Alloxan, a β-cytotoxin, destroys β-cells of islets of Langerhans of pancreas, resulting in a decrease in endogenous insulin secretion and paves the way for the decreased utilization of glucose by the tissues. [29
] In vitro
studies have shown that alloxan is selectively toxic to pancreatic β-cells, leading to the induction of cell necrosis. [32
] The cytotoxic action of alloxan is mediated by reactive oxygen species, with a simultaneous massive increase in cystolic calcium concentration, leading to a rapid destruction of β-cells. [34
] Decreased utilization of glucose by the tissues results in the elevation of BGL.
Expression of elevated fasting BGL confirms induction of diabetes in alloxan-induced experimental rats. The experiment focused on exploring the competence of medium-polar (benzene:acetone, 1:1, v/v) extract from the leaves of S. rebaudiana for medication of diabetes against positive control reference drug glibenclamide. The difference in the initial and final fasting BGLs of different groups in long-term (10-day) studies exposed a significant elevation in BGL in diabetic controls as compared with that of normal control, extract treated and glibenclamide treated rats. Treatment of BGL with Stevia extract indicates the effectiveness of the extract in experimental diabetic animals.
Medium-polar extract from leaves of S. rebaudiana, when administered orally (200 and 400 mg/kg) for 10 days, produced a significant (P < 0.01) dose-dependent reduction in BGL  as well as in the body weight , although body weight was regained by rats treated with both glibenclamide and Stevia extract. Stevia extract exhibited a significant control of BGLs in diabetic rats, together with lowest decrease in the body weight, as compared with glibenclamide. Alternative exogenous treatments to diabetes include dosage of insulin and sulfonylurea drugs (e.g., glibenclamide), which cause hypoglycemia followed by greater reduction in body weight are the most worrisome effects. Treatment with Stevia extract did not cause hypoglycemia as well as significant decrease in body weight of diabetic rats. Stevia extract was found to revitalize β-cells of pancreas, antagonizing β-necrotic action of alloxan.
Effects of Stevia extract on blood glucose level (mg/dL) of diabetic rats
Effects of Stevia extract on body weight of diabetic rats
Excessive hepatic glycogenolysis and gluconeogenesis associated with decreased utilization of glucose by tissue is the fundamental mechanism underlying hyperglycemia in the diabetic state. [35
] Aberration of liver glycogen synthesis or glycogenolysis in diabetes may be due to lack of or resistance to insulin, which is essential to activate glycogen synthase system. The significant increase of liver glycogen level in Stevia
extract-treated groups may be due to reactivation of the glycogen synthase system by improving insulin secretion. Diabetes is associated with weight loss. [36
] The reversal of weight loss in extract-treated diabetic group indicates that the restorative effect of the extract may be due to the reversal of gluconeogenesis and glycogenolysis.
Experimental results also reflect that the Stevia
extract is capable of reducing the oxidative state associated with diabetes. Alloxan produces diabetes by liberating oxygen-free radicals which cause lipid peroxide-mediated pancreatic injury. [37
] The extract may scavenge free radicals and facilitate reconstruction of pancreatic cells to release more insulin and ultimately produces an antidiabetic effect.
Effects on blood glucose level
Administration of benzene:acetone extract (200 and 400 mg/kg) produced a significant (P < 0.01) dose-dependant reduction in BGL of alloxan-induced diabetic rats. Alloxanized rats of group II (negative control) suffered from hyperglycemia as they did not receive any drug, whereas alloxanized rats of group III (positive control) treated with the reference antidiabetic drug glibenclamide showed significant reduction in BGL to the required standard blood glucose level on the 7th day and the levels were continuously maintained up to 10th day. Rats of group IV treated with Stevia extract (200 mg/kg) showed nearly normal BGL (99.00 ± 7.98 mg/dL) value on the 10 th day, whereas group V rats treated with Stevia extract (400 mg/kg) also showed decrease in blood glucose level to nearly normal (93.69 ± 9.33 mg/dL) value, which is very close to 0 day BGL of group V. shows that positive control glibenclamide treated rats attained normalized BGL on the 7th day of treatment, whereas Stevia extract treated rats attained nearby normal BGL on the 10th day.
Effects on body weight
Administration of benzene:acetone extract of Stevia (200 and 400 mg/kg) produced a significant (P < 0.01) dose-dependent reduction in body weight of alloxan-induced diabetic rats. Group II (alloxan-induced negative control) rats revealed 4.46, 32.41 and 36.32% decrease in the body weight on 3rd, 7th and 10th days, respectively, with respect to 0 day control value. Group III (positive control with glibenclamide) rats revealed 5.50, 8.21 and 6.71% decrease in the body weights monitored on 3rd, 7th and 10th days of treatment, respectively. Rats in group IV treated with Stevia extract (200 mg/kg) revealed 10.81, 4.27 and 2.20% decrease in the body weight, while group V (400 mg/kg) rats revealed 16.87, 6.85 and 2.59% decrease in body weight on 3rd, 7th and 10th days, respectively, as compared with 0 day value. Least decrease in body weight was observed in group IV rats (200 mg/kg), i.e., 2.20% on the 10th day .
Glibenclamide versus Stevia extract treatment
The effects of oral administration of medium-polar (benzene:acetone, 1:1, v/v) extract of S. rebaudiana leaves are shown in Tables and . Experimental studies clearly reveal that the medium-polar extract from S. rebaudiana leaves (200 and 400 mg/kg) orally administered for 10 days produced a delayed but significant decrease in the blood glucose level, together with lesser loss in the body weight, as compared with standard positive control drug in the model of alloxan-induced diabetes in rats.
Effects on liver, renal and pancreatic weights
shows the effect of medium-polar extract of S. rebaudiana on renal, pancreatic and hepatic weights of normal, diabetic and diabetic treated rats. A significant intergroup difference (P < 0.05) was observed in glibenclamide treated group and diabetic control group. The liver weight of the normal rats was greater as compared to that of the diabetic control rats and treated diabetic rats. As shown in , administration of alloxan decreased the liver mass to 1.15 ± 0.2 g/100 g body weight, which showed significant difference (P < 0.01) with respect to non-diabetic rats. The liver mass was increased in diabetic treatment groups and glibenclamide treatment groups significantly (P < 0.05) with respect to diabetic control groups. Alloxan administration also caused a decrease in the pancreatic tissue weight. Treatment with the extract caused a significant increase in pancreatic tissue weight (P < 0.05) with respect to diabetic control. S. rebaudiana extract reduced the elevated kidney weight slightly as compared to untreated diabetic rats, although this did not reach statistically significant level.
Effect on liver, pancreatic and kidney weights in diabetic rats
Long-term pretreatment with sulfonylurea glyburide (GB) causes elevated basal insulin secretion (BIS) and decreased glucose-stimulated insulin secretion (GSIS). These characteristics may play an important role in the development of hypoglycemia and secondary failure. Results revealed that stevioside was able to counteract the desensitizing effects of GB and may be a putative new drug candidate for the treatment of type 2 diabetes mellitus. [38
] Abudula et al
. in 2004 [39
] showed that rebaudioside A potentially stimulates insulin secretion from isolated mouse islets in a dose-, glucose- and Ca2+
-dependent manner. According to the study of Dyrskog et al
] rebaudioside A failed to show beneficial effects in diabetic animals. In continuation of the previous study, Abudula et al
. in 2008 [41
] reported the mechanism for the insulinotropic action of rebaudioside A.
According to the study of Gardana et al
] on the metabolism of stevioside and rebaudioside A from S. rebaudiana
extracts by human microflora, both stevioside and rebaudioside A were completely hydrolyzed to their aglycon steviol in 10 and 24 h, respectively. Interestingly, the human intestinal microflora was not able to degrade steviol, which suggests that Stevia
glycosides are zero calorie sweeteners and thus can be utilizable as a dietary supplement by diabetic patients or these sweeteners can also be used for preparing cough syrups.
Stevioside is not absorbed by the human gut; only bacteria of the colon degrade stevioside to steviol. Part of this steviol is absorbed by the colon and transported to the liver by portal blood. In the liver, steviol glucuronide is formed, which is released into the blood and filtered out by the kidneys into the urine. High levels of steviol glucuronide in the urine suggest that there is no accumulation of steviol derivatives in the human body. The steviol glucuronide still present is expected to be excreted in the urine during the next 24 h. Besides steviol glucuronide, no free steviol or any other possible steviol metabolite could be detected in blood or urine. Hepatic metabolism of steviol is extremely low, if existing at all, which is in agreement with the results of Koyama et al
] who demonstrated by their in vitro
experiments that the steviol metabolism by human microsomes was 4 times lower than that by rat microsomes, and the latter one was already very low. [44
A recent in vivo
study by Melis et al
., in 2009, [45
] carried out on 30 male rats, toward evaluation of the renal excretion of steviol suggested that steviol at all doses (0.5, 1.0 and 3.0 mg/kg/h) used, except the lowest (0.5 mg/kg/h), induced a statistically significant increase in glucose clearance when compared to control and exhibited a dose-dependent effect. In our medium-polar extract, the amount of stevioside was 0.45% (dry leaves weight basis) as determined by high-performance thin-layer chromatography (HPTLC) method. Thus, the antidiabetic (hypoglycemic) effects of this extract may be due to the presence of stevioside, rebaudioside A and other sweet glycosides, as was also shown in polar chromatographic signature/profile  of benzene:acetone extract.