicity test of an aqueous extract of the green leaves of morus alba

Fifty male rats were divided into five groups of 10 each and were administered orally with aliquot doses of the aqueous extracts of morus alba (200-1000 mg/kg). Mortality was observed after 72 hours [8].

Hypoglycemic action of morus alba. A total of 25 rats ( 15 STZ-diabetic surviving rats and 10 normal rats) were used in this experiment.

The rats were divided into 5 groups of 5 rats each as follows

group 1, normal control rats.

Group 2 ,normal rats treated with morus alba extract (200 mg/kg body weight) daily by an  intragastric tube for two week.

Group 3, STZ-diabetic control rats.

Group 4.  STZ-diabetic rats treated with gliclazide (100 mg/kg body weight) daily by an intragastric tube for two weeks.

Group 5 . STZ-diabetic rats treated with mours, alba extract (200 mg/kg body weight)  an intragastric tube for two weeks;

At the end of two weeks, the rats were deprived of food overnight and sacrificed by decapitation under ether anesthesia. Blood samples were collected and the livers were immediately dissected out, washed in ice-cold saline, blotted dry and weighed for measuring various biochemical parameters.

Preparation of Homogenates

An accurately weighed piece of liver tissue was homogenized in ice-cold 0.9 % saline using a Teflon pestle connected to a homogenizer motor. The liver homogenate was

diluted to yield a 5 %( W/V )liver  homogenate.

The homogenate was centrifuged at 5000 rpm for 30 minutes at 4 C to remove cell debris and nuclei.

The resulting supernatant was used for biochemical analysis.

Biochemical Analysis

Serum glucose concentrations were estimated by the method of Trinder [9] 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., [10]. Hepatic glucose-6-phosphate dehydrogenase activity was measured b,, applying the method of Chan et al., [11]. Hepatic g]Llcose-6- phosphatase actl%-It% was determined according to the method of’ Rossetti et al.,[ 121.

Statistical Analysis

The results are expressed as means ± SD. Statistical analysis \N,as performed according to the method of Murray [13]. Data were analyzed using unpaired Student’s t-test. P values of < 0.05 were considered to be statistically significant.

Results

The acute oral toxicity of the green leaves o morus alba extract showed neither toxicity nor mortality up to 1000 mg/kg. Thus, the maximum tolerated dose of the extract was found to be 1000 mg/kg body weight. Also no toxic signs were observed over 72 hours of administering the extract.

Table 1, demonstrates serum blood glucose levels in normal control, STZ-diabetic control, STZ-diabetic treated and normal-treated rat with the aqueous extract of mous alba. An extremely significant (P<0.00 I) increase in serum blood glucose levels was observed in STZ­diabetic control rats compared with that of normal-control rat group.

In contrast, an extremely significant (P<0.001) decrease in serum blood glucose levels was observed in either gliclazide (100 mg/kg) or mortis alba extract (200 mg/kg) STZ-diabetic treated rat groups as compared to STZ-diabetic control rat group. No significant changes were observed in serum blood glucose levels of normal-treated rats compared with the corresponding levels of normal-control rat group upon treatment with 200 mg/kg of mortis alba extract (Table 1).

Table 1: Blood glucose levels in normal and STZ-diabetic rats treated with morus alba extract for two weeks treatment.

The results are expressed as mean ± SD for five rats in each group.

Extremely significant (P < 0.001) compared to normal-control.

Extremely significant (P<0.001) compared to STZ-diabetic control rat groups.

NS: Not significant compared to normal-control rat group.

Table 2, illustrates Hepatic glycogen content, glucose-6-phosphate dehydrogenase and glucose-6-phosphatase activities of different experimental rat groups. There were an extremely significant (P<0.001) decrease in glycogen content and glucose-6-phosphate dehydrogenase activity, and an extremely significant (P<0.001) increase in glucose-6- phosphatase activity in the liver homogenates of STZ-diabetic control

rats compared with that of normal-control rat group. In contrast, an extremely significant (P<0.00 I) increase in hepatic glycogen content and glucose-6­phosphate

dehydrogenase activity, and an extremely significant (P<0.001) decrease in glucose-6-phosphatase activity were observed in either gliclazide (100 mg/kg) or mortis alba extract (200 mg/kg) STZ­diabetic treated rat groups as compared to STZ-diabetic control rat group. No significant changes were observed in glycogen content and glucose-6­phosphate dehydrogenase and glucose-6-phosphatase activities of normal-treated rats compared with the corresponding values of normal-control rat group upon treatment with 200 mg/kg of mortis alba extract (Table 2).

Table 2: Effect of two weeks treatment with mortis alba extract on hepatic glycogen content, glucose-6-phosphate dehydrogenase and glucose-6-phosphatase activities in the liver of different experimental rat groups.

The results are expressed as mien ± SD for five rats in each group. ***- Extremely significant (P< 0.001) compared to normal-control.

*** : Extremely significant (P<0.001) compared to STZ-diabetic control rat groups

NS: Not significant compared to normal-control rat group.

Table 3, illustrates the effect of mortis alba extract on the insulin levels in various experimental rat groups. The results of Teble 3 showed that the insulin levels of normal control rat group, STZ-diabetic rat group treated with morns alba extract (200 mg/kg) and STZ-diabetic rat group treated with gliclazide ( 100 mg/kg) were significantly higher than that of STZ-diabetic untreated rat group.

Discussion

Hypoglycemic effect of the aqueous extract of morus alba.

The discovery and development of new and more effective anticllabctic from plants is one of the main goals of present day and chemical research[15]. In the present study, a detailed account has been given to the hypoglycemic effect of the morns alba extract in STZ­induced diabetic rats. The expanded hypoglycemic effect of nzol-lis alba till two weeks may be attributed to its long efficacy on glucose uptake hy the lived cells when compared to the effect of gliclazide (a known hypoglycemic drug).       .

In diabetes mellitus, the disorders in  carbohydrates metabolism plays a   predominant role in diabetic complications [16]. This fact has been observed in the present work in which decreased the levels of hepatic glycogen content, glucose-6-phosphate dehydrogenase activity (glucose oxidation), and increased the activity of hepatic glucose-6-phosphatase (gluconeogenesis) when compared to normal control rat group.

The hepatic glycogen contents were significantly lowered in STZ­control rat group when compared to the normal control rat group. The decrease in glycogen contents in the liver homogenates of STZ-diabetic untreated rats may be attributed to the depression of glycogenesis pathway in the liver of STZ-diabetic untreated rats [17].

In  the present study, it was observed that the activity of G6PD was significantly  liver decreased in the liver homogenates of STZ-diabetic untreated rats when

compared to the corresponding activity of G6PD in the liver homogenates of the normal control rat group. The deficiency of the hepatic G6PD activity (as shown in Table 2) in STZ- diabetic ratsI 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 (Glucose-6-phosphate dehydrogenase (G6PD) is the rate limiting enzyme of the pentose phosphate pathway). These findings are in agreement with the previous

studies which reported that, G6PD activity was decreased in STZ-induced diabetic rats ( [18]. Furthermore, 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 [19].

Glucose-6-phosphatase (G6Pase), an enzyme located mainly III the liver and catalyzes the terminal step in both gluconeogenesis and glycogenolysis. Thus, an increase in G6Pase activity (Table 2) in the liver homogenates of STZ-diabetic untreated rats cells may cause a marked increase in the rate of glucose formation and also decreases glucose usage [20]. 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. These findings are in accordance with that of Ashokkumar and Pari [21] who reported that, the activity of G6Pase was significantly increased, whereas the activity of G6PD was significantly decreased in acute or neonatal STZ-induced diabetic rats.

The present results demonstrated that, the treatment with morus alba significantly ameliorated the adverse influence of streptozotocin,  as:

lowered blood glucose levels leading to increased hepatic glycogen contents,

and a regulation in the metabolic eiizN-i-nes of glucose utilization as compared to rats administered with streptozotocin alone. These results are Yconsistent with those of other studies using different other hypoglycaemic plants [22 – 24].

The present study showed that the aqueous extract of  mores alba  stimulated the secretion of insulin from pancreatic P-cells in different rat groups. Therefore, the hypoglycemic action of morus alba may be similar to the action of gliclazide.

Conclusion

The improvement of glucose metabolism and its blood levels after mores alba treatment of STZ-inhected rats might a treating influence of’ morus alba againstSTZ action. In addition, Our studies indicate that, the aqueous extract of the morus alba   possesses  an ant diabetic  effectn the STZinduced diabetic rats  The hypoglycemic activitv of morus alba may be attributed to either its regulation of metabolic enzymes of glucose utlization or enhance insulin secretion.

Further clinical investigations will be conducted prior utilization as a safe oral  antidiabetic agent.

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